UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The molecular mechanism of copper-containing nitrite reductases Boulanger, Martin J. 2002

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2002-731316.pdf [ 7.07MB ]
Metadata
JSON: 831-1.0103826.json
JSON-LD: 831-1.0103826-ld.json
RDF/XML (Pretty): 831-1.0103826-rdf.xml
RDF/JSON: 831-1.0103826-rdf.json
Turtle: 831-1.0103826-turtle.txt
N-Triples: 831-1.0103826-rdf-ntriples.txt
Original Record: 831-1.0103826-source.json
Full Text
831-1.0103826-fulltext.txt
Citation
831-1.0103826.ris

Full Text

The molecular mechanism of copper-containing nitrite reductases. by Martin J. Boulanger B.Sc , The University of Victoria, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A December 2001 © Martin J. Boulanger, 2001 U B C Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Un i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of th i s thesis f o r sch o l a r l y purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 1/22/02 Abstract Nitrogen is an essential component of all living systems and availability is largely governed by the terrestrial nitrogen cycle. From this system, several forms of nitrogen are available for use in the synthesis of biologically relevant molecules such as proteins and nucleic acids, and for bioenergetic respiratory processes in facultative anaerobes. An imbalance in the nitrogen cycle can lead to many globally harmful events, including toxification of water sources and production of the environmental pollutants nitric and nitrous oxide. One of the crucial enzymes of the nitrogen cycle is nitrite reductase, which catalyses the key environmental step of converting a mineral form of nitrogen (nitrite) into a gaseous form (nitric oxide). The objective of this thesis is to characterize the molecular mechanism of copper-containing nitrite reductases (CuNiR) through the identification and characterization of catalytically important residues of the CuNiR from Alcaligenes faecalis S-6 (AfNiR). In the native nitrite-soaked crystal structure of AfNiR, nitrite bound to the active site copper forms a hydrogen bond with the side-chain of Asp98 that is connected to His255 through a solvent-bridged hydrogen bond. Three variants (D98N, H255D and H255N) of Asp98 and His255 were generated to probe the proton donation role of these residues. Nitrite reductase assays showed large reductions in activity for all three variants relative to native AfNiR suggesting an essential catalytic role for Asp98 and His255. Spectroscopic studies showed that the mutations did not affect significantly copper occupation although small changes in the electronic structures of the metal co-factors were detected, consistent with positional shifts in the ligand solvent observed in the structures. High-resolution nitrite-soaked crystal structures of the D98N and H255N AfNiR ii variants in both the.oxidized and reduced state show clearly that both residues are essential for directing productive nitrite binding in the active site. In the D98N nitrite-soaked structures both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N5 atom of Asn98 to form a hydrogen bond with the bound nitrite indicating that the hydrogen bond between Asp98 and nitrite in the native AfNiR structure is important in anchoring nitrite in the active site for catalysis. In the nitrite-soaked H255N crystal structures, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. The reoriented nitrite serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated simultaneously to the copper. In the native enzyme, an isoleucine residue (Ile257) occludes the active site pocket and packs closely against the bound nitrite. A combinatorial mutagenesis approach was used to generate small library of six variants at position 257 in AfNiR. The activities of these six variants span nearly two orders of magnitude with one variant I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (< 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies show that a bidentate, O-coordinate mode of nitrite binding is required for catalytic productivity and that the nature of the residue at position 257 strongly directs this mode of binding iii Table of contents Abstract » Table of contents iv List of tables vii List of figures viii List of abbreviations x Acknowledgements xii Chapter 1 - Introduction 1 1.1 History and Overview 1 1.2 Bioenergetic respiration 2 1.3 The terrestrial nitrogen cycle 4 1.4 Nitrogen metabolism 6 1.4.1 Nitrification 6 1.4.2 Denitrification 6 1.4.2.1 Enzymes of the denitrification pathway 7 1.5 Dissimilatory nitrite reductases 12 1.5.1 Heme-containing NiRs 13 1.5.2 Copper containing NiRs 15 1.5.2.1 Phylogenetic diversity 15 1.5.2.2 Biological function and regulation 16 1.5.2.3 Structure 21 1.5.2.4 Type I copper site and electron transfer 25 1.5.2.5 Type II copper and the active site 28 1.5.2.6 Proposed catalytic mechanisms 30 1.6 Objectives, hypotheses and outline 31 Chapter 2 - Materials and Experimental methods 33 2.1 Materials 33 2.1.1 Chemical supplies and media 33 2.1.2 Bacterial strains and plasmids 33 2.2 General experimental methods 36 2.2.1 D N A manipulation 36 2.2.2 Basic protein characterization 38 2.2.3 Combinatorial mutagenesis 39 iv 2.2.4 Nitrite reductase activity assay 40 2.2.5 Pseudoazurin based assay 40 2.2.5.1 Cloning, expression and purification of pseudoazurin 41 2.2.6 Basic fuschin assay (Sulfite quantification) 42 2.3 Spectrometry 43 2.3.1 Mass spectrometry 43 2.3.2 Ultraviolet-Vis spectrometry 43 2.3.3 Atomic absorption spectrometry 44 2.3.4 Electron paramagnetic resonance spectrometry 44 2.4 Structure Determination 45 2.4.1 Crystal growth and substrate soaking 45 2.4.2 Crystal manipulation and data collection 46 2.4.3 Structure solution and refinement 47 2.5 Optimization of AfNiR expression and purification 49 2.5.1 Existing periplasmic expression system 49 2.5.2 PCR amplification and cloning of nirK 51 2.5.3 Growth conditions 53 2.5.4 Periplasmic expression of pAfNiR22b 54 2.5.5 Cytoplasmic expression of pAfNiR28a 56 2.5.5 Current expression and purification protocol for AfNiR 58 Chapter 3 - Catalytic role for two water bridged residues (Asp98 and His255) in the active site of copper-containing nitrite reductase 60 3.1 Introduction 60 3.2 Results 61 3.2.1 Characterization of mutants 61 3.2.2 Activity assays 64 3.2.3 Crystallography 64 3.2.3.1 Structure of D98Nmutant 67 3.2.3.2 Structure of H255Nmutant 70 3.2.3.3 Structure ofH255D mutant 71 3.3 Discussion 72 3.3.1 Active site hydrogen bond network 72 3.3.2 RoleofAsp98 73 3.3.2 RoleofHis255 75 3.3.2 Revised mechanism 76 3.4 Conclusion 78 Chapter 4 - Alternate substrate binding modes to two mutant forms of nitrite reductase from Alcaligenes faecalis S-6: Structural model of a transient catalytic intermediate 79 4.1 Introduction 79 v 4.2 Results 80 5.2.1 Native active s ite 80 5.2.2 Mutant active sites 83 5.2.3 Type I copper site 89 4.3 Discussion 92 5.3.1 Role of Asp98 in determining binding mode of nitrite 92 5.3.2 Role of His255 in determining binding mode of nitrite 93 5.3.3 Catalytic mechanism of copper-containing nitrite reductase 96 5.3.3 Type I copper site 99 4.4 Conclusion 101 Chapter 5 - Role of Ue257 in directing the mode of nitrite binding in AfNiR 102 5.1 Introduction 102 5.2 Results 103 5.2.1 Characterization of AfNiR variants 103 5.2.2 Overall structures 104 5.2.3 Bidentate mode of nitrite binding 107 5.2.4 Monodentate mode of nitrite binding I l l 5.2.5 Effects of sulfite on the removal of nitrite 114 5.3 Discussion 120 5.3.1 Native active site and the hydrophobic blanket 120 5.3.2 Role of Ile257 in determining the mode of nitrite binding 120 5.3.3 Mechanistic implications 123 5.3.4 Sulfite directed removal of NO 124 Chapter 6 - The molecular mechanism of copper-containing nitrite reductases: An overview 127 6.1 Summary the proposed catalytic mechanism for CuNiR 127 6.2 Distinct mechanisms for heme cd\ and CuNiRs: supportive evidence 130 6.3 Binding modes of nitrite: A mechanistic dilemma 132 6.4 The essential role for Asp98 134 6.5 Conclusions 134 6.6 Outstanding questions and futures directions 137 Bibliography 140 Appendix - Publications arising from graduate work 156 vi List of tables 1.1 Examples of inorganic electron acceptors 4 1.2 Chemical reactions of the dissimilatory denitrification pathway 9 2.1 Bacterial strains used in this study 34 2.2 Bacterial plasmids used in this study 35 2.3 Degenerate primers used in combinatorial mutagenesis of Ile257 39 2.4 List of oligo-nucleotides used in afnir gene constructs 52 2.5 Primer combination for periplasmic and cytoplasmic gene constructs 53 3.1 Characterization of recombinant native, D98N, H255N and H255D AfNiRs 62 3.2 EPR parameters of native and mutant AfNiRs 64 3.3 Data collection and refinement statistics for D98N, H255N and H255D crystal structures 66 4.1 Data collection and refinement statistics for oxidized and reduced D98N[N0 2"] and H255N[N02"] 81 4.2 Type II copper - ligand bond lengths for oxidized and reduced D98N[N0 2"] and H255N[N02"] 88 4.3 Type I copper - ligand bond lengths and geometries for oxidized and reduced D98N[N0 2"] andH255N[N02"] 91 5.1 Characterization of native and Ile257 AfNiR variants 104 5.2. Data collection and refinement statistics for crystal structures of Ile257 AfNiR variants 105 5.3 Type II copper - ligand bond lengths and geometries for Ile257 AfNiR variants 110 vii List of figures 1.1 Biogeochemical inorganic nitrogen cycle 5 1.2 Cellular localization of the bacterial dissimilatory denitrification enzymes 8 1.3 Phylogenic comparison of CuNiRs 18 1.4 Sequence alignment of select class I CuNiRs 20 1.5 Secondary structure representation of AfNiR 23 1.6 Ligand environment of the type I and II copper centers in AfNiR 24 1.7 Electrostatic surface representation of AfNiR and pseudoazurin from Alcaligenes faecalis S-6 27 1.8 Active site of AfNiR with solvent and nitrite copper ligand 29 2.1 AfNiR crystals 46 2.2 Expression of pUC19q/m> in four different E.coli cell lines 50 2.3 Schematic for cloning of afnir into pET28a and pET22b 54 2.4 Time-course periplasmic expression of pAfNiR22b construct in HMS174(DE3)at37°C 55 2.5 Time-course expression of pAfNiR28a in HMS174(DE3) at 37° C and co-expression with molecular chaperones 56 2.6 Time-course expression of pAfNiR28a in HMS174(DE3) at 30° C 57 3.1 Electron paramagnetic resonance (EPR) spectra of recombinant native, D98N, H255N and H255D AfNiR 63 3.2 Active site structure of native and D98N, H255N and H255D AfNiRs 69 3.3 Proposed active site hydrogen bond network in native AfNiR, D98N, H255N and H255D 73 3.4 Proposed catalytic mechanism for AfNiR 77 4.1 Stick diagram showing the orientation of nitrite in the active site of oxidized nitrite-soaked native AfNiR, D98N[N0 2"] and H255N[N02"] 82 4.2 Active site view of oxidized and reduced nitrite-soaked D98N[N0 2"] and H255N[N02"] 85 4.3 Stick diagram showing the dinuclear type I copper site in reduced nitrite-soaked H255N[N02_] structure 89 viii 4.4 Catalytic mechanism for copper-containing nitrite reductases 97 4.5 Stereo diagram of the dinuclear type I copper site from reduced nitrite-soaked H255N[N02_] AfNiR type I copper site superimposed on C U A site from nitrous oxide reductase 100 5.1 Electron density maps showing the different orientation of nitrite bound to the six Ile257 AfNiR variant crystal structures 109 5.2 The effect of sulfite on the removal of nitrite from solution as measured by a competition based nitrite reductase assay 116 5.3 Monitoring the reoxidation of pseudoazurin in presence of AfNiR nitrite and sulfite 118 5.4 Removal of sulfite from solution monitored with a basic fuschin assay 119 5.5 Space-filled model of the active site cleft of AfNiR and partial amino acid sequence alignment of copper-containing nitrite reductases 121 6.1 Summary catalytic mechanism for CuNiRs 128 6.2 The multiple conformations of nitrite in AfNiR variants 133 ix List of abbreviations NaR Nitrate reductase NaP Periplasmic nitrate reductase NaS Cytoplasmic membrane-bound nitrate reductase NiR Nitrite reductase NoS Nitric oxide reductase NoR Nitrous oxide reductase nirK Gene encoding copper-containing nitrite reductase afnir nirK gene from Alcaligenes faecalis S-6 AfNiR Nitrite reductase from Alcaligenes faecalis S-6 AcNiR Nitrite reductase from Achromobacter cycloclastes AxNiR Nitrite reductase from Alcaligenes xylosoxidans RsNiR Nitrite reductase from Rhodobacter sphaeroides SOD Super oxide dismutase A Angstrom unit (1 A = 0.1 nm) B Crystallographic thermal factor KDa Kilodaltons O.D. Optical density Fo, Fc Observed and calculated structure factors r.m.s Root mean squared FPLC Fast Phase Liquid Chromatography b.p. Base pairs ATP Adenosine Tri-phosphate L B Luria - Bertani broth IPTG Isopropylthio (3 galactoside SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electroph Taq Thermus aquaticus D N A polymerase B S A Bovine Serum Albumin Tris Tris(hydroxymethyl) aminomethane MES (2-[N-Morpholino]ethanesulfonic acid) EPR Electron paramagnetic resonance X E X A F S Extended X-ray Absorption Fine Structure ENDOR Electron Nuclear Double Resonance FT-IR Fourier Transform Infrared Spectroscopy U.V. Ultraviolet Vis Visible PCR Polymerase Chain Reaction E° Standard reduction potential °C Degrees Celsius K Degrees Kelvin K m Michaelis constant KCat Catalytic constant Kan r Kanamycin resistance Amp r Ampicillin resistance MOPS 4-morpholinepropanesulfonic acid dNTP dinucleotidetriphosphate K H z kilohertz mW milliwatt G Gauss Rmerge Ehki Si |L(hkl) - <I(hkl))| / E h k , Ej Ii(hkl) Rwork S ||F0bs| " |Fcalc|| / S |F0bs| The conventions of the IUPAC - IUB Combined Commissions on Biochemical Nomenclature are followed for the three letter and one-letter abbreviations for amino acids (IUPAC-TUB 1965, TUPAC-TUB 1968), for designating atoms and for describing the conformational torsion angles of the polypeptide chain (IUPAC-IUB 1970). Variant proteins are designated by the one letter code for the native amino acid followed by the number of the residue and the one letter code for the variant residue. Nitrite-soaked crystal structures of AfNiR variants are designated with the following nomenclature (variant protein[nitrite]), for example I257T[N02"]. xi Acknowledgements This thesis is dedicated to all those people who have supported and believed in me from the beginning. I would like to thank my wife Kathy, my parents Gail and Lou, and my brother Joey, for their tireless encouragement. A special thank-you goes out to Dr. Edward Ishiguro who was instrumental in developing my interest in science during my undergraduate degree. I am indebted to my supervisor Michael Murphy, who continually pushed me to reach the next level. I have also benefited greatly from my committee members, Drs. Ross MacGillivray, A . Grant Mauk and Natalie Strynadka, each of whom generously opened their labs to me throughout my graduate degree and in their own way helped me become a better scientist. I especially would like to thank Ross for his continual positive reinforcement and the opportunity to meet his friends and colleagues. A special acknowledgment goes out to all the grad students, technicians, and post-docs who provided technical support throughout my graduate degree. Gary Sidhu deserves an award for all the time he spent teaching me crystallography. Thank-you to Drs. Federico Rosell and Houming Zhang who provided invaluable assistance during spectroscopic data collection and to Nham Nguyen for teaching me how to grow and mount crystals. I am grateful to Hung Vo, Gloria Lau, Gord Rintoul, J.P. Heal, Rene Warren, Dan Lim and Derek Knoechel who helped me in more ways than I can count. Mike Page in particular deserves a special mention for teaching me the value of thinking first and acting later. Finally, cheers to all the guys, Mike P., Gary, Vinh, Grant, Darko, Eric, Angus, Derek, big Mike, Iain, and Mike K. for the "extracurricular" activities during grad school. xii Chapter 1 - Introduction Chapter 1 - Introduction 1.1 History and Overview Organic nitrogen is an integral structural and metabolic component of living cells. In living systems, nitrogen is found primarily complexed with hydrogen, carbon, and oxygen in the form of nucleotides and amino acids. Nitrogen makes up nearly 80 % of the earth's atmosphere in the form of a stable dinitrogen molecule that, except for select nitrogen fixing species, is not biologically available. The demands to feed a growing world population of the early twentieth century led to the development of a method for converting stable atmospheric nitrogen into ammonia that is currently used as the basis for most agricultural fertilizers. This chemical conversion, known as the Haber-Bosch process, currently produces more fixed nitrogen in the form of ammonia than all of the denitrifying microorganisms on earth. The introduction of chemically derived ammonia based fertilizers has made possible the feeding of the nearly 6 billion people on this planet, but not without serious health and environmental impacts. Among the more serious environmental concerns is the contamination of water sources by toxic nitrogen compounds. During the development of infants, for example, excess nitrite in drinking water can interfere with the conversion of methemoglobin into hemoglobin, limiting the body's ability to transport oxygen leading to methemoglobenemia or blue baby syndrome (Fisher 2001). The environmental impacts are also severe. In the presence of high levels of nitrogen from agricultural or sewage run-off, algal species can grow uncontrollably effectively depleting water sources of oxygen and nutrients. Some of these algal species can have serious impact on commercial fisheries and environmentally sensitive areas through the formation of red tide, during which devastating chemical toxins 1 Chapter 1 - Introduction are released (Fisher 2001). Denitrifying microorganisms have been used with some success in the bioremediation of contaminated water sources with some of the nitrogen cycle enzymes more recently being extended to the engineering of environmental biosensors for nitrate and nitrite (Aylott et al. 1997, Wu et al. 1997). The removal of excess nitrogen from the environment is also not without problems. During denitrification gaseous intermediates are released that produce a significant environmental impact on a global scale. Nitrous oxide, for example, is a green house gas several hundred times more powerful than carbon dioxide and nitric oxide has been implicated in the destruction of the ozone layer (Averill 1996). The focus of this thesis is to characterize biochemically the copper-containing nitrite reductase from Alcaligenes faecalis strain S-6, which catalyzes the first committed step in the dissimilatory denitrification pathway through the conversion of a mineral form of nitrogen (NCV) into a gaseous form (NO). One of the long-term goals of deciphering the catalytic mechanism of copper-containing nitrite reductase is to engineer enzymes capable of increased efficiency during bioremediation. 1.3 Bioenergetic respiration Many of the microorganisms that inhabit the surface of the earth obtain energy through the oxidation of a variety of energy replete substrates, such as carbohydrates, with the electrons transferred to oxygen reducing it to water. Certain species however, occupy specialized anaerobic environments where oxygen is limiting and therefore must use an alternative electron acceptor to oxygen. Some examples of terminal electron acceptors employed by many of these facultative anaerobes include nitrogen and sulfur oxides, and 2 Chapter I - Introduction carbon dioxide (Table 1.1). The terrestrial nitrogen cycle is driven largely by the chemical reduction and oxidation by denitrifying and nitrifying microorganisms that use nitrogen oxide compounds during respiration. Similar to aerobic respiration, the denitrification pathway is linked to the energy conserving N A D H dehydrogenase and cytochrome bcj complexes and can therefore be thought of as the anaerobic analogue of the oxygen based electron transport and respiration performed by cytochrome c oxidase. During aerobic oxidative phosphorylation, all of the enzymes in the electron transport chain, the cytochrome c oxidase complex, cytochrome aa?, oxidase and the quinol aa^ oxidase, are all capable of translocating protons. In contrast, of the enzymes involved in anaerobic dissimilatory respiration, only the initial enzyme of the pathway, nitrate reductase (NaR), is capable of translocating protons across the membrane. The ability to form a pH gradient across a membrane through proton transport, during electron transfer is related directly to the efficiency of the respiratory process. As a result, the overall efficiency expressed as charge per electron transferred is +5 for oxygen respiration and +3 for nitrogen-based denitrification indicating that oxygen is a more efficient terminal electron acceptor than nitrogen (Table 1.1). The efficiency of aerobic respiration has led many microorganisms to evolve sensors to detect the presence of molecular oxygen (Ferguson 1994, Jordan et al 1997, Melville et al 1990, Schroder et al 1993, Zumft 1997). 3 Chapter 1 - Introduction Table 1.1 Examples of inorganic electron acceptors1 Reaction , d G 0 / H 2 0 2 + 2H 2 -» 2 H 2 0 -238 2N0 3 " + 2 H + + 5H 2 N 2 + 6H 2 0 -225 N 0 3 " + 2 H + +4H 2 -> N H 4 + 3H 2 0 - 150 S0 4 2 " + H + + 4 H 2 -» SET 4 H 2 0 -38 HCO3" + 4H 2 + FT -» CH4 + 3H 2 0 -34 'Table adapted from (Brittain et al. 1992) 1.2 The terrestrial nitrogen cycle The biogeochemical nitrogen cycle consists of several biologically and abiologically linked processes that circulate nitrogen species throughout the environment to be used in the life cycle of many organisms (Figure 1.1). Biologically, a crucial step in the nitrogen cycle is the conversion of atmospheric nitrogen (N 2) into ammonia (NH 3) that is used in the synthesis of biologically productive amines to be incorporated into proteins, nucleic acids and other essential building blocks of life. As organisms decay, these biological amines are released into the environment and oxidized primarily to nitrate and nitrite during nitrification. These higher oxidation species of nitrogen are then used as terminal electron acceptors in the synthesis of ATP during bioenergetic respiration in facultative anaerobic denitrifying microorganisms. 4 Chapter I - Introduction Lightning discharges N2(0) > NO, Nitro en N 2 ° ( + 1 ) Dissimilatory 1 ogen Denitrification f l X a ) 1 0 n NO(+2) 1 V NH/(-3) , A s s i m i l a t o r y N02"(+3) ; Denitrification NH2OH(-l) Nitrification N03'(+5) * Organic nitrogen used in biological amines Figure 1 .1 The biogeochemical inorganic nitrogen cycle. The substrates, intermediates and products of the dissimilatory denitrification pathway are highlighted in blue. Numbers in brackets indicate the formal oxidation state of the nitrogen oxide species. Adapted from (Averill 1994). 5 Chapter 1 - Introduction 1.4 Nitrogen Metabolism 1.4.1 Nitrification Nitrification is an aerobic process utilized by both autotrophic and heterotrophic microorganisms in which ammonia is oxidized to nitrate via hydroxylamine and nitrite intermediates (Figure 1.1). Autotrophic organisms, however, are the only ones to use this process to derive energy where the oxidation of ammonia to nitrite and nitrate involves the donation of electrons to oxygen. In some microorganisms, such as Paracoccus denitrificans, heterotrophic nitrification is not used in respiration, as these species are solely dependant on the oxidation of organic substrates for energy (Moir et al. 1996). In autotrophic microorganisms, the conversion of ammonia to nitrate proceeds by a two-step process catalyzed by different enzymes. In the first step the oxidation of ammonia to hydroxylamine is catalyzed by ammonia monooxygenase (AmMo) with oxygen serving as the exogenous electron acceptor. Little is known about AmMo but recent studies indicate a similarity to a membrane bound methane monooxygenase (Holmes et al. 1995) with the potential to bind both copper and iron (Zahn et al. 1996). In the second step, hydroxylamine is oxidized to nitrite by the heme containing soluble hydroxylamine reductase. 1.4.2 Denitrification Enzymes of the denitrification pathways are generally associated with two different roles; they can be involved in the synthesis of biological molecules (assimilatory denitrification) or in the respiratory pathway (dissimilatory denitrification), which is used to generate a transmembrane proton gradient that is coupled to the generation of ATP. Unlike the dissimilatory pathway, none of the enzymes of the assimilatory pathway conserve energy. 6 Chapter 1 - Introduction The six-electron reduction of nitrite to ammonia during assimilatory denitrification is accomplished with a hexa-heme cytochrome c nitrite reductase (Brittain et al. 1992, Lin 1998). Recently, two of these enzymes have been isolated and crystallized from the microorganisms Sulfurospirillum delenyanum (Einsle 1999) and Wolinella succinogenes (Einsle et al. 2000). The structures show the enzyme to be a homodimer with ten closely packed heme c cofactors. These enzymes are also capable of reducing nitric oxide and hydroxylamine (Costa et al. 1996). Ammonia produced by this reaction is generally used for biosynthetic purposes rather than excreted. The hexa-heme assimilatory NiRs also show structural homology to sulfite reductases and have been shown to catalyze the six electron reduction of sulfite to sulfide in Vibrio fischeri (Hirasawa-soga et al. 1983). Denitrifying bacteria occupy a wide range of habitats where oxygen is limiting, including soil, water and the digestive tract (Knowles 1982, Payne 1981, Tiedje 1988). As such, nitrate and nitrite are used instead of oxygen as electron acceptors during bioenergetic respiration in these microorganisms. The higher oxidation species of nitrite are reduced to gaseous nitrogen oxide compounds (NO, N 2 0 ) and dinitrogen (N 2). The dissimilatory pathway is responsible for generating the only biological source of dinitrogen (N 2) (Cutruzzola 1999) and is the only means of returning large amounts of fixed nitrogen to the atmosphere thereby completing the terrestrial nitrogen cycle (Figure 1.1). 1.4.2.1 Enzymes of the denitrification pathways In dissimilatory denitrification four metallo-enzymes are required to convert nitrogen oxide species between the oxidation states of+5 to 0 (Figure 1.1). These enzymes are a membrane bound or soluble form of nitrate reductase (NaR or NaP, respectively), a heme or 7 Chapter 1 - Introduction copper-containing nitrite reductase (NiR), nitric oxide reductase (NoR) and nitrous oxide reductase (NoS) (Figure 1.2 and Table 1.2). The cellular localization of these enzymes in bacteria (Figure 1.3) has been thoroughly studied using cell fractionation, antibody labeling and electron microscopy (Coyne et al. 1990, Komer 1992). Figure 1.2 Cellular localization of the bacterial dissimilatory denitrification enzymes. Nitrate reductase can exist either as a soluble periplasmic enzyme (NaP) or in a membrane bound form (NaS). Nitrite reductase (NiR) is a periplasmic enzyme that can bind either heme or copper. Nitric oxide reductase (NoR) and nitrous oxide reductase (NoS) represent the final two steps in bacterial denitrification. 8 Chapter 1 - Introduction Table 1.2 Chemical reactions of dissimilatory denitrification and reduction potentials measured at pH 7.0 Reaction E° (V) NCV + 2e" + H20 N02~ + 20H" + 0.42 NCY + le" + H20 NO + 20H" + 0.37 2NO + 2e~ + H+ N 20 + OH" + 1.17 N 20 + 2e" + 2H+ N 2 + H20 + 1.33 a Reduction potential values were obtained from (Averill 1994) Four different classes of nitrate reductase enzymes have been described (Berks 1995), only two of which are involved in dissimilatory denitrification. The remaining two belong to the structurally diverse class of assimilatory nitrate reductases that are found in prokaryotes and eukaryotes (Lin et al. 1998). The two structurally different dissimilatory nitrate reductases found in bacteria are involved in respiration and are either bound in the cytoplasmic membrane (NaR) or are soluble in the periplasm (NaP). These dissimilatory enzymes catalyze the two-electron reduction of nitrate to nitrite. The membrane-bound NaR is proposed to couple a transmembrane proton gradient to the oxidation of the electron transfer molecule quinol. The membrane-bound NaR is comprised of three polypeptide chains. The a and /3 chains associate with the inner surface of the cytoplasmic membrane and the y chain forms a transmembrane cv-helix. The alpha subunit contains the M G D co-factor to which the nitrate substrate binds. Both the a and /3 subunits likely coordinate multiple iron sulfur centers used in the shuttling of electrons. The y subunit contains two heme cofactors that are 9 Chapter 1 - Introduction reduced by ubiquinol (Berks et al. 1995). The periplasmic NaP is a multi-subunit enzyme that coordinates both an iron-sulfur center and a molybdenum bound as a fe-molybdopterin guanine dinucleotide (MGD) cofactor in the active site (Veselov et al. 1998, Watmough et al. 1999). Recently, the first crystal structure solved (PDB code - 2NAP) of a nitrate reductase is of the NaP form isolated from the sulfate reducing bacterium Desulfovibrio desulfuricans (Dias 1999). As nitrate is turned over, the product, nitrite, is transported across the cytoplasmic membrane (Figure 1.2) where it is converted to nitric oxide by a soluble heme or copper based nitrite reductase. An additional cytoplasmic nitrite reductase involved in nitrite assimilation or detoxification in plants and fungi does not show any sequence similarity to the bacterial enzymes. Coordination of a siroheme co-factor by these NiRs suggests certain structural homology with the siroheme containing sulfite reductase (Richardson et al. 1998). The dissimilatory bacterial nitrite reductase enzymes will be discussed more thoroughly in Section 1.5. Three different nitric oxide reductase (NoR) enzymes exist in nature, two in bacteria and the other in fungi that serve different physiological roles. In bacteria, nitric oxide reductases are bound in the cytoplasmic membrane and are either comprised of a NorC and NorB subunits to form cNoR (Carr et al. 1990, Dermastia 1991, Heiss et al. 1989, Kastrau et al. 1994) or a single polypeptide qNoR. In the bacterial chromosome, the norC gene is always located directly upstream of the norB gene. The nomenclature for these enzymes is derived from the nature of the in vivo electron donors. A c-type cytochrome in the bacterial NoRs is thought to accept electrons from in vivo donors, and a d-type cytochrome coupled to an adjacent iron atom forms a dinuclear cluster in the catalytic site that binds two molecules of NO (Arciero et al. 1998). A quinol donates electrons to qNoR from Ralstonia eutropha 10 Chapter 1 - Introduction (Cramm et al. 1997), which lacks the NorC subunit. Both enzymes exhibit structural similarity to the respiratory heme-copper oxidases (Watmough et al. 1999). These enzymes catalyze the two-electron reduction of nitric oxide to nitrous oxide through the formation of a nitrogen - nitrogen bond. A partial denitrification pathway in certain fungal species, such as Fusarium oxypsorum (Ferguson 1998) is connected to the mitochondrial respiratory chain and produces nitrous oxide as the final product instead of dinitrogen. The recent crystal structure of NoR from Fusarium oxypsorum (Park et al. 1997) reveals significant differences from the bacterial NoRs. The fungal enzyme exists as a soluble enzyme that is part of the cytochrome P450 family (Park et al. 1997) and uses N A D H as the in vivo electron donor. The product of NoR is nitrous oxide, a poorly reactive species that undergoes a two-electron reduction to dinitrogen catalyzed by nitrous oxide reductase (NoS). This represents the final step in bacterial dissimilatory denitrification. Recently, the nitrous oxide reductase from Pseudomonas stutzeri was isolated and characterized and shown to be a dimeric protein of 79 KDa subunits that binds 4 copper atoms per monomer (Brown et al. 2000, Prudencio et al. 2000). In the oxidized form, NoS is bright purple consistent with a mixed-valent, thiolate bridged dinuclear copper (CuA) (Neese et al. 1998) similar to the binuclear center found in cytochrome c oxidase. Crystallographic studies shows that a second copper site in NoS from Pseudomonas nautica (Brown et al. 2000) is a tetranuclear Cuz cluster. During catalysis, electrons are passed form the binuclear copper site to the tetranuclear copper that forms the active site of the protein and binds the substrate. 11 Chapter I - Introduction 1.5 Dissimilatory Nitrite Reductases During dissimilatory denitrification, the key environmental step is the conversion of a mineral form of nitrogen (nitrite - NCV) to a gaseous form (nitric oxide - NO) by nitrite reductase. Two genetically distinct dissimilatory NiRs exist that contain either heme c and heme di prosthetic groups or multiple copper centers (CuNiR) (reviewed in Cutruzzola 1999). Several of these NiRs have not yet been purified but alternatively were identified using D N A hybridization techniques (Smith 1992, Ye et al. 1993), PCR based methods (Braker et al. 1998) or inhibition of denitrification with the copper chelator diethyldithiocarbamate (DDC) (Shapleigh et al. 1985). Antibody cross-reactivity studies show a high degree of structural homology within the heme cd\ and CuNiR families (Coyne 1989). Heme and copper-containing nitrite reductases have never been found to coexist in the same microorganism and may represent an environmental adaptation based on the availability of copper and iron. Interestingly, a heme containing NiR is present in Alcaligenes faecalis while a copper-containing NiR has been purified from the closely related Alcaligenes faecalis S-6. Overall, there has been no clear correlation established between the microbial taxonomy of a microorganism and the type of NiR present. Interestingly, the nitrite reductase activity of a heme cd/ N iR deficient strain of Pseudomonas stutzeri can be complemented with a copper-containing nitrite reductase demonstrating that both classes of enzymes perform the same function in vivo (Glockner et al. 1993). Enzymological studies of both the heme or copper based NiRs have been difficult due primarily to potent inhibition of the enzyme by the product, nitric oxide (NO). The product inhibition results in non-linear kinetic plots from which it is difficult to obtain reliable initial 12 Chapter 1 - Introduction velocities. As a result, other techniques such as spectroscopic measurements and isotopic exchange studies have been used primarily to probe the catalytic mechanism of nitrite reductases (Averill 1996). 1.5.1 Heme containing nitrite reductase Cytochrome cd\ denitrifying nitrite reductases are encoded by the nirS gene and are the most common; being found in almost two-thirds of denitrifying species studied (Coyne et al. 1989). These enzymes are synthesized as a pre-protein that is exported to the periplasm where in the mature form they exist as a soluble homodimer of 120 KDa. Each monomer is related by a two-fold axis, coordinates covalently a heme c prosthetic center, and is non-covalently associated with a heme di group (Hochstein 1989, Weeg-Aerssens et al. 1991). The heme c domain is largely a-helical in structure and accepts electrons from soluble cytochrome C551 or azurin (Averill 1996). An eight bladed /3-propeller domain coordinates the heme dj that is unique to denitrifiers containing the nirS gene (Chang et al. 1986). The heme di cofactor is the site where nitrite binding and catalysis occurs. This unusual heme cofactor requires a specific biosynthetic pathway (Zumft 1997) that may be a limiting factor in the variety of species able to utilize the heme cd\ NiR. In the apoprotein, up to 80 % of the catalytic efficiency (Weeg-Aerssens et al. 1991) and spectral properties of the holoprotein (Silvestrini et al. 1992) can be restored following reconstitution with a synthetic heme di moiety. Although the functional role of these NiRs in denitrifiers is the one electron reduction of nitrite to nitric oxide, they are also capable of catalyzing the four-electron reduction of oxygen to water (Silvestrini et al. 1994). The mechanism of heme cd\ nitrite reductases is well characterized and proceeds via 13 Chapter 1 - Introduction the formation of an electrophilic nitrosyl intermediate (Fulop et al. 1995). In the first step, nitrite binds in an N-coordinate fashion to the reduced d\ heme co-factor forming an Fe 2 + -NO2" complex. In the second step, a dehydration reaction removes a water molecule leaving the highly unstable nitrosyl intermediate (Fe 2 + - N O + <—> Fe 3 + - NO), which rapidly decomposes to release NO. Intramolecular electron transport from the heme c moiety completes the catalytic cycle. The formation of the nitrosyl intermediate was first suggested from isotope exchange studies with 1 8 0 and 1 5 N labeled nitrite that showed that heme cd\ N iR could catalyze the nitrosation of hydroxylamine and azide (Fenderson et al. 1991, Kim 1983). The nitrosyl complex is diamagnetic and hence undetectable by EPR, but has been measured successfully with FT-IR for cdi NiRs from Pseudomonas stutzeri (Wang et al. 1996) and Paracoccus pantotrophus (George et al. 2000). The hydration step is supported from isotope exchange of 18 18 2+ O from H2 O into the product nitric oxide. The dead end product, Fe - NO is formed if the electron transfer occurs before the release of NO or via inhibition when the released NO rebinds to the oxidized enzyme (Weeg-Aerssens et al. 1991). Two protonated histidine residues near the heme dj co-factor have been shown to be important in the catalytic activity of heme cd; N iR from Pseudomonas stutzeri (Wilson et al. 2001) and Pseudomonas aeruginosa (Cutruzzola et al. 1997). These histidines are at least in part responsible for the nitrite binding affinity of ferrous heme d\ heme and donation of protons during the dehydration reaction of the bound nitrite. Furthermore, a hydrogen bond network from these histidines is thought to orient correctly a mobile active site tyrosine to favor competitive coordination of a hydroxyl group to the ferric heme d\ group and promote release of NO. Mutations of these histidines (Cutruzzola et al. 1997) results in significantly 14 Chapter 1 - Introduction reduced enzyme activity with the formation of an FeNO dead end product. 1.5.2 Copper-containing nitrite reductases 1.5.2.1 Phylogenetic diversity Copper-containing NiRs (CuNiRs) are encoded by the nirK gene and are observed in approximately one third of the denitrifying species studied. Although less prevalent than the heme containing NiRs, species containing CuNiRs are more taxonomically and physiologically diverse (Averill 1996). CuNiRs have been identified primarily in gram negative (Abraham et al. 1993, Fenderson et al. 1991, Kakutani et al. 1981b, Michalski et al. 1985) denitrifying soil bacteria but also occur in gram positive bacteria (Hoffmann et al. 1998). CuNiRs have also been isolated from eukaryotic species such as the fungus Fusarium oxysporum (Kobayashi et al. 1995), the archeal species Haloferax denitrificans (Inatomi 1996) and the halophile Bacillus halodenitrificans (Denariaz et al. 1991). The aniA gene, which shows weak sequence similarity (-30% identity) to other CuNiRs from soil bacteria has been cloned and sequenced (Hoehn et al. 1992a). Recently, an insertional mutation was made in the aniA locus establishing clearly that AniA is an inducible nitrite reductase essential for anaerobic growth of A. gonorrhoeae (Mellies et al. 1997) . Interestingly, AniA, unlike other CuNiRs, encodes a 5' palmitoylation signal that serves to anchor it to the outer membrane (Hoehn et al. 1992b). Expression of the soluble domain of AniA yields a protein capable of significant nitrite reducing activity (Boulanger 2001c). Despite low sequence identity, structural analysis shows the core cupredoxin fold of AniA to be similar to that found in copper-containing nitrite reductases from soil bacteria (Boulanger 2001c). Notably, the expression of AniA by N. gonorrhoeae has been shown to 15 Chapter I - Introduction provide protection against killing by human sera (Cardinale 2000) and the serum of patients with gonorrhoeae or pelvic inflammatory disease contains antibodies to AniA (Clark et al. 1988). Recently, a comprehensive phylogenic analysis (Figure 1.3) of fifteen different CuNiRs has shown that the family of copper-containing NiRs are actually comprised of two sub families, termed class I and II, that show minimal sequence similarity (Figure 1.4). Several of the class II CuNiRs, including that from N. gonorrhoeae, are derived from pathogens and show significant deletions in the primary sequence that map to surface regions associated with electron transfer properties in the class I CuNiRs (Boulanger 2001c). 1.5.2.2 Biological function and regulation Under oxygen limiting conditions, CuNiRs catalyze the one electron reduction of nitrite to produce nitric oxide and water. Although NO is the main product of CuNiRs, in vitro, N2O is also produced from AcNiR, i f NO is not removed from the reaction (Carr 1989, Jackson et al. 1991, Kim 1984, Weeg-Aerssens 1991). The reduction of nitrite by CuNiRs in the presence of oxygen is proposed to produce peroxides, which through oxidative chemistry can lead to the inactivation of the protein (Kakutani et al. 1981a). CuNiRs are generally present in species that live in low oxygen environments such as Alcaligenes faecalis S-6, which was originally engineered to increase the efficiency of wastewater remediation (Kakutani et al. 1981a). More recently, studies with NiR from Rhodobacter sphaeroides 2 A3 suggest that the product(s) of nitrite reduction, possibly nitric oxide, is required as an effecter molecule to up regulate the expression of the denitrification pathway (Tosques et al. 1997). 16 er 1 - Introduction Figure 1.3 A phylogenic tree of 15 different copper-containing nitrite reductases. Bootstrap values are given for nodes with values less than 75% (based 10,000 replicates). Accession numbers are given for sequence in the GenPep databank. The remaining sequences are preliminary translations from unfinished genome sequencing projects. Sequences were aligned using C L U S T A L W and the dendogram generated using the PROTDIST, NEIGHBOR, SEQBOOT and CONCENSUS programs of the PHYLIP package (Felsenstein 1996). (Figure was taken from (Boulanger 2001c)) 17 er 1 - Introduction o 0) CO CO O CO CO - C O 3 > > % CD <Q" (Q I Si 3 O X w con a o 3 o > > 2. Q O 3 o CT Dl o 3 5 3 M 5i CO CT> ~ 8 8 S 3 cn to 2 § 8 " M 00 O U l > CD ro -• 3 > CD <£> o o 3 Q. 18 er 1 - Introduction Figure 1.4 Amino acid sequence alignment including class I and II copper-containing nitrite reductases generated with C L U S T A L W (Higgins 1996). Sequence codes are as follows: AfNiR, Alcaligenes faecalis S-6 [2120968], AxNiR, Alcaligenes xylosoxidans [3721764], RsNiR, Rhodobacter sphaeroides 1480720], AniA, Neisseria gonorrhoeae and HmNiR, Haloarcula marismortui [CAB93142]. Red boxes indicate conserved residues. The type I and II copper atoms ligands are denoted by the symbols >k and!, respectively. Every tenth residue on a line is denoted by *. We acknowledge the Gonococcal Genome Sequencing Project supported by USPHS/NIH grant #AI38399, and B.A. Roe, L . Song, S.P. Lin, X . Yuan, S. Clifton, Tom Ducey, Lisa Lewis and D.W. Dyer at the University of Oklahoma - ACGT. Figure was taken from (Boulanger 2001c). 19 Chapter I - Introduction AfNiR 1 AxNiR 1 RsNiR 1 AniA 1 HmNiR 1 AfNiR 17 AxNiR 11 RsNiR 49 AniA 17 HmNiR 17 AfNiR 72 AxNiR 66 RsNiR 103 AniA 71 HmNiR 71 AfNiR 127 AxNiR 121 RsNiR 158 AniA 126 HmNiR 126 AfNiR 181 AxNiR 175 RsNiR 213 AniA 172 HmNiR 172 AfNiR 234 AxNiR 228 RsNiR 266 AniA 217 HmNiR 217 AfNiR 287 AxNiR 281 RsNiR 319 AniA 270 HmNiR 270 AfNiR AxNiR 336 RsNiR 374 AniA 325 HmNiR MRKATAA--EIAALPRQK MQDADKLPHTK -MFTRRAALVGAAALASAPLVIRTAGAQ—EAPAQLASAAPVDLSNLPRVK MAAQATAETPAGELPV ETTPQEPAMNAAQQTD VTLVAPPQVHPHEQATKSqPKWEFTf MfT IHEKKMVI DDKGTTLQAMTFNGSMpQP  9 HTLVPPPFAHAHEQVAASGJPp/1NEFEMRI]J EIKE VQLDE-DAYLQAMTfFpGSI  IDAVTTHAPEVPPAIDRDYjPAKVRVHMETMEIKTMKMDD-GVEYRYWT^FPGDWGP VDRIAADPTAIPDPIDRSHPKTVSVHl^TWEbVAEIEP-GVTYTYMTlEi LM^vtaQdDfirLELTLltjpETNTLMHMI  T L wViHE G DKVQ LT L V|Np AT N AM EJHNMDtelH  MlHVtRRQDtrVELTITpSI  LMBVHEGDfirVELTLIDSipPENTMHHMll EEGNSMHHMI MIFhTOEQDpVEVEFS|NNPSSTV^ ;DQI POP pgp DCTiKAflG^liGGGGLTE IMPGEKTILPJFKA AtiGklJGGkKLTNVNpGEQATLPjFK^ DtLHAVHGpGEGkEASMVT Dra^llGfeLPGGGLTLINpGEKVVLPjFKA F K A pGbTKTFPjFKA^ 127 TKPOVFV YHCAP PG-MVPWHpVS GMNG AIM V L PREJGIJHDGKGKALTY DKIYYVGE ]TFV (YHCAPEG -MVP^H|VVS |GM|S]^ DHEGKPVRYDpVYYIGE PKVDKEFYIVQ QDEYV-PRDENGKYKKYEAPGDAYEDTVKVTMRTLTP|THWE]NC^VG|AJLTGDK F D I Y I P K G P D G K Y K D Y A T L A E S Y G D T V Q V M R T L T SDK Y I P K D E D G T Y M R F S T P S E G Y E D M V A V M D T L I GDE Y T K G K K G A Q G L Q P F D H E I Y TTGDTGEKGHHDFD MEAMAAEEL -AM PB HI VEIN GtKVGjAtLT GAN—AL P 3HIVENGAVGALTGEG—AL MDKAVAEqP 3YWE NGHVG AIAGDN—AL TYVLIYNCEKYAITPDRHGSP n * tVWETG 217 KAKAlGfeTVRMYVGNGGPNLVSS EtHlvIl GJE I FtoK V YVE G SMQV|GpTARVYFVTGGPNLDSSETHp|lG|SVWp^  KFHNAPERDLETWFIRGG K—LINENVQSTIVPAG  G S A I V E F K V D I P G N Y T L V D K B I F R A F N K G A L G Q L K V F J G ^ E N P E I M T Q K L S D T A Y A SCAIATLHAEVh?GPIKLVDM^LSRVARKATMA1INREfckANPDVFEPEA 20 Chapter I - Introduction At the genomic level, there does not appear to be a clear clustering of the genes involved in denitrification (Cutruzzola 1999), with the exception of the nir-nor cluster in Pseudomonas aeruginosa (Zumft 1997), which maintains the intramolecular concentration of NO at nanomolar levels (deBoer et al. 1994, deBoer et al. 1996). Sequencing of genomic clones of CuNiRs from several species has identified several redox-linked transcription regulatory sequences similar to the FNR (fumarate and nitrate oxygen sensing) (Ye et al. 1993) and A N R (anaerobic regulation of arginine deiminase and nitrate regulation) (Spiro 1990) promoter regions. The A N R protein in particular is capable of up regulating the expression of several genes including those involved in denitrification (Cuypers 1993, Van Spanning et al. 1997, Ye et al. 1995). These observations show that some facultative anaerobic microorganisms have evolved sensors to detect chemical species besides nitrogen oxides that can be used as terminal electron acceptors during electron transport. Such an adaptation broadens significantly the environmental niches capable of supporting life for these microorganisms. 1.5.2.3 Structure Originally, gel filtration and sedimentation equilibrium studies of several copper-containing nitrite reductases reported the physiologically relevant molecule to be a dimer or tetramer with subunits in the 30 to 40 KDa range (Kakutani et al. 1981b, Masuko 1984, Michalski et al. 1985, Zumft 1987a). The crystal structure of the green NiR from Achromobacter cycloclastes was the first copper-containing nitrite to be solved (Godden et al. 1991). From this study the molecular topology was determined to be a homotrimer with subunits of approximately 37 KDa. Subsequently, the crystal structures of the green CuNiR 21 Chapter 1 - Introduction from Alcaligenes faecalis strain S-6 (AfNiR) (Kukimoto et al. 1994, Murphy et al. 1995) and the blue CuNiR from Alcaligenes xylosoxidans (Dodd et al. 1998, Inoue et al. 1998) were also solved to high-resolution and shown to be homotrimers. The extensive sequence similarities between these three different CuNiRs (> 60%) are consistent with the high degree of structural homology (Suzuki 2000). AfNiR is a green 110 KDa soluble periplasmic homotrimer with each monomer comprising an N-terminal (residues 1 to 161) and a C-terminal domain (residues 171 to 339) (Figure 1.5). Each domain is folded into a Greek key R-barrel motif of the cupredoxin fold and connected through a short linker region (residues 161 to 171). The N-terminal domain of each monomer is located at the exterior of the trimer while the C-terminal domain surrounds the three-fold axis at the trimer core. A stretch of polypeptide at the C-terminus (residues 306 to 314) extends beyond the main monomer - monomer interface and packs against the N-terminal domain of the adjacent monomer. Extensive inter and intra subunit hydrogen bond networks contribute significant rigidity to the overall molecule (Adman et al. 1995). Most of the known class I CuNiRs cross-react with polyclonal antibodies raised against the CuNiR from A. cycloclastes or from R. sphaeroides suggesting that there is substantial structural similarity. No significant global structural changes are observed in the enzyme over the pH range of 5.2 to 6.5, in the presence or absence of nitrite or when the copper atoms are in different redox states consistent with high stability of the molecule (Adman et al. 1995, Murphy et al. 1997b). 22 Chapter 1 - Introduction A B Figure 1.5 Secondary structure representations of the A) CuNiR trimer and B) monomer from A. faecalis S-6. The N-terminal domain of each monomer is shown in yellow and the C-terminal domain shown in blue. The copper atoms coordinated in a type I site geometry are colored green and those incorporated into a type II site are colored rust. 23 Chapter 1 - Introduction Each monomer contains two spectroscopically different copper atoms. The two copper sites are approximately 12.5 A apart and are intimately linked through a Cys-His bridge incorporating the type I ligand Cysl36 and the type II ligand Hisl35 (Figure 1.6). Crystallographic, spectroscopic and functional studies of different CuNiRs from several species have revealed that the type I copper center is the site of electron transfer from a proteaceous electron donor and the type II copper is the site of nitrite reduction (Abraham et al. 1993, Kukimoto et al. 1994, Libby et al. 1992). Wat503i R HislOO His145 ^ f ^ J ^ His306 7 His135 Figure 1.6 The ligand environments of the type I and type II copper sites in CuNiRs are depicted as balls and sticks showing the covalent linkage between them. 24 Chapter 1 - Introduction 1.5.2.4 Type I copper site and electron transfer The type I copper atoms are buried within the N-terminal domains of each monomer approximately 6 A below the surface of the protein. Each type I copper is coordinated by four amino acid residues (His95, Cysl36, Hisl45 and Metl50) in a distorted trigonal planar geometry with the methionine forming a weaker interaction at the axial position. The ligand environment of the type I copper in the green coloured AfNiR differs only slightly from the coordination of the small blue copper protein pseudoazurin, which coordinates the type I copper with trigonal bypyramidal geometry (Murphy et al. 1997a). In this case, there are two axial ligands, the methionine and a carbonyl oxygen from the protein backbone. The traditional blue type I copper sites show an intense absorption in the visible spectrum with a major peak at 600 nm and small hyperfine couplings values of 3 - 7 mT as detected by EPR. The strong absorbance results from an allowed ligand to metal charge transition (LMCT) between the copper atom and the sulfur of the cysteine ligand (Solomon et al. 1976, Suzuki 2000). The absorbance spectrum of blue coloured CuNiRs such as AxNiR is broadened to include less intense peaks at 458 nm and 700 nm. The electronic structure of the green coloured CuNiRs, such as AfNiR and AcNiR, is changed further to show a much more intense absorption at 460 nm. Similar to the absorbance peak at 600 nm, resonance raman spectroscopy of the green CuNiRs have also ascribed the 460 nm peak to an L M C T band between the copper and the sulfur of the cysteine (Han 1993). A weak shoulder peak observed in green CuNiRs at approximately 400 nm is been attributed to an L M C T between the sulfur of the methionne ligand (Lacroix et al. 1996). Recently, the colour difference between the green and the blue type I copper sites has been attributed to the angular geometry of the amino acid copper ligands, in particular the 25 Chapter I - Introduction angle of the Met ligand (Lacroix et al. 1996). A comparison of the structures of blue and green CuNiRs shows a difference in the Xi angle of approximately 100 0 (Boulanger 2001c, Inoue et al. 1998). As a result of the reorientation of the methionine ligand, the angle between the His-Cu-Met is 115 0 in blue CuNiRs and is shifted to 132 ° in the green CuNiRs. Overall, the green AfNiR shows a rhombic symmetry in EPR spectra differing from the more axial type I copper center in blue CuNiRs from Alcaligenes xylosoxidans (Abraham et al. 1993), Pseudomonas aeruginosa (Zumft 1987b) and the small blue proteins azurin and plastocyanin (Ryden 1984). Interestingly, the mutation of the methionine ligand to a threonine in CuNiR from Rhodobacter sphaeroides converts the colour of the type I site from green to blue but with no apparent change in the electronic structure as measured by EPR (Olesen et al. 1998). The differences in the visible absorbance spectra between blue and green CuNiRs is likely due to slight alterations in the angular geometry of the copper ligands, but does not appear to affect redox potential or correlate with enzyme activity. In AfNiR, site-directed mutagenesis studies have identified the charged molecular surface (Figure 1.7) proximal to the type I copper site as the docking site for the electron donor pseudoazurin. Several basic residues on pseudoazurin (Kukimoto et al. 1994) and acidic residues on AfNiR, Glu l 18, Glul97, Asp204 and Asp205, (Kukimoto et al. 1996) were identified that when changed, alter the kinetic parameters of the electron transfer between these two proteins. Replacements of Glul 18, Glul97, Asp201 or Asp205 that are localized to the pseudoazurin binding surface on AfNiR with alanine resulted in greater than a two-fold increase in the K m for pseudoazurin (Kukimoto et al. 1996). At pH 7.0 the measured K m for native pseudoazurin and AfNiR is on the order of 50 juM with a k c a t o f 396 s"1 (Kukimoto et al. 1994). The second order rate constant measured by cyclic voltametry 26 Chapter 1 - Introduction between AcNiR (Kohzuma et al. 1993) or AfNiR (Iwasaki et al. 1992) and pseudoazurin at the optimal pH of 6.2 is similar at 7.3 x 105 NT's"1 and 1.8 x 106 M"V\ respectively. Figure 1.7 Electrostatic surface representation of A) nitrite reductase (PDB code 1AS6) and B) pseudoazurin (1PAZ) from Alcaligenes faecalis S-6. Note the charge localization proximal to the type I copper sites, designated by arrows, in both proteins. Red colouring represents areas of negative potential contoured at - 8 o and blue denoting areas of positive potential contoured at 2.0 a. Figure was prepared using GRASP (Nicholls et al. 1991). 27 Chapter 1 - Introduction Two crystal complexes of NiR and pseudoazurin, the proposed in vivo electron donor, from Achromobacter cycloclastes have been solved at low resolution (Adman 2001, Murphy et al. 1998). Analysis of these structures does not contradict the site-direct mutagenesis studies performed by Kukimoto, but weak electron density maps of the pseudoazurin are difficult to interpret accurately. These authors suggest that a significant rotational freedom may exist between the molecular partners. 1.5.2.5 Type II copper and the active site Three mononuclear type II coppers are located at the bottom of 16 A pockets at each interface between the N-terminal domain of one monomer and the C-terminal domain of an adjacent monomer in the trimer. These copper atoms are coordinated with distorted tetragonal geometry by a solvent molecule and three histidines, two of which (His 100, Hisl35) are derived form one monomer with the third (His306) from the adjacent monomer (Figure 1.8). Since the type II copper center does not possess a cysteine ligand, it does produce a significant absorbance in the visible spectrum. However, visible absorption spectra of AfNiR variants that are no longer able to bind a type I copper suggest that the type II copper is at least partially responsible for the very weak absorption at 700 nm (Suzuki 2000). The paramagnetic signal of the type II copper as measured by EPR spectroscopy shows large hyperfme coupling constants of 12 - 20 mT for the type II copper center. A similar redox potential between the type I and II coppers as measured by pulse radiolysis and cyclic voltametry is in the range of + 240 to +260 mV (Iwasaki et al. 1992). In the oxidized state, the solvent ligand forms a hydrogen bond with the side-chain of a nearby aspartate residue (Figure 1.8). This water appears to assist in orientating Asp98 and 28 Chapter 1 - Introduction likely acts as part of a proton shuttle pathway to this residue (Adman et al. 1995, Murphy et al. 1997b). In the nitrite-soaked AfNiR structure, the ligand water is displaced by nitrite, which also forms a hydrogen bond with the side-chain of Asp98. Figure 1.8 Active site of oxidized AfNiR with A) solvent molecule or B) nitrite serving as the apical ligand the to the type II copper coloured in rust. The backbone of the A monomer is coloured yellow and the B monomer coloured blue. Hydrogen bonds are shown as dotted lines. Oxygen atoms are coloured red, nitrogen atoms blue and carbon atoms black. A second protonatable active site residue, His255, forms a solvent bridged hydrogen bond with the side-chain of Asp98. Although the side-chain of His255 is positioned within 3.6 A of the bound nitrite in the native nitrite-soaked structure (Murphy et al. 1997b), poor 29 Chapter 1 - Introduction geometry limits the formation of a hydrogen bond. The solvent network that connects these two active site residues forms a channel extending to His260 that sits in small pocket on the surface of the enzyme. Activities show two maxima at approximately pH 5.7 and 5.2. The side-chains of Asp98 and His255, the only two protonatable residues in the active site, are likely responsible for these activities (Abraham et al. 1997). The active site cavity of AfNiR is lined with a distinct polar and apolar face suggesting a possible route for escape of NO (Adman et al. 1995). The hydrophobic residues that form one side of the cavity extend the length of the cavity and forming a hydrophobic blanket surrounding the type II copper. At the surface the side-chains of a phenylalanine residue directly occludes the mouth of the active site cavity. 1.5.2.7 Proposed catalytic mechanisms The proposed molecular mechanisms of copper-containing nitrite reductases described in the literature fall into two distinct classes. These classes differ fundamentally in two main aspects; the required binding mode of nitrite to the type II copper atom and the nature of the chemical intermediates through which the reduction of nitrite proceeds. To date, no clear molecular mechanism for CuNiRs exists that is broadly accepted by the biological and chemistry communities. The first catalytic model was proposed by Hulse and Averill (Averill 1996, Hulse et al. 1989) and recently revised by Dodd and coworkers (Dodd et al. 1997). In this mechanism, the reduction of nitrite to nitric oxide proceeds through the formation of an electrophilic nitrosyl intermediate (Cu(I) - NO + ) similar to the mechanism of the heme-containing NiRs. The formation of the nitrosyl intermediate requires nitrite or nitric oxide to 30 Chapter I - Introduction be N-coordinated to the type II copper atom through the nitrogen atom. The chemical relevance of this binding mode for nitrite is supported by studies where nitrite bound in a In-coordinate fashion to copper in biomimetic complexes results in stoichiometric production of NO (Halfen et al. 1994). A distinct second model describing the catalytic process for CuNiRs has been proposed where nitrite binds in an O-coordinate fashion to an oxidized type II copper atom. The O dependent coordination of nitrite precludes the formation of a chemically defined nitrosyl species suggested by the first mechanism. The chemically unusual O-coordinate binding mode for nitrite was originally suggested by ENDOR spectroscopy (Howes et al. 1994) and later confirmed with E X A F S spectroscopy and (Strange et al. 1995, Strange et al. 1999) crystallography (Murphy et al. 1997b). To date, all experimental evidence derived from studying nitrite binding to the protein is consistent with an O-coordination. 1.6 Objectives, Hypotheses and Outline Nitrite reductase is a crucial enzyme in the terrestrial nitrogen cycle catalyzing the key environmental step of converting a mineral form of nitrogen (nitrite) into a gaseous form (nitric oxide). Despite the importance of this enzyme, the mechanism for copper-containing nitrite reductases remains poorly defined with preliminary evidence suggesting a mechanism distinct from the cd\ NiRs. The goal of my thesis is to identify and characterize catalytically important residues in the active site of the copper-containing nitrite reductase from Alcaligenes faecalis S-6 with the ultimate goal of better understanding the molecular mechanism of CuNiRs. Traditionally, crystallography and spectroscopy have been the primary methods for studying CuNiRs. To this I have added D N A manipulation techniques 31 Chapter 1 - Introduction such as combinatorial mutagenesis. Collectively, these approaches are used throughout this thesis. The high-resolution crystal structures of the nine AfNiR variants presented in this thesis are the only structurally characterized variants of any CuNiR reported in the literature and as such provide a unique perspective from which to study the molecular mechanism of CuNiRs. The primary hypothesis of my thesis is that the absolutely conserved protonatable active site residues Asp98 and His255 (Figure 1.8) are intimately involved in the molecular mechanism of CuNiRs. Chapter 3 describes the cloning, expression and purification of three AfNiR variants (D98N, H255N and H255D) at these two positions, as well as the associated functional, spectroscopic and preliminary crystallographic analysis. Chapter 4 presents a more thorough crystallographic approach in studying the binding mode of nitrite to the oxidized and reduced forms of the D98N and H255N AfNiR variants. A secondary hypothesis is that an active site isoleucine, Ile257, is responsible for directing a catalytically productive mode of nitrite binding. Chapter 5 describes the construction of six variants at position 257 in AfNiR; several modified functional assays and high-resolution nitrite-soaked crystal structures. Collectively, the data presented in these chapters increases significantly our understanding of the catalytic requirements of CuNiRs The studies presented in Chapters 3 and 4 have been published recently in the Journal of Biological chemistry (Boulanger et al. 2000) and Biochemistry (Boulanger 2001a), respectively. The work described in Chapter 5 has been written in manuscript form to be submitted to Protein Science (Boulanger 2001b). 32 Chapter 2 - Materials and Methods Chapter 2 - Materials and Methods 2.1 Materials 2.1.1 Chemical supplies and media A l l chemicals were purchased from Fisher Scientific, Sigma Chemical Co, or Boehringer Mannheim unless otherwise specified. Restriction endonucleases, IPTG and T4 D N A ligase were purchased from Life Technologies (Grand Island, N.Y.) . PFU Turbo high fidelity polymerase used in the combinatorial mutagenesis experiments was purchased from Stratagene (La Jolla, CA.). Taq polymerase and buffer E were kindly provided by Dr. Ross MacGillivray (University of British Columbia). Factor Xa used to remove the six-histidine tag from recombinantly expressed AfNiR was purchased from Haematologics (Essex Junction, VT). Acrylamide and other electrophoresis reagents were obtained from Bio-Rad laboratories (Hercules, CA). ProBond nickel resin was purchased from Invitrogen (Burlington, ON). Bacterial media components were purchased from DIFCO Laboratories (Sparks, MD.). Luria-Bertani (LB) broth with appropriate antibiotics was used for overnight inoculation of bacteria; 2xYT media (with the modification of lOg of NaCl per liter of culture) was used for bacterial protein expression. Antibiotics were used at the following concentrations: ampicillin, 100 ug/ml; kanamycin, 25 ug/ml. 2.1.2 Bacterial strains and plasmids Tables 2.1 and 2.2 list the strains of Escherichia coli and plasmid constructs used in this study, respectively. The E. coli strain DH5a was used as the host strain in genetic manipulations. HMS174(DE3), BL21(DE3), TOPP1, TGI , JM101 and JM109 E. coli strains were used for protein expression. Bacterial cell stocks were stored at -80 °C in L B medium 33 Chapter 2 - Materials and Methods containing 15% glycerol. pBluescript® II SK- and the pET vectors were obtained from Stratagene and Novagen Inc., respectively. Drs. Makoto Nishiyama, Mutsuko Kukimoto and Michael Murphy carried out initial site-directed mutagenesis of Asp98 and His255 in the lab of Dr. Sueharu Horinouchi at the University of Tokyo. The mutagenic synthetic oligonucleotides used for the D98N mutant was 5' CTG A T G C A T A A T A T C A A T TTC CAT G C G 3' and incorporates an Eco T22I restriction endonuclease site. The primer for the H255D mutant was 5' CGC GAT A C G CGT C C A GAT CTG A T C G G G 3' and encodes an internal Mlu I cleavage site. A similar primer was used for the H255N mutant with the exception that the G A T codon for His255 was changed to A A T . Mutations were confirmed with M l 3 dideoxy nucleotide sequencing (Sanger et al. 1977). Prior to the studies presented in this thesis, these variant proteins had not been expressed and thus, were uncharacterized. Table 2.1 Bacterial strains used in this study Strain Relevant characteristic(s) Reference E. coli DH5a E. coli TGI E. coli TOPP1 E. coli JM101 E. coli JM109 E. coli HMS174(DE3)and BL21(DE3) General host strain used for plasmid propagation General protein expression strain General protein expression strain General protein expression strain General protein expression strain Strains for high-level protein expression encoding the T7 R N A polymerase Novagen (Sambrook 1989) (Sambrook 1989) (Sambrook 1989) (Sambrook 1989) (Sambrook 1989) 34 Chapter 2 - Materials and Methods Table 2.2 Bacterial plasmids used in this study Strain, or plasmid Relevant characteristic(s) Source pBluescript® II SK-® pET24c' pET22b® pET28a® pUC19q/w'r pAfNiR22b pAfNiR28a pD98N28a pH255N28a pH255D28a pI257G28a pI257A28a pI257V28a pI257L28a pI257M28a pI257T28a p U B l ppAz24c E. coli cloning vector used in genetic manipulations , Amp r E. coli expression vector, Kan r E. coli expression vector, Amp r E. coli expression vector, Kan r nirK gene from A. faecalis S-6 cloned in pUC19 HinDIII / PstI sites afnir cloned in frame with pelB leader sequence for periplasmic expression afnir cloned in pET28a (Ncol / Xhol) in frame with a C-terminal 6 x His tag D98N afnir variant in pET28a (Ncol / Xhol) H255N afnir variant in pET28a (Ncol / Xhol) H255D afnir variant in pET28a (Ncol / Xhol) I257G afnir variant in pET28a (Ncol / Xhol) I257A afnir variant in pET28a (Ncol / Xhol) I257V afnir variant in pET28a (Ncol / Xhol) I257L afnir variant in pET28a (Ncol / Xhol) I257M afnir variant in pET28a (Ncol / Xhol) I257T afnir variant in pET28a (Ncol / Xhol) Pseudoazurin gene (paz) from A. faecalis S-6 in pUC19 (Hindll l /Sai l) paz subcloned from pUB 1 into pET24c (Nhel / Xhol) Stratagene Novagen Novagen Novagen (Nishiyama et al. 1993) This study This study This study This study This study This study This study This study This study This study This study (Nishiyama 1992) This study 35 Chapter 2 - Materials and Methods 2.2 General experimental methods 2.2.1 D N A manipulation Ligation reactions. Intact plasmids and digested products were electrophoresed on a 1% agarose gel run at 100 mA. Gels were soaked in a solution of ethidium bromide and the D N A samples visualized by exposure to ultraviolet light. Digested D N A fragments used in ligation reactions were excised from the agarose gel and purified using the QIA gel extract Kit (Qiagen, Inc.) as described by the manufacturer. Each ligation reaction contained 1 pi of T7 D N A polymerase (Life Technologies), 2 pi T7 D N A polymerase reaction buffer, 1 pi 10 mM ATP, and sterile distilled water added to a final volume of 20 pi. Ligation reactions using D N A inserts with cohesive ends were incubated at 24 °C for 4 to 6 hours and included a three-fold molar excess of insert to vector. Blunt end ligations were carried out at 16 °C overnight using a five-fold molar excess of insert to vector. Competent cell preparation. An overnight bacterial culture (500 pi, DH5a for cloning and HMS174(DE3) for protein expression) was inoculated into 500 ml of Luria-Bertani (LB) broth and incubated at 37 °C in a shaking incubator until an O.D.600 of approximately 0.55 was reached. Cells were transferred to two sterile centrifuge bottles, cooled on ice for 30 minutes, and harvested by centrifugation at 2,000 g for 15 minutes at 4 °C. The supernatant was decanted, and each bottle of cells was resuspended gently in 20 ml cold sterile RF1 solution (100 mM RbCl, 10 mM CaCl 2 , 30 mM potassium acetate, 50 mM M g C l 2 , 15% glycerol, pH 5.8). The bacterial suspensions were incubated on ice for 15 minutes and centrifuged at 1,500 g for 15 minutes at 4 °C. Each cell pellet was resuspended with 3.5 ml cold sterile RF2 solution (75 mM CaCl 2 , 10 mM RbCl, 10 m M MOPSNa, 15% glycerol, pH 6.8). The cells were cooled on ice for 15 minutes, aliquoted (150 pi), flash-36 Chapter 2 - Materials and Methods frozen in a dry ice/ethanol bath, and stored at -80 °C. Transformation and isolation of plasmid DNA. Frozen competent cells were thawed on ice. D N A samples (7.5 pi of ligation products) were incubated with 75 ul of competent cells, cooled on ice for 30 minutes followed by a heat shock at 37 °C for 90 seconds. L B broth (200 pi) was added to the transformation mixture followed by a 45-minute incubation at 37 °C in a shaking water bath. Different quantities of bacterial suspension were plated on L B agar plates with appropriate antibiotic and incubated at 37 °C overnight. Crude plasmid purification from bacterial cells to screen for successful transformation was performed by the alkaline lysis method as described in (Sambrook 1989). Isolated plasmids were analyzed by restriction enzyme digestion to confirm the presence of the desired insert. Sequencing grade plasmids were prepared using the QIAprep Miniprep Kit (Qiagen, Inc.) as described by the manufacturer. DNA sequence analysis. Sequence reactions were performed using the Big Dye® Terminator cycle sequencing kit purchased from Applied Biosystems (Foster city, CA). The PCR reaction mix consisted of 2 p\ of Terminator® ready reaction mix, 2 pi (~ 500 ng) of template D N A and 1 pi (~3 pmol) sequencing primer. The amplification protocol used is as follows; denature at 96 °C for 30 seconds, anneal at 50 °C for 15 seconds and extend at 60 °C for 4 minutes. Extension products were purified with an ethanol - salt precipitation prior to sequencing. The products of the sequencing reactions were visualized on an Applied Biosystems model 377 automated sequencer at the University of British Columbia N.A.P.S. Unit. The T3 (5' - ATT A A C CCT C A C T A A A G G G A - 3') and T7 (5' - T A A T A C G A C T C A C T A T A G G G - 3') primers were used for D N A sequencing in pBluescript® II SK-. The T7 forward (5' - ATT A A C CCT C A C T A A A G G G A - 3') and T7 reverse (5' - ATT 37 Chapter 2 - Materials and Methods A A C CCT C A C T A A A G G G A - 3') primers were used for sequencing directly from the pET vectors. No mutations were detected when using either Taq or the high fidelity Vent® polymerase during PCR amplification. 2.2.2 Basic protein characterization SDS PAGE. Samples were heated at 95 °C in 5 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer for 5 minutes. After denaturation, samples were resolved on 12 - 15 % polyacrylamide gels electrophoresed in running buffer (Tris base 30g, glycine, 144g, SDS lOg in total of 2L pH 8.3) at 40 mA for approximately 45 minutes. Protein bands were visualized following staining of the gel in Coomassie blue R solution followed by destaining with an acetic acid methanol wash. Bradford standard assay. Purified protein samples were diluted to an appropriate concentration, estimated by absorption at 280 nm, and quantified by the method of Bradford (Bradford 1976) using the Bio-Rad Protein Assay Dye Reagent® Concentrate (Bio-Rad) as described by the manufacturer. Highly purified samples of bovine serum albumin (BSA) purchased from Sigma were used as the standards in the Bradford assay. N-terminal amino acid sequence analysis. Purified AfNiR at a concentration of 20 mg/ml dissolved in 10 mM Tris pH 7.0 was subjected to N-terminal sequencing using the Perkin Elmer ABI 476A automated sequencer at the University of British Columbia nucleic acid and protein sequencing unit. The first 6 amino acids were found to be identical to those predicted from the translation of the afnir gene sequence following removal of the amino terminal methionine. 38 Chapter 2 - Materials and Methods 2.2.3 Combinatorial mutagenesis A combinatorial mutagenesis procedure based on the "Quick change" system (Stratagene) was developed to generate a small library of six mutations at position 257 in the afnir gene cloned in pET28a (pAfNiR28a). The degenerate primers (Table 2.3) were designed using The Combinatorial Codons program (Wolf 1999) to maximize the likelihood of replacing Ile257 with aliphatic amino acids. Dr. John Hobbs carried out primer synthesis in the N.A.P.S. unit at the University of British Columbia. The polymerase chain reaction (PCR) mixture contained 5 ng of double stranded template DNA, 125ng of each mutagenic primers, 1/xl of a lOmM dNTPs, 5 p\ reaction buffer supplied by Stratagene and distilled water to a final volume of 50 /d. Key to the success of the mutagenesis was the use of dimethyl sulfoxide (DMSO) to a final concentration of 0.5 % and using the thermostable PFU Turbo® D N A polymerase purchased from Stratagene. Mutations of Ile257 to Leu, Val, Ala, Gly, Met and Thr were confirmed by D N A sequence analysis as described in Section2.2.1. Table 2.3 Degenerate primers used for combinatorial mutagenesis of Ile257 % X , % x 2 % x 3 Forward primer 5'- G A G A C C A C A TCT G X i X 2 X 3 G G G G G G C A TGG G G A TTA TG - 3' A G T C a21 46 0 33 A G T C 0 17 41 42 A G T C 33 33 33 0 Reverse primer 5'- CAT A A T CCC GCC C X , X 2 X 3 C A GAT GTG GTC TC - 3' A G T C 33 33 0 33 A G T C 41 42 0 17 A G T C 0 33 21 46 These numbers represent the % by weight of each nucleotide at the X i , 2 and 3 positions within the mutated codon 39 Chapter 2 - Materials and Methods 2.2.4 Nitrite reductase activity assays Nitrite reductase activity was measured at 30 °C in 5 ml test tubes with a final reaction volume of 1 ml as described by Kakutani et al. (Kakutani et al. 1981a). Methylviologen reduced with an excess of dithionite buffered in 100 m M sodium bicarbonate was used as the artificial electron donor to AfNiR. The starting solution contains 500 p\ dHbO, 2 mM sodium nitrite (100 /xl), 0.1 mM methylviologen (100 jul) and 20 mM potassium phosphate (KP0 4 ) buffer pH 7.0 (200 /zl). AfNiR (100 p\) was added such that the final nitrite concentration was between 0.4 (20%) and 1.6 mM (80%) after 5 minutes. The reaction was started with the addition of sodium dithionite to a final concentration of 5 mM. Residual nitrite was detected in a 15 ul sample from the first step using the Griess reagents (500 ul each of 0.02% N-(l-naphthyl) ethylenediamine and 1% sulfanillic acid in 25% HC1. For the control, 1.95 mM (98%) nitrite remained. Units are defined as the amount of AfNiR required to reduce 1 /miol of N02~ per minute. Sulfite competition assay. Experiments to show the effect of sodium sulfite on the removal of nitrite from solution (Chapter 5), the traditional nitrite reductase assay described above was used with the sole modification that sodium sulfite was added along with nitrite in the first stage of the assay to a final concentration of 0, 2, 3.5, 5 and 10 mM. 2.2.5 Pseudoazurin based assay In this assay, pseudoazurin from Alcaligenes faecalis S-6 was subcloned, expressed and purified in this study as described in Section 2.2.5.1. Throughout this assay, all solutions were degassed, stored in anaerobic vials and transferred using Hamilton syringes. In the oxidized form, pseudoazurin is deep blue in colour. Reduced pseudoazurin was obtained by 40 Chapter 2 - Materials and Methods adding a few crystals of dithionite, which resulted in immediate loss of colour indicating successful reduction of the protein. To remove excess dithionite, the reduced pseudoazurin was buffer exchanged with 100-fold degassed potassium phosphate (KPO4) buffer (pH 7.0) under a nitrogen atmosphere, transferred to an anaerobic vial and degassed for a further 15 minutes with argon. The assay was performed in a 3 ml anaerobic cuvette sealed with a rubber septum under a positive argon atmosphere. The starting solution for the assay contained 20 mM potassium phosphate (KPO4) buffer (pH 7.0), 10 mM sodium sulfite, 20 nM of AfNiR and 50 pM of reduced pseudoazurin. In the absence of AfNiR, no significant reoxidation of pseudoazurin was observed over the duration of the experiment when incubated alone or in the presence of sulfite. Absorbencies were measured on a C A R Y 50 Bio spectrophotometer. 2.2.5.1 Cloning and expression of pseudoazurin The pseudoazurin gene from Alcaligenes faecalis S-6 was amplified from the p U B l vector kindly provided by Dr. Makoto Nishiyama at the University of Tokyo. The following oligo-nucleotide primers were used: 5' primer, 5'A7AG-CTT G/CT-AGC G A A A A T A T C G A A GTT C A T A T G CT 3' and the 3' primer, 5'CAT C/TC-GAG T C A TTT A G C GCT GGC GAT G A C 3'. A similar amplification protocol used for AfNiR was used to amplify pseudoazurin with the exception that the annealing temperature was changed to 56 °C. The PCR product was cloned into the Hindlll and Xhol restriction sites of pBluescript® II SK-, followed by subcloning into the Nhel and Xhol sites of the expression vector pET24c to produce ppAz24c (Table 2.2). A fresh transformant of the ppAz24c clone in HMS174(DE3) cells was inoculated 41 Chapter 2 - Materials and Methods into 5 ml of 2xYT media supplemented with kanamycin and grown overnight at 37 °C. The following day, the 5 ml culture was subcultured into IL of 2xYT media and grown at 37 °C until an O.D.600 of 1.0 was reached at which time IPTG was added to a final concentration of ImM and the culture grown for a further 6 hours. Cells were harvested, lysed with a French press and the insoluble debris removed by centrifugation. Copper acetate was added to the supernatant to a final concentration of 5 mM and the supernatant loaded onto a carboxymethyl cellulose (CM) column equilibrated with 20 mM phosphate buffer pH 7.0. Pseudoazurin was eluted as an intense blue band at a concentration of 200 mM NaCI. The blue fractions were concentrated and loaded directly onto a Superdex G75 gel filtration column equilibrated in 20 mM phosphate buffer pH 7.0 and 250 mM NaCI. The purity of the blue pseudoazurin fraction was monitored by UV-Vis spectroscopy (A277/A593=1.92). This protocol yielded approximately 10 mg of purified pseudoazurin / L of culture. 2.2.6 Basic fuschin assay (sulfite quantification) To quantify the amount of sulfite remaining in solution, the pseudoazurin-based assay described above was combined with a fuschin-based method as described previously (Leinweber 1987). The fuschin reagent was prepared by adding 1.6 ml of a 3% ethanolic solution of basic fuschin to 93.6 ml of water containing 4.4 ml of sulfuric acid (H2SO4) followed by the addition of 0.4 ml of 40 % formaldehyde. The solution was decolorized with the addition of 200 mg of activated carbon, shaken for 15 minutes and clarified by filtration. A 250 p\ sample from the pseudoazurin based assay (Section2.2.5) was added to a test tube containing 50 ul of 1 % alcoholic potassium hydroxide (KOFf) followed by the addition of a 50 p\ solution of saturated mercuric chloride (HgCb). This solution was 42 Chapter 2 - Materials and Methods thoroughly mixed and the insoluble particulates removed by centrifugation. A 50 /xl sample of the clear supernatant was added to 4.0 ml of the fuschin reagent and the increase in absorbance at 580 nm was monitored on a C A R Y 50 Bio UV-Vis spectrophotometer. Total remaining sulfite was interpolated from a standard curve of sodium sulfite standards ranging from 0.1 to 1.5 mM. 2.3 Spectrometry. 2.3.1 Mass spectrometry. Samples of native recombinant AfNiR in 10 mM MES buffer pH 6.0 were prepared to a concentration of 2 pM, prior to injection onto a reverse phase column interfaced to an electrospray mass spectrometer. The protein was eluted with increasing concentrations of acetonitrile pH 2.0 and injected directly into the carrier stream (90% acetonitrile / water) at a flow rate of 50 uL/min into a PE-Sciex API 300 quadrupole mass spectrometer. Dr. Shouming He, in the lab of Dr. Stephen Withers, kindly carried out all mass spectrometry measurements. 2.3.2 Ultraviolet-Visible spectrometry Optical scanning spectra were recorded at 25 °C on a Cary 3E UV-Vis spectrophotometer fitted with a thermostated circulating water bath in the lab of Dr. A . Grant Mauk. Protein samples were analyzed in 10 mM Tris buffer pH 7.0. Initially, one and three ml sample volumes of protein concentrated to at least 10 mg/L were tested and found to give the identical absorbencies. A l l remaining samples were measured in a final volume of 1 ml. 43 Chapter 2 - Materials and Methods 2.3.3 Atomic absorption spectrometry Copper content of native and variant AfNiRs were measured by graphite furnace atomic absorption spectroscopy with a copper detection lamp at a wavelength of 327.4 nm. A standard curve was generated using 12.5, 25 and 50 parts per billion copper standards made up in 2% nitric acid. AfNiR samples were diluted in 2% nitric acid and duplicate 15 /xL samples were injected into the furnace via an autosampler. The concentration of copper in the AfNiR samples was interpolated from the standard curve and the molar ratio of copper calculated using protein concentration values determined by the method of Bradford (Bradford 1976) using a B S A standard. 2.3.4 Electron paramagnetic resonance (EPR) spectrometry. EPR spectra were recorded at X-band frequencies on a Bruker ESP 300E electron paramagnetic resonance spectrometer equipped with a Hewlett Packard Model 532B frequency counter in the lab of Dr. Mauk. For liquid helium spectra (19.5 K) the EPR instrument was fitted with an Oxford Instruments Model 900 continuous flow helium cryostat and an ITC4 temperature controller. Liquid nitrogen spectra were collected at 87 K . AfNiR samples were concentrated to approximately 1 mM and buffer exchanged with 10 mM MES and 40 mM K P 0 4 p H 7.2. Parameters used for data collection at 19.5 K were: modulation frequency 100,000 KHz, modulation amplitude 6.428 G, microwave frequency 9.450 GHz and microwave power 0.3975 mW. A modulation amplitude of 0.708 G and a microwave power of 1.992 mW was used for data collected at 87 K . 44 Chapter 2 - Materials and Methods 2.4 Structure determination 2.4.1 Crystal growth and substrate soaking. Crystals of native and variant samples of AfNiR were grown at room temperature by the hanging drop vapor diffusion method with equal volumes (3ul) of mother liquor and protein solution. Crystal screens I and II® (Hampton Research, Laguna Niguel, CA) and Wizard I and II® (Emerald Biosystems, Bainbridge Island, WA) were used to screen initially crystal growth conditions. Typically, crystals were grown in mother liquor consisting of 0.1 M sodium cacodylate pH 5.5, 0.1 M sodium acetate pH 4.7 and 10 to 15% polyethylene glycol 4000, 6000 or 8000. High concentrations of zinc ion (50 mM) in the crystallization mix produced AfNiR crystals in space group R3; however, lower zinc ion concentrations (< 5mM) combined with equimolar amounts of copper ion result in orthorhombic crystals of space group V2{2{2\ (Chapter 4) that are isomorphous with previous AfNiR crystals (Kukimoto et al. 1994, Murphy et al. 1997b, Murphy et al. 1995). The protein stock used in crystallization was 10 mg/ml buffered in 10 mM Tris pH 7.0. These conditions resulted in green crystals that grew typically to dimensions 0.3mm X 0.4mm X 0.4 mm within two weeks (Figure 2.1). Nitrite-soaked oxidized crystals were obtained by placing crystals in mother liquor supplemented with 5 mM sodium nitrite over 45 minutes at room temperature. The crystals were then transferred to fresh mother liquor supplemented with 5 mM nitrite and 30 % glycerol as a cryo-protectant. Initial attempts at reducing nitrite-soaked crystals with ascorbate at room temperature resulted in the crystals reoxidizing and turning green within 20 minutes. Reduced, colorless nitrite-soaked crystals were ultimately obtained by soaking crystals in mother liquor supplemented with 5mM nitrite at 0 °C for 45 minutes followed by 45 Chapter 2 - Materials and Methods an increasing stepwise gradient of freshly prepared ascorbate from 1 m M to 5 m M to 20 m M over one hour. Reduced nitrite-soaked crystals were fragile and required a 10 % stepwise addition of glycerol to a final concentration of 30 % over 5 minutes. Figure 2.1 Crystals of AfNiR with approximate dimensions of 1 mm x 0.5 mm x 0.4 mm. The green colour is derived from the type I copper center. 2.4.2 Crystal manipulation and data collection To collect X-ray diffraction data under ambient conditions, approximately 20 °C, crystals of D98N, H255N and H255D were mounted in glass capillaries (Charles Supper Company, Natick, M A ) . Initially, crystals were transferred from the cover slip to a glass depression plate containing fresh mother liquor. Using a pipette at one end of the capillary, crystals were taken up into the capillary. Thin sections of Whatman® filter paper were used to wick away moisture from the crystal and the ends of the capillary were sealed with wax. The capillary was mounted in a small copper adapter and secured into the goniometer head 46 Chapter 2 - Materials and Methods with Crazy Glue®. For cryogenic data collection, crystals (oxidized and reduced D98N[N0 2"] and H255N[N02"] and oxidized I257V[N02"], I257L[N0 2 '], I257A[N02"], I257T[N02"], I257M[N02"] and I257G[N02"]) were incubated with fresh mother liquor and 30 % glycerol and mounted into small loops and placed directly into a nitrogen stream at 100 K generated by a cryostat (Oxford Cryo Systems, Oxford, U.K.) . The crystal loops used for cryogenic data collection were initially constructed from teased out dental floss, syringe needles and Crazy Glue® and later purchased from Hampton Research. X-ray data was collected on a Rigaku R-AXIS lie image plate system with C u K a radiation generated by a Rigaku R U 300 rotating anode operating at 100 mA and 50 kV and focused with either a graphite monochromoter (D98N, H255N and H255D) or Osmic confocal max-flux optical mirrors (all nitrite-soaked variant structures). The diffraction data frames for the former crystals were exposed for 35 to 40 minutes with a crystal oscillation angle of 0.75°. Focusing the X-ray beam with the mirrors increased the intensity allowing a data collection time of 8 to 10 minutes with a 1° oscillation. Diffraction data was processed with the program DENZO (Otwinowski 1997). Typically, an initial frame was collected for each data crystal and processed. Using the indexed data frame, the program STRATEGY (Raveli et al. 1997) was used to calculate spatial positions of the axes in the crystal. Whenever possible data collection was started on a crystal axis to maximize data collection efficiency. 2.4.3 Structure solution and refinement The D98N, H255N and H255D mutants crystallized in space group R3 with one monomer in the asymmetric unit resulting in the functional trimer being generated by the 47 Chapter 2 - Materials and Methods crystallographic three-fold. These crystals are isomorphous with those obtained previously for the type I copper site AfNiR variant M150E (Murphy et al. 1995). The D98N variant structure was solved by molecular replacement using the program AmoRe (Navaza et al. 1997) with chain A of the nitrite-soaked native AfNiR structure (Murphy et al. 1997b) following removal of the solvent atoms, nitrite and the side-chain of residue 98. The correlation coefficients calculated from the rotation and translation functions were 24.6 and 69.0, respectively and represented the highest peaks. The D98N mutant structure was refined to a resolution of 1.9 A by the maximum likelihood method using the program CNS (Brunger et al. 1998). The D98N structure was then used as the starting model for the H255D structure, which was used subsequently to solve the H255N structure. Initially, refinement of the H255N and H255D structures did not yield a free R-factor below 30 %. A self-rotation function showed that a 2-fold symmetry was present perpendicular to the crystallographic 3-fold axis. Submission of the reflection data to the Merohedral Crystal Twinning web server (Yeates 1997) confirmed that these crystals are indeed twinned. The H255N and H255D data sets were corrected using the program DETWIN from the CCP4 suite (Collaborative Computational Project 1994), and subsequent refinement reduced the free R-factor to below 22 %. A l l of the nitrite-soaked variant structures presented here grew in a primitive orthorhombic lattice (P2i2i2i) and contain the assembled trimer in the asymmetric unit. These crystals are isomorphous with the native nitrite-soaked structure (Murphy et al. 1997b), which when used as the model resulted in an R w o r k of less than 26 % in each case following 75 cycles of positional refinement. Manual intervention was accomplished using the visualization program O (Jones et al. 48 Chapter 2 - Materials and Methods 1991) and the program PROCHECK (Laskowski et al. 1993) was used to identify regions of the structure requiring manual fitting. For each refined crystal structure presented in this thesis over 90 % of the residues in each structure occupy the most favorable position with the remaining residues in the allowed regions in the Ramachandran plot as described by PROCHECK (Laskowski et al. 1993). 2.7 Optimization of AfNiR expression and purification 2.5.1 Existing periplasmic expression system Dr. Makoto Nishiyama kindly provided the clone containing the 1 Kb open reading frame encoding the nirK gene from Alcaligenes faecalis S-6 cloned into pUC19. Over expression of AfNiR from this construct in JM109 yielded approximately 2 mg of protein / L (Kukimoto et al. 1994) of culture following an initial osmotic shock procedure to release periplasmic proteins. Briefly, IL of cells were harvested and resuspended in 200 ml of 10 m M Tris-HCl (pH8.) and NaCI to 0.85 %. Cells were again harvested and resuspended in 20 ml 30 mM Tris-HCl pH 8.0 with 20 % sucrose. To this solution, 1.6 ml of 0.25 mM EDTA was added and the cells were gently shaken at room temperature for 10 minutes. The cells were harvested and resuspended in 200 ml ice-cold water and shaken at 4 °C for 15 minutes. The supernatant from a final spin at 10,000 x g for 30 minutes contained the periplasmic proteins. The soluble periplasmic proteins were subjected to a four column chromatographic purification (Kukimoto et al. 1994) with column fractions testing positive for the ability to reduce nitrite (Section2.2.4) pooled after each step. The nature of this procedure resulted in significant losses of protein contributing to a low overall yield. To obtain sufficient protein to probe effectively the biochemical properties of AfNiR 49 Chapter 2 - Materials and Methods an efficient recombinant expression and purification system was developed. Figure 2.2 Expression trials of native AfNiR from the original periplasmic pUC19 (pUC19a/m>) in A) JM109 and B) additional protein expression strains. A) Lanes 1 and 2 contain the soluble extracts from induced and uninduced JM109 cells with the pUCT9a/w> construct, respectively. Lane 3 contains a JM109 control without plasmid. Lane 4 contains a sample of purified AfNiR. B) Lanes 1 and 2 contain pUC19q/mr induced and uninduced in JM101 cells. Lanes 3 and 4 contain pUCT9q/w> uninduced and induced in JM109 cells. Lanes 6 and 7 contain the induced soluble extracts of pUC19q/wr in TOPP1 and TGI cells, respectively. High M.W. markers were used and cells were lysed with lysozyme. 50 Chapter 2 - Materials and Methods Several cytoplasmic and periplasmic gene constructs (Table 2.2) and bacterial expression strains (Table 2.1) were tested for the ability to produce soluble AfNiR. The initial expression system for AfNiR from the pUC19a/m> construct used a modified native signal sequence to facilitate export to the periplasm (Kukimoto et al. 1994). Despite a reported yield of 2 mg/L (Kukimoto et al. 1994), no detectable expression of AfNiR in was observed by SDS-PAGE analysis (Figure 2.2 A). In addition to JM109, three strains of E.coli (Figure 2.2 B) were also tested for the AfNiR expression. Unfortunately, no significant increase in the expression of AfNiR was observed in these strains tested (Figure 2.2 B). Recloning of the afnir gene with the engineered signal sequence (Kukimoto et al. 1994) into pTRC99A® was also unable to produce notable protein expression. The lack of measurable periplasmic expression of afnir using the engineered signal sequence suggested that further optimization of the signal sequence was required. The following Sections outline the construction of an improved recombinant over expression system for AfNiR that yields 40 - 50 mg of pure protein / L of culture. For the sake of clarity, only the gene constructs using the pET28a® and pET22b® vectors will be presented here. 2.5.2 P C R amplification and cloning of afnir The coding region of afnir was amplified by PCR using primers listed in Table 2.4 from the 1 Kb afnir operon cloned into the Hindlll and PstI restriction sites in the pUC19q/m> vector. In total, five different vectors were engineered (table 2.5) including periplasmic constructs with different signal sequences, and cytoplasmic constructs. 51 Chapter 2 - Materials and Methods Table 2.4 List of primers used in afnir gene constructs Primers DNA sequence and relevant restriction sites afnir 5'2 5' A T G A / A G C T T C/CATGG CC G A A C A G A T G C 3' Hindll l Ncol afnir 5'3 5' A T G A / A G C T T G/CTAGC G C A A C T G C G G C A G A A 3' Hindll l Nhel afnir 5'4 5' A T G A / A G C T T C/CATGG C A A C T G C G G C A G A A 3' Hindlll Ncol afnir 3'2 5' C A T C/TCGAG TTA CGT GCC A G A TGG TGC 3' Xhol Stop afnir 3'3 5' CAT C/TCGAG A A T CCT TCC CTC G A T CGT A C C A G A TGG TGC 3' Xhol Factor Xa cleavage site Amplification of the afnir gene was accomplished with the following thermal cycling program; an initial hot start denaturation step for 2 min at 94 °C at which time Taq polymerase was added followed by 1 min steps of denaturation at 94 °C, annealing temperatures ranged from 55-57 °C and extension at 72 °C for 30 cycles. In all cases, the amplified PCR products were digested with Hindlll and Xhol restriction enzymes and cloned into pBluescript® II SK- for sequencing. The use of Vent® D N A polymerase resulted in amplified PCR products devoid of mutations as determined by D N A sequencing. To clone the afnir gene from pBluescript® II SK- into the pET28a or pET22b vectors, a triple ligation was required due to an internal Ncol restriction site. As depicted in the cloning scheme (Figure 2.3), the afnir gene in pBluescript® II SK- was digested with Ncol, EcoRI and Xhol, which resulted in two fragments of approximately 100 and 900 b.p. Following gel 52 Chapter 2 - Materials and Methods purification, both fragments were ligated with the target vector previously digested with Xhol and either Ncol (pET28a®) or Nhel (pET22b®). The resulting pAfNiR28a and pAfNiR22b constructs were characterized for protein expression efficiency in both the cytoplasm and periplasm, respectively. Table 2.5 Primer combination for periplasmic and cytoplasmic afnir gene constructs Construct Primers used Vector Periplasmic / Cytoplasmic pAfNiR28a afnir 5'4 afnir 3'3 pET28a® Cytoplasmic pAfNiR99c afnir 5'3 afnir 3'2 pTRC99A® Cytoplasmic pAfNiR99p afnir 5'2 afnir 3'2 pTRC99A® Periplasmic (afnir)3 pAfNiR22b afnir 5'4 afnir 3'3 pET22b® Periplasmic (pelB) pAfNiR22bn afnir 5'2 afnir 3'3 pET28a® Periplasmic (afnir) a Identifies source of signal sequence 2.5.3 Growth conditions AfNiR transformants were picked from L B agar plates and grown overnight (approx 18 hours) in 5 ml 2xYT broth at 30 °C. A 2 liter flask containing 1 liter of 2xYT media was inoculated with 2 ml of the overnight culture and grown at 30 °C until an O.D.600 of 0.9 at which time IPTG was added to a final concentration of 1 mM and the culture grown for a further 16 hours. 53 Chapter 2 - Materials and Methods • I Figure 2.3 The afnir gene in pBluescript® II SK- digested with Hindll l and Xhol giving (A) ~ 1Kb gene fragment. Following gel purification, this fragment was further digested with EcoRl . The resulting two bands were purified from an agarose gel (B) and used in a triple ligation with pET28a® and pET22b® target vectors digested with Ncol and Xhol. Hindlll /Xhol 1 Kb.p . EcoRl/XhoI 900 b.p. NcoI/EcoRl 100 b.p. 2.5.4 Periplasmic expression of pAflViR22b Increased expression efficiency was observed with the pAfNiR22b (pelB signal sequence) construct expressed in HMS174(DE3) cells (Figure 2.4). Unfortunately, most of the AfNiR protein appeared to be insoluble with no significant yields following osmotic 54 Chapter 2 - Materials and Methods shock procedure. No attempts were made to purify the AfNiR protein in the cytoplasmic fraction because the signal sequence would not have been removed rendering the protein unsuitable for further studies. A small increase in what is likely soluble AfNiR is apparent following the 19 hr post-induction time-point (inset box). ^3hrs^ ^_8hrs^ 19 hrs 1 2 3 4 5 6 7 8 43 KDa 29 KDa Figure 2.4 Time course expression trials for AfNiR using periplasmic pAfNiR22b construct that incorporates the pelB leader sequence. An osmotic shock procedure was used to release the periplasmic proteins. Induction was performed at 32 °C in HMS174(DE3) cells. Lanes 1 and 8 contain the high M.W. markers. The left lane of each time point contains the cytoplasmic fraction and the right lane contains the periplasmic fraction. Lane 9 is a control containing purified AfNiR. 55 Chapter 2 - Materials and Methods 2.5.5 Cytoplasmic expression of pAfNiR28a As a mature protein, AfNiR is a relatively large trimeric complex of 110 KDa, which may pose significant problems for efficient periplasmic secretion. As such expression of a cytoplasmic AfNiR construct (pAfNiR28a) in HMS174(DE3) grown at 37 °C was tested with encouraging results (Figure 2.5). Despite significant intracellular expression of AfNiR there was limited soluble protein following lysis of the cells (Figure 2.5 - A). Attempts to increase solubility of AfNiR through the co-expression of molecular chaperones (GroEL/ES) (Figure 2.5 - B) designed to aid in protein folding showed little effect. AfNiR Figure 2.5 Cytoplasmic Expression of pAfNiR28a in HMS174(DE3) cells at 37 °C. (A) Lanes 1 and 2 contain the soluble and insoluble extracts from an overnight expression of pAfNiR28a. (B) Time-course co-expression of GroEL/ES chaperones at 37 °C to increase solubility of AfNiR. Lane 1 contains the M.W. marker. In lanes 2,3,5 and 6 the leftmost lane contains the soluble fraction and the rightmost lane contains the insoluble fraction. Only the soluble fraction from the four-hour time point is shown (lane 4). 56 Chapter 2 - Materials and Methods Altering the basic parameters of the protein expression proved significantly more effective than the co-expression of the molecular chaperones. Overall, decreasing the growth temperature to 30 °C and increasing the cell density to O.D. 6oo of 0.9, from 0.6, prior to induction with IPTG provided the most effective results. A time course expression trial pAfNiR28a (Figure 2.6) shows clearly the increase in the ratio of soluble to insoluble AfNiR. 3h rs 7.5 hrs 19hrs - > v . * . A < AfNiR Figure 2.6 Time-course cytoplasmic expression trial of AfNiR for pAfNiR28a in HMS174(DE3) cells at 30 °C. Lane 1 contains the high M.W. markers. The leftmost lane of each time point contains the soluble extract and the rightmost lane contains the insoluble extract. Pelleted cells were solubilized with lysozyme to release the soluble proteins. Note the increase in soluble AfNiR protein after 19 hrs. 57 Chapter 2 - Materials and Methods 2.5.6 Current expression and purification protocol for AfNiR Cloning. The afnir fragment (approximately 1 Kb) amplified by the synthetic oligonucleotide primers afnir 5'4 and afnir 3'3 (Table 2.5) was purified by agarose gel electrophoresis, digested with HinDIII and Xhol and cloned into pBluescript® II SK- for sequencing. Using T3 and T7 primers, successful D N A sequence was obtained showing that no mutations had occurred during amplification. The pBluescript® II SK- construct containing the afnir gene was digested with Ncol and Xhol and cloned into pET28a® in frame with a C-terminal six-histidine tag generating the pAfNiR28a construct. Expression. Freshly picked transformants of pAfNiR28a in HMS174(DE3) cells are inoculated into 5 ml of 2xYT broth supplemented with kanamycin to a final concentration of 25 /ig/ml and grown overnight at 37 °C. The following day, the overnight culture is subcultured into 1 L of 2xYT media and grown at 30 °C until the O.D.600 reaches approximately 0.9 at which time IPTG is added to a final concentration ImM. Purification. The HMS174(DE3) cells are harvested for 15 minutes at 5000 rpm in a GSA rotor in a Sorvall RC-5B centrifuge. The pellet, which should be white in colour, was suspended in 40 ml native binding buffer (20 m M N a 2 P 0 4 pH 7.8 and 500 mM NaCI) and 50 tiM CuCl 2 . The cells are broken with a French press, the insoluble debris pelleted in the centrifuge and the soluble fraction applied directly to the Pro-Bond® nickel resin (Invitrogen). A dark green band of AfNiR is formed at the top of the column. The column is washed with wash buffer (20 mM N a 2 P 0 4 pH 6.0 and 500 mM NaCI), 50 and 100 mM imidazole. AfNiR is eluted at 300 mM imidazole followed by a final wash step at 500 mM imidazole. From a 1 L preparation, the concentration of AfNiR eluting from the Ni-column is approximately 5 mg/ml with a total of 8 to 10 ml. AfNiR is dialyzed against 10 mM Tris 58 Chapter 2 - Materials and Methods pH 7.0 followed by an overnight (~ 20 hrs) incubation at 4 °C with Factor Xa (1:500 w/w Factor Xa/AfNiR ratio) to remove the His tag. Finally, AfNiR is applied to a high Q anion exchange column (Biorad Ltd, Hercules CA) in binding buffer (Tris-HCl pH 8.0) and eluted with a continuous gradient of NaCl. Purified AfNiR elutes at approximately 250 mM NaCl. Yields of purified AfNiR mutants are approximately 35 to 45 mg/L of culture. Basic characterization. Dialysis against Tris buffer pH 7.0 supplemented with 10 to 50 mM copper followed by a final dialysis step against 10 mM Tris buffer pH 7.0 results in full occupation of the copper sites as determined by graphite furnace atomic absorption spectroscopy. The recombinant AfNiR produced by this procedure begins at residue Ala4 and includes an extra 4 residues at the C-terminus (He Glu Gly Arg) that are part of the Factor Xa cleavage sequence. Mass spectrometry analysis shows that the recombinant AfNiR is within 5 Da of the calculated mass following cleavage of the N-terminal methionine residue consistent with N-terminal sequencing. 59 Chapter 3 - Catalytic roles for Asp98 and His255 CHAPTER 3 - Catalytic role for two water bridged residues (Asp98 and His255) in the active site of copper- containing nitrite reductase 3.1 Introduction Two protons are required in the reduction of nitrite to nitric oxide by nitrite reductase. Interestingly, there are two highly conserved residues in the active site (Asp98 and His255) with ionizable side-chains that are suggested to be involved during catalysis (Adman et al. 1995, Murphy et al. 1997b). Crystal structures derived from nitrite-soaked oxidized crystals of both native AcNiR and AfNiR reveal that nitrite bound to the type II copper forms a hydrogen bond with the side-chain of Asp98 (Adman et al. 1995, Murphy et al. 1997b). Furthermore, the side-chains of Asp98 and His255 are intimately connected through a single water-bridged hydrogen bond. The hydrogen bond between the side-chain carboxylate of Asp98 and the oxygen atom of the nitrite suggest a direct role for this residue in protonation of intermediates in the reaction pathway. The role of His255 in the reaction is more controversial. Early modeling by Strange et al. suggested that bound nitrite might form a hydrogen bond to the side-chain of His255 enabling a direct proton transfer by this residue during catalysis (Strange et al. 1995). Alternatively, His255 has been suggested to donate a proton indirectly to reaction intermediates via a proton shuttling pathway using the bridging water to Asp98 (Adman et al. 1995). Additionally, a mutation to glutamate of the residue equivalent to His255 in RsNiR suggested that this residue might be important in electrostatic stabilization of nitrite binding (Olesen et al. 1998). In this Chapter, I report the first crystallographic, spectroscopic and functional characterization of three variants (D98N, H255N and H255D) of Asp98 and His255 in 60 Chapter 3 - Catalytic roles for Asp98 and His255 AfNiR reported in the literature. Nitrite reduction activity assays of these variants show that both Asp98 and His255 are essential for high-level NiR activity. In the D98N and H255D variants, slight perturbations are observed in the electronic structure of the active site copper by EPR spectroscopy. Analysis of the high-resolution crystal structures of these mutants suggest a direct proton donation role for Asp98, while His255 is likely responsible for providing indirectly a proton during catalysis and directing the binding of nitrite to the active site. 3.2 Results 3.2.1 Characterization of mutants Copper content was measured by atomic absorption spectroscopy to ensure that the mutations did not affect occupation of copper. The copper content of the samples was found to be 1.8 ± 0.1 atoms of copper per AfNiR monomer, except for the H255N mutant that has lower copper occupation of 1.5 atoms of copper per monomer (Table 3.1). The variation in the copper content is likely due to small differences in copper occupation between preparations and errors involved in the determination of protein concentrations using the Bradford assay. UV-visible spectroscopy was used to probe changes in the electronic structure of the type I copper site. The visible absorbance maxima of native AfNiR and the three mutants are at the same three characteristic wavelengths of 458, 585 and 680 nm as seen in previous preparations of native AfNiR (Kakutani et al. 1981b, Kukimoto et al. 1994). Also, the ratio of the 458 and 585 nm bands (1.3 ± 0.05) is well conserved among the three mutants and native AfNiR (Table 3.1). 61 Chapter 3 - Catalytic roles for Asp98 and His255 Extinction coefficients for the mutants were calculated to be 3.0 x 103 M " 1 cm"1 for the trimer using absorbance at 458 nm and protein concentrations determined by the method of Bradford (Bradford 1976). Table 3.1 Characterization of native and mutant AfNiRs Parameter Native D98N H255D H255N Mass (Da) 36843 (36838)a 1(1) 20 (22) 18 (21) Copper contentb 1.7 1.8 1.9 1.5 A2so / A458 nm 17.2 16.1 17.2 17.5 A458 / A 5 8 5 nm 1.29 1.28 1.31 1.32 Specific activity 0 330 3.5 0.68 0.20 a The absolute mass of the native is given along with the theoretical mass in parentheses. The values for the mutants are the observed and theoretical mass (in parentheses) differences relative to the native. b Copper content per monomer. 0 Specific activity values are reported as an average of at least two trials and are reproducible within 10% error. A more rigorous analysis of both the type I and type II copper sites was performed with EPR spectroscopy which showed a slight perturbation in the type II copper sites in the D98N and H255N mutants, but not in the H255D mutant (Figure 3.1). Overall, the EPR parameters are similar to previously published values (Kukimoto et al. 1994, Olesen et al. 1998, Suzuki et al. 1999) with the exception of a slightly diminished type II copper coupling 62 Chapter 3 - Catalytic roles for Asp98 and His255 (A||) of 100 + 5 G in the D98N mutant and an increased g-value of 2.53 for the type II signal from the H255N mutant (Table 3.2). These perturbations may be the result of small shifts in the positions of the ligand waters observed in the crystal structures. I ' 1 ' 1 ' ' — ! ' I ' 1 2.6 2.4 2.2 2.0 1.8 1.6 g - values Figure 3.1 Electron paramagnetic resonance (EPR) spectra for native variant AfNiR. A l l spectra were recorded at 19.5 K except for H255N AfNiR that was run at 87 K. 63 Chapter 3 - Catalytic roles for Asp98 and His255 Table 3.2 EPR parameters of native and mutant NiRs NiR Type I Type II gll A,, (G) gll A,, (G) Native 2.19 12+ 10 2.33 135 ± 5 D98N 2.21 70 ± 5 2.36 100 ± 5 H255N 2.21 82 ± 10 2.53 160+10 H255D 2.19 70 + 5 2.33 140 ± 15 3.2.2 Activity Assays The specific activity measured for native AfNiR is within experimental error of that reported for native AfNiR from a previous periplasmic recombinant expression system (Kukimoto et al. 1994). The specific activities of the mutants are much lower than the native enzyme (Table 3.1). Of the three mutants, D98N AfNiR is the most active with a specific activity 94 fold less than native AfNiR. The H255D and H255N mutants are a further 5 and 18 fold less active, respectively. 3.2.3 Crystallography A l l three mutants crystallized in space group R3 with one monomer in the asymmetric unit resulting in the functional trimer being generated by the crystallographic 64 Chapter 3 - Catalytic roles for Asp98 and His255 three-fold. These crystals are isomorphous with those obtained previously for M150E AfNiR; however, previous crystals of the native protein are of space group V2\2\2\ and contain the complete trimer in the asymmetric unit (Murphy et al. 1997b, Murphy et al. 1995). Data collection and refinement statistics are presented in Table 3.3. The residues modeled begin at Ala4 and end at Gly339 (D98N structure) or Glu342 (H255N and H255D structures). Ramachandran plots of the mutant structures shows that over 90 % of the residues are in the most favored conformation with the remaining residues occupying allowed conformations. In each of the mutants there is a short region of poor density that correlates to a surface exposed disordered loop starting at residue 187 and ending at residue 192. A least-squares fit of a carbon atoms (r.m.s. deviation < 0.2 A for main-chain atoms) shows that overall folds of the three mutant structures are similar to the previously determined native AfNiR structure (Murphy etal. 1995). The models include additional metal binding sites that are occupied by either zinc ions (D98N, H255N) or cadmium ions (H255D). Interestingly, one of these sites is located at the trimer axis near residue Asp275, and may function to stabilize subunit interactions. The other two metal sites are located at crystal contacts. Although unlikely, the presence of zinc in the crystallization buffer may result in substitution of the copper sites. The green color of the crystals indicates that the type I site contains primarily copper. Crystal structures of preparations of type II depleted AxNiR do not show the presence of zinc in the active site despite E X A F S evidence consistent with zinc binding to surface residues (Strange et al. 1999). Furthermore, anomalous scattering experiments of M150E AfNiR showed the presence of zinc bound to the mutated type I site with negligible binding at the type II site (Murphy et al. 1995). 65 Chapter 3 - Catalytic roles for Asp98 and His255 Table 3.3 Data collection and refinement statistics Crystal D98N H255D H255N Cell Dimensions (A) a=b=l 27.94 a=6=127.64 a=Z>=127.66 c=66.43 c=67.45 c=67.22 Resolution (A) 1.90 (2.05- 1.90)a 1.70(1.78- 1.70) 1.80 (1.91 - 1.80) R-merge 0.074 (0.241) 0.050 (0.237) 0.060 (0.259) W / Mi)}b 7.56 (2.90) 13.6(3.14) 17.5 (4.27) Completeness (%) 92.4 (92.8) 94.2 (81.1) 99.9 (100.0) Unique Reflections 29123 (5853) 42465 (4552) 37771 (6282) Working R-factor 0.184 0.188 0.180 Free R-factor0 0.214 0.211 0.211 R.M.S.D bonds (A) 0.0085 0.0090 0.0091 Overall B-factor (A) 28.9 27.4 28.8 Type I Cu B-factor (A2) 27.2 23.9 27.9 Type II Cu B-factor (A2) 22.7 18.5 23.6 No. solvent atoms 270 241 286 PDB entry code3 1ET5 1ET7 1ET8 a Values in parentheses are for the highest resolution shell. b {1} / {(T(I)} is the average intensity divided by the average estimated error in intensity. c A randomly selected fraction of the data (10%) was selected for calculation of the free R-factor (Brunger 1997). 66 Chapter 3 - Catalytic roles for Asp98 and His255 The type I and II copper ligand distances and bond geometries are in reasonable agreement with previously published values of native AfNiR structures (Murphy et al. 1997b, Murphy et al. 1995) except for a small increase in the type II copper ligand distances observed in the H255D structure. The longer ligand bond lengths may be due to partial occupation of the copper site by cadmium, an additive in the crystallization mix. 3.2.3.1 Structure of the D98N mutant The type II copper in the active site of native NiR is situated at the interface between two monomers and is coordinated in a tetrahedral arrangement by the imidazole rings of three histidine residues and a solvent water molecule (Wat503). In the native AfNiR structure, the Asp98 side-chain forms a well-defined hydrogen bond to this ligand water via atom 061. A second hydrogen bond is formed from atom 052 to His255 NeE2 via a bridging water molecule (Figure 3.2). In the D98N mutant, the Asn98 side-chain amide is rotated by approximately 40° about the xz torsional angle resulting in the N52 atom being displaced by about 1.00 A relative to the 051 atom of Asp98 in the native AfNiR structure. Furthermore, Asn98 N52 is located 3.42 A away from the ligand water and has poor geometry to form a hydrogen bond (Figure 3.2). The ligand water has also shifted (-0.8 A) from the location of the equivalent water in the native AfNiR structure (Murphy et al. 1997b) (our unpublished data). Instead of an interaction with the ligand water, the N52 atom of Asn98 is involved in a hydrogen bond (3.07 A) to Wat582 (not found in the native AfNiR structure or shown in Figure 3.2). 67 er 3 - Catalytic roles for Asp98 and His255 Figure 3.2 Structures of native and mutant forms of AfNiR. (A) The active site of native AfNiR is depicted with the type II copper atom as a brown sphere and the water molecules as aquamarine spheres. The side-chains of the three histidine copper ligands and His255 are blue. Asp98 is colored red. The backbone secondary structure elements are light blue (N-terminal domain) and yellow (C-terminal domain of the neighboring subunit). Potential hydrogen bonds are in dotted lines. (B) An analogous representation of D98N AfNiR in which Asn98 is colored salmon. (C) The structure of H255N AfNiR is depicted with Asn255 in light red. (D) Asp255 is drawn in red in the H255D AfNiR structure. 68 Chapter 3 - Catalytic roles for Asp98 and His255 Chapter 3 - Catalytic roles for Asp98 and His255 In contrast, the amide oxygen (051) of Asn98 is located in the same approximate position as the carboxylate oxygen (052) atom of Asp98 (A 0.40 A) such that the hydrogen bond (3.15 A) to the bridging water is retained. Analogous to the native AfNiR structure, Asn98 051 also participates in a hydrogen bond (3.14 A) with the backbone amide of Phe99. As a result of these extensive hydrogen bond interactions, the Asn98 side-chain is well defined in the structure and has an average B-factor of 25.7 A2. The orientation of the Asn98 side-chain amide was assigned based on crystallographic B values, 26.8 A2 for N52 and 25.7 A2 for 051, and on the hydrogen bond donor and acceptor properties of the amide nitrogen and oxygen atoms. 3.2.3.2 Structure of the H255N mutant In native AfNiR, His255, like the copper ligand His306, is situated on the adjacent monomer on the opposite side of the active site from His 100, His 135 and Asp98. The Ne2 atom of His255 is linked through a water bridged water hydrogen bond to the side-chain of Asp98 (Figure 3.2). The most obvious effect of replacing His255 with an Asn is an opening of the active sight that allows room for an extra water molecule (B-factor of 42.0 A2) that is not observed in the native AfNiR structure (Figure 3.2). This new solvent molecule (Watl099) is located between Asn255 051 (2.86 A), and the bridging water (Watl098, 2.58 A) and maintains a rigid hydrogen bond network and proton shuttling pathway between residue 255 and Asp98. Water 1099 is also situated 2.33 A from the ligand water (Wat 503) and 3.20 A from the type II copper. The short distance between Watl099 and the ligand water is due to a shift in the position of the latter water of about 1.4 A such that the hydrogen bond to Asp98 051 is retained. The conformation of Asp98 is unaffected by the mutation. 70 Chapter 3 - Catalytic roles for Asp98 and His255 In the native AfNiR structure, the N<51 atom of His255 is involved in a bifurcated hydrogen bond to the backbone carbonyl of Glu279 and Thr280 Oy l . As a result of a small rotation about the Xx torsion angle of Asn255, the N52 atom is directed towards only the side-chain hydroxyl of Thr280. The orientation of the Asn255 side-chain was chosen to maintain this hydrogen bond interaction and to minimize the difference in the B-factors of the 051 and N52 atoms. The average B-factor of the Asn255 side-chain is 23.3 A2. 3.2.3.3 Structure of the H255D mutant The conformation of the Asp255 side-chain is almost identical to that of Asn255 (Figure 3.2). The 051 atom of Asp255 is directed towards Thr280 051 that in turn forms a hydrogen bond to the main-chain carbonyl of Gly286. As observed in H255N AfNiR, an additional solvent water molecule (Wat 1099) is situated close (-2.5 A) to the type II copper atom (Figure 3.2). This water is clearly defined in omit difference maps and has a B-factor of 29.8 A2. Wat509 is located 3.14 A from Asp255 052, 2.87 A from the bridging water, and 2.87 A from the ligand water. Unlike the H255N structure, the ligand water does not shift significantly from the position found in the native structure. The interaction of Asp98 with the ligand water (2.8 A) and the bridging water (3.3 A) are largely unchanged from that observed in native AfNiR. 71 Chapter 3 - Catalytic roles for Asp98 and His255 3.3 Discussion 3.3.1 Active site hydrogen bond network In the CuNiR structures determined to date a bridging water molecule links the side-chains of Asp98 and His255 (Adman et al. 1995, Godden et al. 1991, Kukimoto et al. 1994, Murphy et al. 1995). This water appears to assist in orientating Asp98 and has been suggested to act as part of a proton shuttle pathway to this residue (Adman et al. 1995). As shown in Figure 3.3, the likely location of the hydrogen atoms of the bridging water can be inferred based on the available protein hydrogen bond donors and acceptors. The bridging water donates a proton to Asp98 052 and is a proton acceptor from His255 Ne2. Gly259 N (not shown) and another water occupy the remaining hydrogen bond capacity of the bridging water completing a rigid hydrogen bond network (Figure 3.3). Surprisingly, the D98N, H255N and H255D mutant structures show little perturbation in the position of this water molecule despite mutations of these residues that alter hydrogen bond interactions. 72 Chapter 3 - Catalytic roles for Asp98 and His255 His 255 C T h r 280 o... / c B279 Asn255 — C C u 2 H H O \ V C — Asp98 H H H 6 b c c B259 A102 AfNIR C u 2 * /° \ ; N H ^ H . YY X H CX. _ C Asp98 H O o o I I c c B259 A102 H255N H \ His 255 C T h r 280 o... / N H . H S— Asn98 C B279 Asp255 — C \ T h r 2 8 0 O . . H H O H H 6 b c c B259 A102 D98N F K X H CX \ .• - C Asp98 ° ^ H . O H H 6 b c c B259 A102 H255D Figure 3.3 The proposed active site hydrogen bond networks in native AfNiR and the D98N, H255N, and H255D mutants. 3.3.2 RoIeofAsp98 The positioning of protons in the active site as shown in Figure 3.3 assumes that Asp98 is negatively charged and His255 is positively charged as would be expected at pH ~6, the optimum for activity and at pH ~5 under which crystals were obtained. In native AfNiR, Asp98 serves as a proton acceptor from both the ligand and bridging waters. The D98N mutation results in a side-chain that acts as both a hydrogen bond acceptor (atom 051) 73 Chapter 3 - Catalytic roles for Asp98 and His255 and donor (atom N62) resulting in the loss of one of these hydrogen bonds to residue 98. From the crystal structure of this mutant, the Asn98 side-chain is reoriented such that N52 is further away from the ligand water (Figure 3.2). Thus Asn98 retains the hydrogen bond to the bridging water at the expense of losing the hydrogen bond to the water bound to the type II copper site suggesting that the former interaction is stronger in the native enzyme. In the absence of a stabilizing interaction with residue 98 in the D98N mutant, a shift in the ligand water results in a small perturbation in the electronic environment of the type II copper site as detected by EPR (Table 3.2). The lack of interaction between Asn98 and the ligand water is consistent with a model of Asp98 not being protonated, favoring the binding of substrates that can act as proton donors. A key feature of a proposed mechanism for nitrite reduction by AfNiR is the direct role played by Asp98 as the proton donor to the nitrite (Adman et al. 1995, Murphy et al. 1997b). The loss of this critical interaction between residue 98 and the ligand water and the inability of Asn98 to act as a proton donor are the likely causes of the low activity of the D98N mutant. The proposed protonation state of Asp98 is supported by the examination of carbon monoxide bound to reduced native and D98N AfNiR by Fourier transform infra-red spectroscopy (Zhang et al. 2000). The vibrational band observed for CO bound to native AfNiR is unaffected by changes in pH or the presence of deuterated solvent, presumably because Asp98 is not protonated and thus cannot form a hydrogen bond with CO. In contrast, similar spectra of CO bound to D98N AfNiR show strong pH dependence and are consistent with a protein group, likely the side-chain amide of Asn98, forming a hydrogen-bound to the CO. 74 Chapter 3 - Catalytic roles for Asp98 and His255 3.3.3 RoleofHis255 In the hydrogen bond model (Figure 3.3), His255 is protonated with the Ne2 atom acting as a proton donor to the bridging water. The mutation of His255 to Asn or Asp would be expected to eliminate this interaction; however, for each mutant an additional water molecule is introduced into the active site and acts as an adapter by donating protons to either Asn255 or Asp255 and the bridging water (Figure 3.2). This additional water overlaps with the location of nitrite bound to the type II copper in the native enzyme (Murphy et al. 1997b) and would need to be displaced for substrate to bind. In addition, the hydrogen bond between this additional active site water (Wat1099) and the ligand water may further limit or alter the binding of nitrite. Not surprisingly, these mutations of AfNiR at Ffis255 are more than 450 fold less active than native AfNiR and are even less active than D98N AfNiR. Furthermore, the higher activity of D98N AfNiR may be due to His255 donating protons via the bridging water and stabilizing intermediates in the reaction mechanism as an alternative to Asp98. The copper content of H255N AfNiR is slightly lower than observed for the other mutants; however, the difference is not likely to change the specific activity by more than two-fold. Recently, the analogous residue to His255 in RsNiR (His287) has been mutated to a glutamate and characterized functionally and spectroscopically (Olesen et al. 1998, Veselov et al. 1998). The H287E mutant is 100 fold less active than native RsNiR as measured by the ability to oxidize cytochrome c. ENDOR and EPR spectroscopy show greatly reduced nitrite binding to the type II site in the H287E mutant leading to the suggestion of a charge repulsion effect between the negatively charged Glu287 and the nitrite molecule (Olesen et al. 1998, Veselov et al. 1998). To correlate the RsNiR mutation (Olesen et al. 1998) with the crystal structures of the 75 Chapter 3 - Catalytic roles for Asp98 and His255 His255 AfNiR mutants, a structural model of the H255E mutation was generated from the nitrite-soaked native AfNiR structure. This model shows that the side-chain carboxylate of Glu255 may be positioned within hydrogen bonding distance of both the bridging water and the bound nitrite. Nitrite bound to the active site copper is likely protonated; however, the proximity of a second negative charge in addition to that of Asp98 could be expected to alter nitrite binding. Comparison of the H255E model with the H255D and H255N AfNiR crystal structures reveals that a steric clash exists between the position of the Glu255 side-chain and the additional active site water (Wat 1099). 3.3.4 Revised mechanism Figure 3.4 presents a revised catalytic mechanism for CuNiRs incorporating roles for the bridging water and His255. The resting oxidized enzyme has water bound to the type II copper forming a hydrogen bond with the negatively charged Asp98 side-chain. The nitrite substrate displaces the ligand water and is protonated when bound to the type II copper and also serves as a proton donor in the hydrogen bond with Asp98. Reduction of the type II copper is followed by reduction and decomposition of the nitrite resulting in the formation of a transient complex with a hydroxyl and NO bound to the copper. Protonation of the hydroxyl ion by His255 via the bridging water and release of NO returns the enzyme to the resting state. 76 Chapter 3 - Catalytic roles for Asp98 and His255 C u 2+ H ^ H „ ""O V H i s 2 5 5 ^ ^ \ - ^ C - A s p Q S + ; N — H % / % ii" O N — ^ ' ? HO-N C L C u 2+ O H i s 2 5 5 . \^Jj^~ H * " " » I > I I 0 / H ' " ' Q / C — A s p 9 8 H + , - N O e-C u 2+ o o N . H H ""in, H i s 2 5 5 . -X C - A s p 9 8 i - J CT I H ^HiiiiiiiiimO C u + O H i s 2 5 5 . l ^ ^ ^ H % % / , _ / H O" C — A s p 9 8 / I H Figure 3.4 Proposed catalytic mechanism of nitrite reductase from native AfNiR An attempt to model His255 in the nitrite-soaked structure in an orientation such that a geometrically favorable hydrogen bond could be formed with the nitrite substrate was unsuccessful. An alternative role for Ffis255 may be to provide a positive charge in a location that assists in the reduction step leading to the formation of the transient complex of NO and water bound to the copper (Figure 3.4). Thus, the loss of this positive charge rather than direct donation of a proton may contribute to the large reduction in activity of the H255N and H255D mutants. Analysis of the previously determined nitrite-soaked structure (Murphy et al. 1997b) shows that the bridging water is too distant (3.7 A) to form a hydrogen bond to the 77 Chapter 3 - Catalytic roles for Asp98 and His255 oxygen of the bound nitrite that interacts with Asp98. The position of this nitrite oxygen in the transient complex may be such that the bridging water could act to donate the second proton in conjunction with His255. 3.4 Conclusion The large loss of activity shows clearly that both Asp98 and His255 play a critical role in the catalytic mechanism of CuNiR. A stabilizing hydrogen bond from Asp98, absent in the D98N mutant, donated from the ligand water, and later from the nitrite substrate, is required for full activity of the enzyme and is consistent with a role in donating protons during the reaction. The structural conservation of the bridging water in all three mutants and the proximity to the substrate binding site suggests an important catalytic role, likely by acting as a conduit through which protons are shuttled and in orientating Asp98. The dramatic reduction in activity in the H255N and H255D mutants, suggests a catalytic role for residue 255, possibly through donation of a proton via the bridging water and the positioning of a positive charge to stabilize electrostatically reaction intermediates. 78 Chapter 4 - Nitrite-soaked D98N and H255N structures CHAPTER 4 - Alternate substrate binding modes to two mutant (D98N and H255N) forms of nitrite reductase from Alcaligenes faecalis S-6: Structural model of a transient catalytic intermediate 4.1 Introduction The catalytic requirement of both Asp98 and His255 is demonstrated clearly from site-directed mutagenesis and functional studies (Chapter 3). Mutations of these residues have also been studied recently in RsNiR and AxNiR. From these studies, greater than one hundred-fold reduction in specific activity and significantly reduced affinity for nitrite (Kataoka et al. 2000, Olesen et al. 1998, Veselov et al. 1998) was measured. To understand better the roles of the active site residues Asp98 and His255 in determining the mode of nitrite binding and catalysis in AfNiR, I report the nitrite-soaked crystal structures of D98N and H255N, in both the oxidized and reduced forms. Both Asp98 and His255 are found to be essential in orienting correctly the binding of nitrite in the active site of AfNiR. The oxidized D98N[N02"] crystal structure indicates that Asp98 in the native enzyme serves as a proton acceptor in forming a hydrogen bond with nitrite and is essential for productive substrate binding. In the oxidized H255N[N02_] structure, a restructured solvent network results in a unique pentacoordinate binding mode of nitrite that provides a structural model for a proposed transient catalytic intermediate. In the reduced D98N[N02~] and H255N[N02~] structures a unique dinuclear site is formed in the type I copper site and shows structural similarities to the traditional C U A site. 79 Chapter 4 - Nitrite-soaked D98N and H255N structures 4.2 Results The D98N and H255N crystals used for the soaking experiments grew in a primitive orthorhombic lattice and contain the assembled trimer in the asymmetric unit. The nitrite-soaked native AfNiR structure (Murphy et al. 1997b) was used as the starting model for both the oxidized D98N[N02"] and H255N[N02*] crystal structures following removal of the mutated residues, all solvent atoms and the nitrite. These oxidized mutant structures were used as the starting models for their respective reduced nitrite-soaked structures. The final structure of each mutant begins at Ala4 and ends at Glu339. Data collection and refinement statistics are presented in Table 4.1. 4.2.1 Native active site. The active site of AfNiR is located at the bottom of a 16 A deep cavity at the interface of two adjacent subunits. The type II copper is coordinated by two histidines (His 100, Hisl35) from one subunit, a third histidine (His306) from the adjacent subunit, and a solvent ligand. In the native enzyme, Asp98 is oriented to form a hydrogen bond with the ligand water and interacts with His255 through a single water bridged hydrogen bond (Kukimoto et al. 1994, Murphy et al. 1995). This bridging water completes a solvent network extending to His260 that sits on the surface of AfNiR (Murphy et al. 1995). In the nitrite-soaked AfNiR crystal structure (Murphy et al. 1997b), nitrite displaces the ligand water, interacts with the type II copper in an asymmetric manner through the oxygen atoms, and hydrogen bonds with Asp98 (Figure 4.1 A). No hydrogen bond between nitrite and His255 is observed in the native nitrite-soaked structure. 80 Chapter 4 - Nitrite-soaked D98N and H255N structures Table 4.1: Data collection and refinement statistics Crystal Oxidized D98N[N0 2"] Reduced D98N[N0 2"] Oxidized H255N[N02"] Reduced H255N[N02"] Cell Dimensions (A) A=61.92 a=61.43 a=62.00 a=61.63 6=102.5 6=102.3 6=102.4 6=102.2 c=145.8 c=145.9 c=146.3 c=146.0 Resolution (A) 1.65(1.75-1.65)a 2.00(2.13-2.00) 1.90(2.02-1.90) 1.95(2.07-1.95) R-merge 0.064 (0.251) 0.062 (0.294) 0.066 (0.143) 0.071 (0.292) {1} / MD}b 13.4 (2.63) 19.6 (4.25) 19.8(5.35) 14.6(3.96) Completeness (%) 88.6 (83.3) 97.6 (95.5) 90.0 (70.1) 96.5 (98.2) Unique Reflections 84714(14072) 61451(9867) 66707 (8527) 67729(11357) Redundancy 5.31 9.68 7.71 7.74 Working R-factor 0.178 0.186 0.164 0.179 Free R-factor 0.210 0.225 0.203 0.218 R.m.s.d. Bond length (A) 0.010 0.006 0.008 0.008 Overall B-factor (A2)c 17.6 26.2 17.8 21.8 PDB entry code* 1J9Q 1J9R 1J9S 1J9T 3 Values in parentheses are for the highest resolution shell. b {1} / {a(I)j is the average intensity divided by the average estimated error in intensity. c B-factors are an average from all three monomers 81 Chapter 4 - Nitrite-soaked D98N and H255N structures Two hydrophobic residues (Ile257 and Leu308) are within 5 A of the bound nitrite and may serve as important determinants in substrate binding (Adman et al. 1995, Murphy et al. 1995). Residues Alal37, Vall42, Vall46, and Phe312 complete the active site hydrophobic pocket that extends out along one side of the cavity to the molecular surface. Figure 4.1 Stick diagram showing the orientation of nitrite in the active site of (A) nitrite-soaked native AfNiR (Murphy et al. 1997b), (B) oxidized D98N[N0 2"J and (C) H255N[NC>2"] structures. Hydrogen bonds are depicted as dotted lines. 82 Chapter 4 - Nitrite-soaked D98N and H255N structures 4.2.2 Mutant active sites. In the oxidized D98N[N02~] structure, nitrite is also coordinated to the type II copper in a bidentate manner via the oxygen atoms (Figure 4.IB). Although the coordination is similar to that observed in the native structure, the position of the nitrite N atom relative to the copper is altered. In the mutant, the nitrite nitrogen is positioned almost perpendicular to the plane defined by the oxygen atoms and the type II copper resulting in a severely bent conformation (Figure 4. IB). In the native nitrite-soaked AfNiR structure (Figure 4.1 A) only a slight nitrite bend is observed of approximately 30° from the vertical plane defined by the type II copper and the oxygen atoms of the nitrite (Murphy et al. 1997b) differing from the D98N[N(V] structure by approximately 60°. A rotation of approximately 20° about the %2 torsional angle of Asn98 in the oxidized D98N[NC»2"] structure results in the N52 atom being displaced by about 0.6 A relative to the 051 atom of Asp98 in the native structure. Although 2.6 A away, the reoriented side-chain of Asn98 displays poor hydrogen bond geometry with the bound nitrite. The 01 and 02 atoms of the bound nitrite form hydrogen bonds with Wat2098 (2.45 A) and the bridging water, Watl098 (3.50 A), respectively (Figure 4.1B, Table 4.2). Asn98 081 is positioned to form a 3.38 A hydrogen bond with the bridging water through which it is linked with His255 Ns2 (Figure 4.IB). The Ne2 atom of His255 sits 3.32 A from the closest nitrite atom (02) but poor geometry limits the formation of a hydrogen bond. An average B-factor of 36.4 A2 and omit difference electron density maps indicate that nitrite is poorly ordered in the oxidized D98N[N02_] structure (Figure 4.2 - D98N(ox)). Furthermore, the side-chain of Asn98 is also disordered occupying two distinct conformations. 83 er 4 - Nitrite-soaked D98N and H255N structures Figure 4.2 Active sites of oxidized D98N[N02~], reduced D98N[N0 2"], oxidized H255N[N02"] and reduced H255N[N02"] AfNiR mutants. The three histidine type II copper ligands are drawn in blue as is His255 in the top panels. Asn98 (top panels) is colored in light red, as is Asn255 in the bottom panels. Asp98 (bottom panels) is colored in red. Water molecules are drawn as aquamarine spheres. The aliphatic residues Ile257 and Leu308 Val304, Vall46 and Vall42 are shown in green. Copper atoms are colored gray; nitrogens are colored dark blue and oxygen atoms red. The backbone of monomers C and B are shown in burgundy and teal respectively. Nitrite bound in the active site is colored. Omit Fo - Fc electron density maps of the nitrite are contoured at 4a and drawn as a yellow wire mesh. Note the presence of density for two distinct conformations of Asn98 in the oxidized and reduced D98N[N02~] structures. 84 Chapter 4 - Nitrite-soaked D98N and H255N structures Chapter 4 - Nitrite-soaked D98N and H255N structures The occupancy of each conformer varies between the three different active sites in the crystal asymmetric unit, but in each case the predominant position is directed towards the type II copper. The nitrite may also bind in multiple conformations in the oxidized D98N[N02~] structure; however, only one orientation could be modeled reliably into the somewhat diffuse density. The altered binding orientation of nitrite in the oxidized D98N[NG*2"] structure results in a small shift of Ile257 towards the nitrite such that the closest atom, Ile257 C51, is positioned 2.96 A from the nitrite nitrogen. The shift of Ile257 results in a slightly more occluded substrate-binding site. With the exception of the C51 and C52 atoms of Leu308 that are 4.76 and 4.01 A from the closest atom of nitrite (01) respectively, all other active site hydrophobic residues are greater than 5.00 A away (Figure 4.2 - D98N(ox)). The most surprising feature of the oxidized H255N[N02~] crystal structure is that nitrite binding to the type II copper does not displace the ligand water (Wat503). Instead, nitrite forms a monodentate coordination to the copper through a single oxygen atom, 02 (Figure 4.1C). In this new binding mode, the nitrite N atom is displaced approximately 90° out of the plane defined by the oxygen atoms and type II copper. Relative to the nitrite-soaked native AfNiR structure, the nitrite is displaced away from Ile257 and towards Alal37 by approximately 1.5 A (Figure 4.2 - H255N(ox)). The nitrite 01 atom contacts Ile257 C51 (3.49 A) and the 02 atom is positioned near Alal37 CP (3.42 A). As a result of nitrite binding, the ligand water (Wat503) is shifted approximately 1 A toward the bridging water (Watl098). The pentacoordinate type II copper adopts a distorted square pyramidal geometry with His306 at the sole axial position. 86 Chapter 4 - Nitrite-soaked D98N and H255N structures The hydrogen bond network surrounding nitrite bound to the active site is altered drastically. The shift of the ligand water allows the formation of hydrogen bonds to the bridging water (3.47 A) and to the 081 atom of Asn255 (3.49 A) (Figure 4.1C). The ligand water maintains a 2.67 A hydrogen bond with the 082 of Asp98 (Figure 4.1C). The oxygen atom of nitrite not coordinated to the copper (01) overlaps approximately with the location of Wat2098 in the nitrite soaked native structure and is anchored through a hydrogen bond (2.61 A) to Asp98 082 (Figure 4.1C). Two new solvent atoms are introduced into the active site, Wat3098 and Watl099. Wat3098 (B-factor 25.9 A2) is located near the position of the displaced Wat2098 and is close enough to the nitrite 01 atom (2.70 A) to form a hydrogen bond. Watl099 (B-factor 18.5 A2) results from replacing His255 with the smaller asparagine residue. This extra water hydrogen bonds with Asn255 and Watl098 effectively maintaining the solvent network bridge between Asp98 and residue 255 (Figure 4.1C). Data for reduced D98N[N02_] and H255N[N02"] crystals were collected to attempt to identify structures of catalytic intermediates. In these structures, increased B-factors and weaker electron density from omit maps (Figure 4.2 - D98N(red), H255N(red)) suggest that nitrite is bound at lower occupancy or is more disordered. The average B-factors for nitrite are increased from 32.4 A2 to 37.3 A2 and from 23.3 A2 to 45.8 A2 upon reduction of the D98N[N02_] and H255N[N02~] structures, respectively. In each structure, however, nitrite adopts a similar position in the active site relative to the oxidized structure (Figure 4.2). Comparing the reduced and oxidized D98N[N02"] structures, the B-factor of the bridging water (Watl098) that connects Asn98 with His255 shows the greatest increase (35.8 A2 versus 16.9 A2). 87 Chapter 4 - Nitrite-soaked D98N and H255N structures Table 4.2 Type II Copper - ligand and copper - nitrite bond lengths Parameter Oxidized D98N[N02~] Reduced D98N[N0 2"] Oxidized H255N[N02"] Reduced H255N[N02"] I. Type II Cu ligand distances (A) Cu502- 100N' 2 2.00 (0.01)a 2.00 (0.04) 2.04 (0.06) 2.01 (0.02) Cu502- 135N e 2 2.08 (0.04) 2.14(0.01) 2.19(0.02) 2.10(0.03) Cu502 - 306N e 2 2.06 (0.03) 2.16(0.02) 2.19(0.01) 2.16(0.02) Cu502-503OH 2 N / A N / A 2.15 (0.09) 1.70 (0.25) II. Type II Cu Nitrite distances (A) C u 5 0 2 - N 2.32(0.18) 2.27 (0.014) 3.21 (0.13) 3.70 (0.05) Cu502 - O l 2.42 (0.05) 2.89 (0.48) 3.60 (0.15) 4.02 (0.16) Cu502 - 02 2.21 (0.08) 2.21 (0.19) 2.16(0.05) 2.57 (0.07) II. Active site ligand H-bond distances (A) 504O2- 1098OH2 3.50 (0.27) 3.47 (0.24) N / A N / A A s p 9 8 o 8 2 - 5 0 4 O l N / A N / A 2.79 (0.19) 2.81 (0.08) Asp98 0 6 2 - 503OH 2 N / A N / A 2.91 (0.24) 2.36(0.01) 5 0 3 O H 2 - 1098OH2 N / A N / A 3.45 (0.06) 4.27b (0.22) a Values in brackets show range of measurements between three non-crystallographically related monomers in the asymmetric unit In the reduced H255N[N02~] structure, the side chain of Ile257 adopts a different conformation in the reduced state with the C81 atom directed down toward the type II copper positioned 2.95 A from the 02 atom of the nitrite (Figure 4.2 - H255N(red)). Wat3098, which is involved in a hydrogen bond with nitrite in the H255N[N02"] structures shows a 88 Chapter 4 - Nitrite-soaked D98N and H255N structures near two-fold increase (46.2 A2 vs. 26.7 A2) in B-factor in the reduced H255N[NC>2"] structure as does the ligand water, Wat503 (47.1 A2 vs. 28.3 A2) indicating increased disorder or reduced occupancy. The ligand water, Wat503, is no longer within hydrogen bonding distance to the bridging water, Wat 1098, in the reduced H255N[N02"] structure (Table 4.2). 4.2.3 Type I copper site In the oxidized D98N[N02~] and H255N[N02~] crystal structures the mononuclear type I copper sites are coordinated in a distorted trigonal pyramidal geometry by four protein ligands (Hisl45, His95, Cysl36 and Metl50) (Figure 4.3A) as observed in native AfNiR structures (Godden et al. 1991, Kukimoto et al. 1994, Murphy et al. 1995). Figure 4.3 Stick diagram of type I Cu site of (A) oxidized H255N[N0 2"] and (B) reduced H255N[N0 2"] AfNiR. The Fo-Fc omit electron density maps surrounding the type I coppers are contoured at 5<7. 89 Chapter 4 - Nitrite-soaked D98N and H255N structures However, the type I copper site in the reduced D98N[N02~] and H255N[N02~] crystal structures reveals an unexpected copper coordination. In the presence of 20 mM ascorbate, 2 mM copper chloride and 1 mM zinc acetate the type I ligands rearrange to incorporate a second metal atom forming a dinuclear site (Figure 4.3B). The second metal was modeled as a copper in the structure. Crystallographic B-factors of 21.8±0.1 A2 are observed for both copper atoms in the modified type I copper site in the reduced H255N[N02~] structure (Figure 4.3B). The B-factors for the analogous copper atoms in the reduced D98N[N02~] structure are increased approximately 30% (32.7 A2 and 35.8 A2) suggesting reduced metal occupancy. In the reduced H255N[N02"] structure the side-chain of His 145 is shifted approximately 0.6 A in the opposite direction of the original type I copper and is rotated approximately 35° about the X2 angle such that the N51 atom serves as a ligand to a second copper atom (Cu500) (Figure 4.3B). Cysl36 Sy becomes a shared ligand between the two coppers (Cu500 and Cu501) while the Metl50 S8 and His95 N81 ligands remain unperturbed. A loop incorporating residues Alal37 through Hisl45 that packs against the type I site is displaced slightly with a root mean squared deviation of 0.4 A for Cot atoms relative to the oxidized H255N[NC>2~] structure to accommodate the second copper atom (Cu500). Pro 138, the proximal residue from this loop, adopts a different pucker conformation such that the Cy atom is 3.11 A from Cu500. Apart from the shift in this surface loop, the tertiary structure in the modified type I copper site remains largely unchanged. Ligand copper distances and geometries are presented in Table 4.3. 90 Chapter 4 - Nitrite-soaked D98N and H255N structures Table 4.3: Type I Copper - ligand bond lengths and geometries Parameter Oxidized D98N[N0 2"] Reduced D98N[N0 2"] Oxidized H255N[N02"] Reduced H255N[N02"] I.Type I Cu ligand distances (A) C u 5 0 0 - 145N 5 1 N / A 2.01 (0.24) N / A 1.83 (0.13)a C u 5 0 0 - 136SY N / A 2.08 (0.02) N / A 2.12(0.07) C u 5 0 0 - C u 5 0 1 N / A 2.31 (0.02) N / A 2.45 (0.05) Cu 501 - 136SY 2.23 (0.03)a 2.25 (0.1) 2.26 (0.04)a 2.55 (0.45) Cu 501 - 9 5 N 5 1 2.07 (0.02) 2.09 (0.06) 2.12(0.06) 2.21 (0.2) Cu501 - 150S5 2.44 (0.01) 2.38 (0.16) 2.41 (0.05) 2.42 (0.24) Cu 501 - 145N 8 1 2.07 (0.06) N / A 2.00 (0.08) N / A II.Type I Cu ligand angles (°) 145N 5 1 -Cu501 - 150S5 130(3.0) 124(1.0) 131 (2.0) 126 (2.0) 136SY-Cu501 - 9 5 N 8 1 132(1.0) 138(5.0) 131 (1.0) 136 (6.0) 95N 5 1 -Cu501 - 150S5 87.7 (0.3) 96.0 (5.0) 89.8 (2.0) 98.2(1.8) 136SY-Cu501 - 150S5 106(1.0) 116(5.0) 105 (2.0) 121 (3.0) 136S y-Cu501 - 145N 5 1 105 (2.0) 84.2 (0.4) 105 (2.0) 77.5 (2.5) 145N 8 1 -Cu501 - 9 5 N 8 1 97.6(1.5) 96.7(8.8) 96.7(1.3) 94.6(2.1) 1 4 5 N 5 1 - C u 5 0 0 - 150S8 N / A 103 (5.5) N / A 113(2.0) 1 4 5 N 5 1 - C u 5 0 0 - 136SY N / A 164(12) N / A 171 (5.0) 1 4 5 N 5 1 - C u 5 0 0 - 9 5 N 8 1 N / A 88.0 (4.7) N / A 98 (4.7) 136S Y -Cu500-Cu501 N / A 58.8 (4.4) N / A 54.7(1.6) 136SY-Cu501 -Cu500 N / A 53.0 (0.7) N / A 53.0(2.5) 9 5 N 5 1 - C u 5 0 0 - 150S5 N / A 46.9 (3.1) N / A 46.0 (0.9) Bond distances and angles are an average of all three monomers 91 Chapter 4 - Nitrite-soaked D98N and H255N structures 4.3 Discussion In this study, nitrite-soaked crystal structures of two active site mutants of AfNiR (D98N and H255N) have been determined in both the oxidized and reduced forms to a resolution sufficient to observe the precise binding mode of the substrate to the copper. The observed alterations in binding mode in the oxidized structures provide significant insight into the roles of Asp98 and His255 in determining the mode of nitrite binding in the native enzyme. The reduced mutant structures are discussed primarily with respect to the surprising observation of the presence of dinuclear type I copper sites. 4.3.1 Role of Asp98 in determining the mode of nitrite binding. Of the residues in close proximity to the type II copper in the structure of native AfNiR in the resting state, Asp98 is the least ordered (Murphy et al. 1997b). The binding of nitrite results in a decrease in the B-factors of Asp98 resulting from the formation of a direct hydrogen bond to the substrate. The bidentate coordination of nitrite through the oxygen atoms to the type II copper in the D98N[N02~] crystal structures is similar to that observed in the native nitrite-soaked AfNiR crystal structure; however, higher B-factors and omit difference electron density maps indicate that both nitrite and the side-chain of Asn98 are disordered (Figures 4.2 - D98N(ox), (red)). In the structure of D98N AfNiR in the oxidized resting state, Asn98 is also poorly ordered (Chapter 3). The binding of substrate to D98N AfNiR has little effect in stabilizing the conformation of this residue. Clearly, from a comparison of these structures, Asn98 is not able to form a hydrogen bond to water or substrate bound at the active site. The conformational disorder of Asn98 has been suggested by recent FT-IR CO 92 Chapter 4 - Nitrite-soaked D98N and H255N structures studies of the reduced D98N AfNiR variant (Zhang et al. 2000). From these experiments, two CO stretching frequencies were measured and identified as representing different modes of CO binding. The D98N crystal structures in the presence and absence of substrate suggest that these two CO stretching frequencies likely result from two conformations of Asn98, one of which interacts with bound CO. Similar FT-IR CO experiments of native AfNiR recorded over a pH range of 6.0 to 8.0 suggest that Asp98 is deprotonated thereby requiring nitrite to bind in the protonated form to the native enzyme (Zhang et al. 2000). These observations are consistent with five crystal structures of AcNiR solved at pH values between 5 and 6.8 that show minimal structural change in the active site (Adman et al. 1995). Recent steady state kinetics show an increase in K m for nitrite to the D98N and D98E AcNiR mutants of 200 fold and 15 fold, respectively, indicating that a negatively charged Asp98 may be required for high affinity binding of the substrate (Kataoka et al. 2000). Taken together, the available data indicate that the disorder observed for nitrite and Asn98 in the D98N[N02~] crystal structures is a result of the inability of the N52 atom of Asn98 to form a hydrogen bond with nitrite bound in the protonated form. Furthermore, the data presented here identifies the hydrogen bond between Asp98 and nitrite in the native structure as essential in anchoring nitrite in the active site for productive catalysis. This hydrogen bond is also likely to serve as a direct link through which protons are exchanged during catalysis. 4.3.2 Role of His255 in determining the mode of nitrite binding. The lack of a direct interaction with bound substrate suggests that His255 plays a limited role in nitrite binding; however, mutagenesis studies have shown that this residue is 93 Chapter 4 - Nitrite-soaked D98N and H255N structures critical for nitrite binding and function (Kataoka et al. 2000, Olesen et al. 1998). His255 is thought to complement electrostatically the negative charge on nitrite, donate protons to the substrate directly during catalysis or be indirectly involved in nitrite binding through correct positioning of Asp98 through the bridging water (Adman et al. 1995, Boulanger et al. 2000, Kataoka et al. 2000, Murphy et al. 1997b, Olesen et al. 1998, Veselov et al. 1998). In the crystal structure of H255N AfNiR in the resting state, steric constraints indicate that an additional active site water (Wat1099) bound near the copper must be displaced for correct binding of nitrite in the active site (Chapter 3). Surprisingly, in the oxidized H255N[N02_] structure neither the ligand water (Wat503), nor the additional active site water, Watl099, are displaced upon nitrite binding (Figures 4.2 - H255N(ox)). Instead, nitrite adopts a novel-binding mode in the active site displacing the weakly bound Wat2098 and coordinating to the type II copper via a single oxygen atom (Figure 4.2). The resulting pentacoordinate copper displays a distorted square pyramidal geometry that is similar to a synthetic analogue where two nitrogen atoms and two oxygen atoms, analogous to the ligand water and the 01 atom of nitrite, are in plane with the copper and a third nitrogen, analogous to His306, which is positioned as the apical ligand (Halfen et al. 1994). As shown by the low enzyme activity of the H255N mutant, the observed alternate binding mode for nitrite observed in the oxidized H255N[N02~] structure is likely catalytically unproductive. However, this change in nitrite binding mode clearly identifies His255 as an essential residue in determining the productive nitrite binding observed in the native AfNiR crystal structure. Three major factors may determine the alternate binding mode of nitrite. First, the displacement of Wat2098, which shows high B-factors and is only hydrogen bonded singly to the rest of the structure, is more energetically favorable than displacing both the additional 94 Chapter 4 - Nitrite-soaked D98N and H255N structures active site water (Watl099) and the ligand water (Wat503) that are part of an extensive solvent network (Figure 4.1C). Second, a hydrogen bond between nitrite and Asp98, responsible for the low average B-factor for nitrite and the well-defined electron density, limits the number of favorable positions of nitrite in the active site. Third, analysis of the native nitrite-soaked AfNiR crystal structure (Murphy et al. 1997b) suggests that the positively charged His255 may serve to lower the pKa and orient Asp98 through the bridging water (Watl098) for optimal interaction with the bound nitrite. The H255N mutant would likely increase the pKa of Asp98 such that this residue is now protonated and uncharged when nitrite is bound. The change in protonation state of Asp98 may result in the altered binding mode of nitrite. The ability of a mutation at position 255 to affect nitrite binding was observed recently from histidine and water proton ENDOR and EPR spectroscopy of H287E RsNiR (Olesen et al. 1998, Veselov et al. 1998). In these experiments, displacement of the ligand water by nitrite alters the spectral properties of the native enzyme. In the H287E mutant no spectral change is observed following the addition of nitrite suggesting a limited ability for nitrite to bind. One explanation suggested by Olesen et al is that a charge repulsion effect exists between the negatively charged carboxylate of Glu287 and nitrite (Olesen et al. 1998). FT-IR CO data (Zhang et al. 2000) and steady state kinetics (Kataoka et al. 2000) suggest a more indirect role for His255. These experiments indicate that a deprotonated, negatively charged Asp98 residue is required for high affinity nitrite binding by serving as a proton acceptor in forming a hydrogen bond with the protonated, uncharged form of the molecule. The absence of a formal charge on nitrite likely results in limited electrostatic interactions of bound nitrite with charged active site residues such as His255. Modest increases in measured 95 Chapter 4 - Nitrite-soaked D98N and H255N structures K m values for nitrite, combined with a logarithmic decrease in enzyme activity of His255 AcNiR mutants are interpreted as His255 being involved indirectly in coordinating nitrite but essential in orienting Asp98 through the bridging water (Kataoka et al. 2000). 4.3.3 Catalytic mechanism of copper-containing nitrite reductase. The distinctive manner in which nitrite is bound in the H255N nitrite-soaked structures provides unique insight into the catalytic mechanism of copper-containing nitrite reductases. In the current mechanistic model for CuNiRs, a protonated nitrite molecule displaces the ligand water and coordinates to the type II copper prior to electron transfer from the type I copper (Chapter 3). From Figure 4.4, reduction of the type II copper is followed by the formation of a transient complex in which the N - 0 bond of nitrite proximal to Asp98 is broken leaving a hydroxyl group and nitric oxide (NO) simultaneously bound to a pentacoordinated copper (steps 2 and 3). In the transient pentacoordinate complex, as drawn in Figure 4, the 01 and N atoms of NO adopt similar positions to the 01 and N atoms of nitrite observed in the H255N[N02~] crystal structure (inset box). In this transient complex, both Asp98 and His255 are uncharged. The subsequent protonation of the active site, possibly initiated through Ffis255, results in the release of NO and regeneration of the water ligand to the type II copper. 96 Chapter 4 - Nitrite-soaked D98N and H255N structures His255 Figure 4.4 Catalytic mechanism for copper-containing nitrite reductases 97 Chapter 4 - Nitrite-soaked D98N and H255N structures The oxidized H255N[NC>2"] structure provides evidence that the proposed transient intermediate state of the enzyme is energetically accessible. The positioning of NO and the hydroxyl bound to the type II copper modeled from the oxidized H255N[N02_] structure (Figure 4.4 - inset box) are directed favorably with respect to the active site cavity that shows a distinct polar and hydrophobic face. The positioning of nitrite in the oxidized H255N[N02~] structures suggests that in the native enzyme NO is oriented for diffusion out of the active site along the side devoid of charged and polar residues and lined with hydrophobic residues beginning at Alal37, Leu308, Vall46, and extending through to Vall42 and Phe312, which are located on the surface of AfNiR. This model of diffusion limits the interaction of NO with polar residues thereby minimizing non-specific side reactions. The hydroxyl group positioned proximal to the bridging water (Wat 1098) is directed towards the polar side of the active site cavity that assists to stabilize the transient negative charge. Despite the lack of noticeable reoxidation following nitrite soaking and reduction at 0 °C, the type II copper site may be partially oxidized contributing to the observed disorder in the active site. Interestingly, the substantial increase in the B-factor of the bridging water (Watl098) upon reduction of the nitrite-soaked native AfNiR structure (Murphy et al. 1997b) is consistent with a disruption in the solvent network linking residues Asp98 and His255. This proposed disruption would permit the side chain of His255 the freedom to approach reaction intermediates and directly donate a proton during catalysis (Kataoka et al. 2000). However, modeling studies (Chapter 3) have shown that His255 cannot be oriented to form a hydrogen bond with nitrite nor does His255 show increased B-factors indicative of increased mobility in reduced AfNiR crystal structures. An alternative explanation is that the proton is 98 Chapter 4 - Nitrite-soaked D98N and H255N structures shuttled through the bridging water (Watl098). In the oxidized H255N[N02"] structure, the ligand water to the type II copper is positioned within hydrogen bonding distance to the bridging water (Watl098) (Figure 4.2 - H255N(ox)) creating a defined pathway through which protons can exchange during catalysis. A proton shuttled through the bridging water (Wat 1098) during catalysis likely originates from the bulk solvent where structural data shows a well-defined solvent network extending from Wat1098 to His260 that sits on the surface of NiR (Murphy et al. 1995). 4.3.4 Type I copper site. Several different copper sites exist in proteins ranging from the mononuclear type I and II centers to the recently discovered tetranuclear Cuz cluster (Brown et al. 2000). A cupredoxin like fold in cytochrome c oxidase (Blackburn et al. 1994) and nitrous oxide reductase (Brown et al. 2000) coordinate a mixed valent dinuclear copper cluster termed the C U A site represented by the consensus sequence { C (X) 3 C (X) 3 H (X) 2 M} (Zumft et al. 1992). The reduced D98N[N0 2"] and H255N[N02"] crystal structures show that a second metal atom has been incorporated into the type I copper site generating a unique dinuclear metal center (Figures 4.3A and B). This site is structurally similar to the dinuclear C U A site (Figure 4.5), but shows unusual copper coordination. The proximity of the two metal atoms (Table 4.3) is consistent with spectroscopic measurements of a traditional C U A site that identifies a 2.5 A metal-metal bond (Blackburn et al. 1994). The coordination of the copper atoms in a traditional C u A site is that of a distorted tetrahedral geometry. In this site, there are two shared Cys Sy ligands, with His N81 and Met S8 atoms completing the coordination for one copper, and His N81 and a carbonyl oxygen from either a Trp (Brown et al. 2000) or a 99 Chapter 4 - Nitrite-soaked D98N and H255N structures Glu (Tsukihara et al. 1995) coordinating the second copper atom. The pseudo-dinuclear site presented here lacks the overall symmetry of a traditional C u A site as it incorporates only four ligands instead of six in the coordination of the two coppers. Figure 4.5 Stereo diagram of the modified type I Cu site of reduced H255N[N02_] AfNiR (light gray) superimposed on the C u A site of nitrous oxide reductase from P. nautica (dark gray) (Brown et al. 2000). E X A F S spectroscopy (Blackburn et al. 1994) has provided a model to compare the topological similarities and the evolutionary link between the mononuclear type I and dinuclear C u A sites. This connection has been demonstrated further through protein engineering studies of the blue copper protein quinol oxidase (Wilmanns et al. 1995). In this study, extensive site-directed mutagenesis of the type I site loop resulted in the successful formation of a dinuclear C u A center from a type I copper site. To our knowledge, the dinuclear pseudo C u A sites in AfNiR are the first examples of a non-engineered expansion of 100 Chapter 4 - Nitrite-soaked D98N and H255N structures a type I site to a dinuclear copper site similar to a CUA. The small changes in structure and the flexibility of the surface loop incorporating the His 145 ligand suggests that this novel dinuclear site may be physiologically attainable in AfNiR. Lastly, the introduction of such a site would alter the redox chemistry of NiR especially i f the coppers existed in a mixed valence state as observed in the C U A site. Such a site could potentially be used by AfNiR as a mechanism to regulate activity. 4.3 Conclusion The ability of active site residues to mediate the correct mode of nitrite binding is shown clearly in the crystal structures presented here. Despite the lack of an interaction between His255 and nitrite in the native enzyme, the mutation of His255 to an Asn results in a restructured solvent network and a completely novel mode of nitrite binding. We suggest that this alternate binding mode mimics the electronic and structural properties of a proposed catalytic intermediate in native AfNiR. The surprising appearance of a non-engineered dinuclear type I metal site in the reduced mutant structures may be a general model for the evolution of the C U A site. This unusual metal coordination may also represent a physiologically relevant change during catalysis potentially used in regulating the redox chemistry and overall activity of copper-containing nitrite reductases. Further work is currently in progress to determine whether the formation of the dinuclear site is attainable in the native enzyme under physiological concentrations of copper. 101 Chapter 5 -1257 AfNiR variants Chapter 5 - Role of Ile257 in directing the mode Of nitrite binding in AfNiR 5.1 Introduction A blanket of hydrophobic residues in both the heme cd; (Williams 1997) and CuNiRs define the topology of the active site. In the cdj NiR from Thiosphaera pantotropha the hydrophobic patch is comprised of Leu443, Leu460 and Phe444, which are positioned within 5 A of the bound nitrite (Williams et al. 1997). In CuNiRs, Ile257, Leu308, Vall41 and V a i l 44 (numbering is that of AfNiR) form the equivalent hydrophobic blanket. The proximity of these hydrophobic residues to the metal co-factor, suggest a role in defining the mode of substrate binding. In the native nitrite-soaked AfNiR structure (Murphy et al. 1997b) nitrite binds to the copper in a bidentate fashion through the oxygen atoms, of which the 01 atom forms a hydrogen bond with Asp98. Attempts to model nitrite in an N-coordinate form similar to several biomimetic models (Halfen et al. 1996, Halfen et al. 1994) failed due to steric clashes with the side-chain of Ile257. This observation suggests a potentially important role for Ile257 in directing a productive mode of nitrite binding during catalysis. To study the catalytic role of Ile257, a combinatorial mutagenesis approach was taken to construct a small library of six variants. Functional studies of these variants reveal a nitrite reducing ability spanning nearly two orders of magnitude indicating that this active site isoleucine is involved intimately in the catalytic mechanism. Interestingly, a mutation of isoleucine to a valine, which is the only observed natural substitution for the active site isoleucine, shows greater activity than the native enzyme. The altered activities show a strong correlation with alternate binding modes of nitrite observed in the high-resolution (< 102 Chapter 5 -1257 AfNiR variants 1.8 A) nitrite-soaked crystal structures. Collectively these data indicate a requirement for the bidentate mode of nitrite binding for full catalytic activity and are discussed with respect to the proposed mechanistic models. 5.2 Results 5.2.1 Characterization of AfNiR variants Using a combinatorial mutagenesis approach, a small library of mutations was generated at position 257 in the active site of NiR from Alcaligenes faecalis S-6. A l l mutations were confirmed with D N A sequence analysis, which showed that no one mutation was dominant in the library. Mass spectrometry showed each purified variant protein to be within 4 Da of the expected molecular mass following removal of the N-terminal methionine (Table 5.1). UV-visible spectroscopy was used to probe the electronic structure of the type I copper site in each of the six variants. In all cases, absorbance maxima are at the same three characteristic wavelengths of 458, 595 and 680 nm as seen in previous preparations of native AfNiR (Kakutani et al. 1981b, Kukimoto et al. 1994). Also, the ratios of absorbencies of 458 to 595 nm (1.31 ± 0.05) and at 277 to 458 nm (18.0 ± 1.3) are well conserved between the variant AfNiR species reported here (Table 5.1) and in previous preparations of recombinant native (Table 3.1) and native AfNiR (Kakutani et al. 1981a). Using dithionite reduced methyl viologen as the artificial electron donor, the specific activity measured for native AfNiR is within experimental error (10%) of that measured previously (Table 3.1) and reported in the literature (Kukimoto et al. 1994). The activities of the variant AfNiR species span nearly two orders of magnitude (Table 5.1). The replacement of Ile257 with a valine results in a slightly elevated specific activity relative to the native 103 Chapter 5 -1257 AfNiR variants enzyme, while the conservative mutation, I257L, shows a three to four-fold decrease in activity. A reduction of 25 to 40-fold in specific activity is observed for the I257A, I257G and I257M variants. The replacement of Ile257 with a threonine results in the most significant loss of activity of greater than 98% relative to the native enzyme. Table 5.1 Characterization of native and Ile257 variant AfNiRs AfNiR A458/595 A277/458 Mass (Da) a Specific activity (U/mg)b Native 1.30 16.6 36847 (36843) 417 (44)c I257V 1.33 18.5 15(14) 521(50) I257L 1.26 19.1 1(1) 108 (14) I257M 1.35 17.0 22(18) 17.9(3.4) . I257A 1.32 17.9 43 (42) 15.6(0.8) I257G 1.36 17.0 57 (56) 10.4 (0.9) I257T 1.29 17.1 12 (12) 5.90 (0.7) a The absolute mass of the native is given along with the theoretical mass in parentheses. The values for the mutants are the absolute observed and theoretical mass (in parentheses) differences relative to the native. b Specific activity values are reported as an average of at least three trials. c Values in parentheses represent the standard error 5.2.2 Overall structures Each of the nitrite-soaked variant AfNiR crystal structures is refined to a resolution of at least 1.78 A and shows excellent stereochemistry indicated by over 90% of the residues adopting most favorable conformations as determined by a Ramachandran plot (Laskowski et 104 Chapter 5 -1257 AfNiR variants al. 1993) with the remaining residues in the allowed regions. The root-mean-squared (r.m.s.) deviations from ideality for bond lengths is less than or equal to 0.011 A for each structure (Table 5.2). The crystallographic RWOrk and Rf r e e for these high-resolution structures are less than 18% and 21%, respectively. Data collection and refinement statistics are presented in Table 5.2. Table 5.2 Data collection and refinement statistics Crystal I257L [N021 I257V [N02j I257A [N0 2 J I257G [N02-] I257M [N0 2 J I257T [N0 2 J Cell Dimensions (A) o=61.95 6=102.5 a=61.73 6=102.3 a=62.00 6=102.4 o=62.05 6=102.4 o=61.77 6=102.4 o=61.48 6=102.0 c= 146.2 c=145.9 c=146.2 c=146.2 c=146.0 c=146.1 Resolution (A) 1.70 1.75 1.70 1.75 1.78 1.78 R-merge 4.7(21.6)a 6.2(26.2) 6.8(21.6) 5.7(32.4) 8.2(30.6) 6.6(27.4) {1} / MD}b 23.6(4.4) 18.3(3.6) 16.8(4.1) 19.6(2.8) 16.0(3.2) 15.1(4.1) Completeness (%>) 91(64.2) 95(83.5) 93(74.5) 98(90.7) 95(77.6) 87(71.9) Unique Reflections 93657 89868 95952 92678 84883 77077 Redundancy 7.9 11.7 7.9 7.6 9.6 9.8 Rwork 0.156 0.166 0.158 0.159 0.154 0.157 Rfree 0.188 0.208 0.192 0.194 0.197 0.197 # solvent atoms 1345 1324 1352 1340 1465 1428 R.m.s.d. Bonds (A) 0.011 0.010 0.011 0.010 0.010 0.009 Overall B-factor (A2)c 21.3 26.6 21.1 23.0 26.1 23.8 a Values in parentheses are for the highest resolution shell. b {1} / {ff(I)} is the average intensity divided by the average estimated error in intensity. c B-factors are an average from all three monomers 105 Chapter 5 -125 7 AfNiR variants Each refined structure contains the assembled physiological homotrimer of a total of 1005 residues and six copper atoms in the asymmetric unit of the orthorhombic cell. The variant structures begin at residue Ala4 and end at Gly339. In the final model, well-defined electron density permits modeling of all N-terminal residues. At the C-terminus, five residues (TIEGR) that include part of the factor Xa cleavage site remain unmodeled due to disorder. Generally, the surface loops of the variants are well ordered with the exception of two loops extending from Glyl65 to Alal69 and Aspl88 to Glyl91 that show average B-factors of 30 - 40 A2. The overall r.m.s.d of the a-carbon atoms between the variant and the native enzyme structures does not exceed 0.21 A indicating that no global structural rearrangement has resulted from the mutations. The dark green color of the crystals indicates that copper occupies primarily the type I site and comparison of crystallographic B-factors between copper atoms and their ligands are consistent with both the type I and type II copper sites being nearly fully occupied. The coordination geometry of both the type I and II copper sites are in reasonable agreement with previously published values for native AfNiR structures (Table 5.3). A least-squares superposition of the a-carbon atoms of the three histidine ligands and the catalytically essential residues Asp98 and His255 to the native structure show an r.m.s.d. of less than 0.14 A for each variant. A hydrogen bond bridge through Watl098 that connects Asp98 with His255 likely required for activity in the native enzyme (Chapters 3 and 4) is conserved in the six nitrite-soaked variant structures. The positions of the side-chains of the active site residues that lie within 8 A of the copper (Leu308, Phe312, V a i l 42, Alal37 and Val304), with the exception of 257 are equally well conserved. 106 Chapter 5 -1257 AfNiR variants 5.2.3 Bidentate mode of nitrite binding In the nitrite-soaked I257V[N02_] and I257L[NG"2"] crystal structures well-defined omit electron density maps (Figure 5.1) indicate that nitrite is coordinated to the type II copper in an asymmetric bidentate fashion via the oxygen atoms. Bond distances are presented in Table 5.3. Thermal motion parameters ranging from 30 - 35 A2 are also consistent with a well-ordered nitrite molecule. In both structures, nitrite is oriented in a bent conformation with the nitrite nitrogen being displaced approximately 20° out of the plane defined by the type II copper atom and the oxygen atoms of nitrite. By comparison, nitrite in the native nitrite-soaked structure shows a displacement of approximately 45° out of the plane. In the I257V[NC»2"] structure (Figure 5.1), the nitrite is rotated approximately 15° and is shifted toward the side-chain of Asp98 such that the nitrite nitrogen is displaced 0.5 A from the analogous position in the native AfNiR structure (Murphy et al. 1997b). Despite the positional shift in nitrite, no significant displacement of the side-chain of Asp98 is observed. The 02 atom of the bound nitrite forms a 2.65 A hydrogen bond with the 052 atom of Asp98. The Cy2 atom of Val257 is closest to the nitrite nitrogen (3.27 A) with the C y l approaching to within 3.6 A of the 02 atom of nitrite. The side-chain of Leu257 adopts an alternate rotameric conformation from Val257 (I257V[N02"]) and Ile257 in native AfNiR with a change in the Xi angle of nearly 60°. Attempts to model the rotameric conformations of Leu257 such that the C51 atom is positioned analogously to the C51 atom of Ile257 in the native structure were unsuccessful due to steric clashes with the side-chain of His306 (2.69 A) and the bound nitrite (1.55 A). 107 Chapter 5 -1257 AfNiR variants Figure 5.1 Crystal structures of the six nitrite-soaked 1257 variant AfNiRs. Hydrogen bonds are shown as dashed gray lines and ligand bonds are drawn as solid dark gray lines. Water molecules are drawn as aquamarine spheres. Copper atoms are colored gray; nitrogen's are colored dark blue, oxygen atoms red and sulfur atoms yellow. The backbone of monomers B and C are shown in burgundy and teal, respectively. Bonds of the nitrite molecule bound in the active site are colored dark grey. Omit Fo - Fc electron density maps are contoured at 4a and drawn as a green wire mesh. 108 Chapter 5-1257 AfNiR variants Chapter 5 -1257 AfNiR variants In the appropriately modeled conformation, the Cyatom of Leu257 is positioned approximately 0.5 A from the analogous C y l atom of Val257 (I257V[N02~]). Overall, the side-chain of Leu257 is shifted toward the bridging water (Watl098) such that the C51 atom of Leu257 is positioned within 3.4 A. In this conformation, the C51 atom of Leu257 is positioned 3.14 A from the nitrite nitrogen atom and the C52 atom approaches the 02 atom of nitrite to within 3.8 A. The C51 atom of Leu257 sits 2.80 A from the 052 atom of Asp98, which is shifted approximately 0.25 A in the opposite direction relative to the native structure. Overall, the side-chain of Asp98 and the bound nitrite in the I257L[N02~] structure are well defined with average B-factors of 22 A2 and 29 A2, respectively. Despite a 2.60 A hydrogen bond between the 01 atom of nitrite and the side-chain 052 atom of Asp98, the B-factor for the 01 atom is roughly 20 % greater than for the 02 atom and displays somewhat weaker electron density. Table 5.3 Type II copper - ligand bond lengths Parameter I257L I257V I257A I257G I257M I257T [N02"] [N02"] [N02"] [N0 2"] [N02~] [N02"] I. Atomic distances (A) a504O' - C u 2.3(0.1) b 2.4 (0.1) 2.0 (0) 2.0 (0) 2.2(0. If 2.1 (0.3) 2.2 (0.2) 504O2 - C u 2.1 (0.1) 2.0 (0.1) 3.4 (0.2) 3.8 (0.1) 2.0(0) 4.0 (0.4) 3.1 (0.1) 504O2 - 980 5 2 2.6 (0) 2.5 (0.1) N / A 2.5 (0.2) N / A N / A 503O-- 980 5 2 N / A N / A 2.5 (0) 2.6(0.1) 2.5 (0.0) 2.4 (0.1) 503O- Cu N / A N / A 2.1 (0.2) 2.3 (0.2) 2.2 (0.1) 2.1 (0.1) a Residues 503 and 504 represents the solvent ligand and bound nitrite, respectively. b Values in parentheses represent the range of measurements of the three monomers. 0 Values in italics represent alternate conformation of nitrite in I257G[N02"] structure. 110 Chapter 5 -1257 AfNiR variants 5.2.4 Monodentate mode of nitrite binding Replacement of Ile257 with an alanine, threonine, methionine or glycine results in an unusual reorientation of the bound nitrite (Figure 5.1). In these variants, nitrite adopts a monodentate coordination through the 01 atom to the type II copper with the 02 atom directed approximately toward residue 257. The reorientation of nitrite allows a solvent molecule to occupy the position of the 02 atom of nitrite observed in the bidentate coordination. Although the positions of the catalytically important active site residues, Asp98 and His255, are shifted slightly in the monodentate nitrite bound structures, the active site hydrogen bond network remains largely unperturbed. In the I257A[N02_] crystal structure the non-ligand 02 atom of nitrite is positioned 3.0 A from the /3-carbon atom of Ala257. Omit electron density maps (Figure 5.1) and average B-factors of less than 30.0 A2 for nitrite along with little variation between the three sites in the asymmetric unit suggest that the new binding mode is well defined. The solvent ligand (Wat503) is also well defined with average B-factors of 23.5 A2 and sits 2.12 A from the type II copper (Table 5.3). Wat503 forms three hydrogen bonds, one with the 02 atom of nitrite (2.45 A), a second with the 052 atom of Asp98 (2.40 A) and the third with Watl098 (B-factor of 13.5 A2, 3.25 A) that bridges Asp98 with His255 (Figure 5.1). The a-carbon atom of Ala257 is displaced nearly 0.5 A toward the type II copper. The /3 strand incorporating residues Tyr301, Ala302 and Tyr303 that packs against residue 257 is also displaced 0.5 - 0.6 A toward the copper co-factor relative to the native structure. I l l Chapter 5 -1257 AfNiR variants The repositioned ce-carbon atom of Ala257 approaches to within 3.7 A of the side-chain of His255, which shows a 30° rotation about the Xz angle and a shift of the Ne2 atom of 0.35 A. The side-chain of Asp98 also shows a rotation about the X2 angle of approximately 15° such that the 05 atom is also displaced 0.5 A from the analogous position in the native structure. Despite the reorientation of the side-chains of His255 and Asp98 the active site hydrogen bond network including the copper ligand (Wat503) and bridging (Watl098) waters is maintained. The monodentate binding mode of nitrite in the I257T[N02~] structure is surprisingly similar to that of the I257A[N02"] rather than the bidentate mode of binding observed in the crystal structure of the more structurally similar I257V variant (Figure 5.1). The 01 atom of nitrite is directed towards Thr257 and is oriented to form a hydrogen bond (2.45 A) with the O7I atom of the side-chain. The conformation of the threonine side-chain in the I257T[N02" ] crystal structure was assigned based on omit electron density maps. The bound nitrite is well defined with clear omit electron density maps and an average B-factor of 28 A2. The solvent ligand (Wat503 B-factor 17 A2) maintains the tri-coordinate hydrogen bond network with the 01 atom of nitrite (2.79 A), the 052 atom of Asp98 (2.49 A) and Watl098 (3.04 A) observed in the I257A[N02"] structure. The side-chain of Asp98 is displaced approximately 0.5 A away from Thr257 relative to the native structure; however, no significant reorientation of His255 is observed. Despite the introduction of the extended methionine side-chain at position 257 in the I257M variant, omit electron density maps indicate clearly that nitrite is capable of binding to the type II copper (Figure 5.1). High B-factors averaging 40 A2, however, suggests that nitrite is disordered and likely binds at a reduced occupancy. The side-chain of Met257 also 112 Chapter 5 -1257 AfNiR variants shows disorder with B-factors averaging nearly 50 A2 between the three non-crystallographically related active sites in the unit cell. In the I257M[N02_] structure, a solvent molecule serves as the fifth ligand to the type II copper (2.26 A) and forms a hydrogen bond with the 01 atom of nitrite (2.72 A), the 052 atom of Asp98 (2.45 A) and Watl098 (3.11 A). The nitrite is oriented such that the N-01 bond is roughly parallel with the S5-Ce bond of Met257. The 02 atom of nitrite is positioned approximately 3.3 A from the S5 atom of Met257 but poor geometry limits the formation of a hydrogen bond. Well defined omit electron density maps and moderate B-factors (avg. 25 A2) indicate that nitrite binding in the I257G[N02~] structure adopts both a monodentate and bidentate coordination (Figure 5.1). Refinement of the structure indicates that the occupancy of the two different conformations varies between the three non-crystallographically related active sites; although the monodentate form is always predominate. A solvent molecule was refined as the fifth ligand to the type II copper analogous to that of the I257A[N02-] structure when nitrite adopts the monodentate coordination. In this conformer, the 02 atom of nitrite is positioned 3.8 A from the ocarbon atom of Gly257. In the bidentate binding mode, the 01 and 02 atoms of nitrite are positioned 2.29 A and 2.05 A from the type II copper with the 02 atom of nitrite also forming a 2.55 A hydrogen bond with the 052 atom of Asp98. In this mode of binding, nitrite has shifted away from Gly257 relative to the native structure such that the 01 and N atoms of nitrite are positioned 4.09 A and 4.25 A from the a-carbon atom of Gly257. Based on the topology of the electron density maps, acetate, which is present in the mother liquor, was also modeled as a potential exogenous ligand. Steric clashes arising from inappropriate ligand-metal interactions between the carbon atoms of acetate and the type II copper eliminated acetate as a possible ligand. 113 Chapter 5 -1257 AfNiR variants 5.2.5 Effects of sulfite on the removal of nitrite The activity of the I257A variant, with the enlargened active site pocket was studied in the presence of several potential substrates and inhibitors. The specific activity of the I257A variant in the presence of low mM (1-10) concentrations of chloride or azide was not changed appreciably similar to previous studies with native AfNiR (Kakutani et al. 1981b). The addition of sulfite (SO32") to native AfNiR resulted in little change in the removal of nitrite from solution as monitored with the Griess reagents at 535 nm (Figure 5.2 A). However, repeating the same assay with the I257A variant provided an unexpected result. Using concentrations of sulfite ranging from 1 to 10 mM, the removal of nitrite from solution in the I257A mutant was increased significantly, with over 90 % removal of nitrite at a sulfite concentration of 3.5 mM (Figure 5.2 A), which amounts to nearly a ten-fold increase in the specific activity. Extending these studies to the remaining five variants produced a wide range of altered activities (Figure 5.2 B). The addition of sulfite to a final concentration of 3.5 mM resulted in a significant increase in the removal of nitrite from the I257A, I25G and I257T variants (Figure 5.2 B). A small increase was observed with the I257V variants and no significant change was measured for the 1257L variant or the native enzyme. Several control experiments were carried out to determine accurately the effect of sulfite. A l l controls with sulfite in the presence of enzyme were also carried out with a mixture of sulfite and nitrite showing essentially the same results. The addition of sulfite to samples of the native or variant AfNiRs did not result in a change in the visible spectrum of the type I copper site indicating that sulfite alone is not capable of reducing the type I copper center. 114 Chapter 5 -1257 AfNiR variants Figure 5.2 The effect of sulfite on the removal of nitrite from solution as measured by the nitrite reductase assay supplemented with (A) different concentrations of sulfite with native AfNiR and the 1257A variant and (B) 3.5 mM sulfite with all six variants. (A) • Represents the control assay using native AfNiR and no sulfite. * Represents the assay performed with native AfNiR in the presence of sulfite. A Represents the assay performed with I257A and varying concentrations of sulfite. B) In each case, measurements are normalized to the reaction performed with nitrite alone by choosing concentrations of enzyme such that 80 - 90 % of the nitrite remained in solution in the absence of sulfite. Control #1 contained no enzyme and no sulfite. For control #2, sulfite was added to a final concentration of 3.5 mM. Error bars represent a range over three trials. 115 Chapter 5 -1257 AfNiR variants Chapter 5 -1257 AfNiR variants Furthermore, in the absence of reducing agents dithionite and methyl viologen, a mixture of sulfite, nitrite and either the I257A variant or native AfNiR, did not result in the measurable removal of nitrite from solution indicating that sulfite did not reduced directly the type II copper site. In the absence of NiR, no significant removal of nitrite from solution was detected, regardless of the presence of sulfite (Figure 5.2B - controls #1 and 2). To determine whether sulfite could act as a substrate to I257A in the absence of chemical reductants, a modified assay using the in vivo electron donor pseudoazurin was carried out. Initially, the gene encoding pseudoazurin was subcloned, expressed and purified as described in Chapter 2. A l l assays using reduced pseudoazurin were performed in anaerobic cuvettes sealed with rubber septa under a positive argon atmosphere. Transfer of solutions was carried out with airtight Hamilton syringes. This assay was monitored by two different methods; electron transfer reactions were monitored through the reoxidation of reduced pseudoazurin measured at 593 nm, and a fuschin based sulfite assay monitored at 580 nm was used to detect removal of sulfite from solution. As expected, in the control reaction consisting of nitrite and native AfNiR, a clear reoxidation of pseudoazurin is observed, consistent with productive electron transfer and turnover of the enzyme (Figure 5.3). No significant re-oxidation of pseudoazurin was observed when incubated solely in the presence of sulfite in the absence of AfNiR. In the presence of sulfite, minimal reoxidation of pseudoazurin is observed when incubated with either the I257A variant or native AfNiR. More importantly, there is little difference in the overall reoxidation of pseudoazurin between the experiments with I257A and native AfNiR (Figure 5.3). 117 Chapter 5 —1257 AfNiR variants 0.11 6 Time (min) Figure 5.3 Monitoring the reoxidation of pseudoazurin at 593 nm. Each of the curves represents experiments containing reduced pseudoazurin (50 uM), nitrite reductase (20 nM -native or I257A AfNiR) and sulfite to a final concentration of 10 mM. The red curve (top) is the control with native AfNiR (20 nM) and nitrite (2 mM). The burgundy curve (bottom) is the second control of reduced pseudoazurin and sulfite. The blue (2 n d from top) and green (2 n d from bottom) curves represent the reaction with I257A and native AfNiR, respectively. To monitor the removal of sulfite from solution, the anaerobic pseudoazurin based assay described above was coupled to a second step that incorporated a basic fuschin assay to detect sulfite. In this modified assay, a sample of reaction mixture from the first step was removed at different time points with an airtight syringe and injected into a test tube containing the fuschin reagent (Chapter 2). Despite a thirty-minute incubation including 118 Chapter 5 -1257 AfNiR variants reduced pseudoazurin, sulfite and either I257A or native AfNiR, no significant removal of sulfite from solution was measured (Figure 5.4). In an attempt to visualize directly the potential interaction of sulfite with I257A, a sulfite-soaked crystal structure was solved. During refinement reasonable B-factors (< 40 A2) for the sulfite ligand were obtained when modeled at 60 % occupancy. The structure of acetate, which is present in the mother liquor, is similar to sulfite. Acetate was therefore modeled in at full occupancy into the electron density proximal to the type II copper and refined B-factors of 30 A2 are similar to the surrounding atoms. As a control, the crystal structure of the I257A variant was solved in the absence of sulfite or nitrite. In this structure, clear omit electron density for acetate is observed within ligand distance to the type II copper. This observation suggests that the electron density observed in the sulfite-soaked structure likely also represents acetate. 03 IT) < 2.5 T 2 1.5 1 0.5 Control I257A - 0 I257A -10 I257A - 30 Native - 0 Native - Native -min min min min 10 min 30 min Figure 5.4 Basic fuschin assay to detect the removal of sulfite from solution with the I257A variant and native AfNiR. The control reaction contained reduced pseudoazurin and sulfite but no NiR. Error bars represent the average of three trials. 119 Chapter 5 -125 7 AfNiR variants 5.3 Discussion 5.3.1 Native active site and hydrophobic blanket The active site pocket in native AfNiR lies at the bottom of a 16.5 A cavity formed at the interface between two adjacent monomers (Figure 5.5 A). Several bulky residues form a hydrophobic blanket that surrounds the type II copper and restrict access to the active site. Of these residues, Ue257 is positioned within 5 A of the type II copper and directly occludes the nitrite-binding site. In the native AfNiR nitrite-soaked structure, nitrite is bound in a bent conformation that is likely due to the close packing of the side-chain of Ile257 (Figure 5.5 B). The isoleucine at position 257 (numbering is that of AfNiR) is highly conserved amongst almost all copper-containing nitrite reductases suggesting an important role in defining the active site pocket. The two exceptions from the class II family of CuNiRs are observed from Bacillus strearothermophilus and Corynebacterium diptheriae, which both encode a valine (Figure 5.5 C). 5.3.2 RoleofIle257 The altered activities of the 1257 AfNiR variants indicate clearly an intimate catalytic role for Ile257 consistent with the high degree of sequence conservation (Figure 5.5 C). Furthermore, the high-resolution nitrite-soaked variant crystal structures show that the function of residue 257 is related largely to directing the binding mode of nitrite in the active site. Overall, a strong correlation exists between the orientation of the bound nitrite and the accompanying specific activity. 120 Chapter 5 -1257 AfNiR variants I II 'AcNiR 272 AfNiR 270 RsNiR 271 { PsNiR 273 AxNiR 252 RsNiR 262 -AniA 249 HmNiR 258 * BsNiR 185 .CdNiR 220 TAAVGERVLVVHSQ—ANRDTRPHIJI TAAVGEKVLIVHSQ--ANRDTRPHLI TAAVGERVLIVHSQ—ANRDTRPHLI QAKVGDRVLILHSQ--ANRDTRPHLI TAKVGETVLLIHSQ--ANRDTRPHLI KAKVGDNVLFVHSQ—PKRDSRPHLI KAKAGETVRMYVGNGGPNLVS S FHVI SMQVGETARVYFVTGGPNLDSSFHPI LAKVGEKIRLYVNNVGPNEVS S FHV V DVKVGERVRFWVLDAGPNVPLSFHlM GHGDYVWATGKFRN GHGDYVWATGKFNT pGHGEYVWRTGKFVN pGHGDYVWATGKFAN GHGDWVWETGKFAN pGHGDLVWETGKFHN EIFDKVYVEGGK— pSVWDEVWQQGSIAG 3TVFDDVYLDGNP-S GQFDTTWTEGAYTL Figure 5.5 Space-filled models depicting A) the monomer interface showing the occlusion of the nitrite (red) molecule bound to the type II copper (yellow) by the side-chain of Ile257 (green) and B) the active site cleft of AfNiR showing interaction between the side-chain of Ile257 (green) and the bound nitrite (purple). (C) Partial amino acid sequence alignment of copper-containing nitrite reductases generated with C L U S T A L W (Higgins et al. 1996). The outlined box highlights the analogous residues to Ile257 in AfNiR. See figure 1.3 for complete alignment. The bidentate mode of nitrite binding observed in the I257V and I257L variants is similar to that of the native enzyme and accordingly, these variants display the smallest 121 Chapter 5 -1257 AfNiR variants reductions in specific activity. Interestingly, the substitution of Ile257 with valine, which is the only identified natural substitution (Figure 5.5), produces a more catalytically active enzyme than native AfNiR (Table 5.1). From the I257V[N02_] and native structures, the mode of nitrite binding differs primarily in the degree of bend (Figure 5.1), which may be responsible for the increased activity. Alternatively, the reduction in steric hindrance observed with the substitution of an isoleucine with the smaller valine may facilitate access to the copper site while maintaining a sufficient presence to direct the catalytically productive bidentate binding mode for nitrite. Although substitution of Ile257 with a leucine is conservative like the valine mutation, the change in the rotameric conformation of Leu257 directed by the side-chain of His306 likely disrupts nitrite binding as shown by the somewhat diffuse electron density (Figure 5.1). Additionally, this new conformation appears to induce a steric effect on the side-chain of the catalytically essential Asp98, which may also contribute to the reduced activity. Substituting Ile257 with the alanine, glycine, threonine or methionine results in an unusual monodentate mode of nitrite binding. In this form, the nitrite oxygen atom that participates in a hydrogen bond with the side-chain of Asp98 in native AfNiR is repositioned such that it is directed toward residue 257 (Figure 5.1). This unusual coordination is likely catalytically unproductive consistent with a reduced specific activity of 25 to 30 fold for these variant AfNiRs. Characterization of the I257T variant provides an interesting perspective of the stability on the monocoordinate nitrite-binding mode. The side-chains of threonine and valine differ only in the nature of the 7 I atom, which is a carbon for valine and an oxygen for threonine. Despite the structural similarity, the specific activity for I257T is the lowest 122 Chapter 5 -1257 AfNiR variants of all the six variants (Table 5.1), whereas I257V variant is more active than the native enzyme. Analysis of the nitrite-soaked crystal structure revealed the presence of a hydrogen bond between the O7I atom of Thr257 and the 01 atom of the bound nitrite. This interaction appears to stabilize the monodentate coordination of nitrite that impairs catalytic activity. The structural data suggests that in the absence of an appropriate residue at position 257, nitrite prefers to bind in a monodentate fashion via a single oxygen atom. Importantly, the hydrogen bond between nitrite and the side-chain of Asp98 known to be essential for catalytic activity (Chapters 3 and 4) (Zhang et al. 2000) is not conserved in this unusual binding mode. Instead, a solvent molecule ligand (Wat503) completes the hydrogen bond with Asp98. Overall, the role of Ile257 appears to direct the catalytically productive bidentate mode of nitrite binding to the type II copper such that the hydrogen bond with Asp98 is maintained. 5.3.3 Mechanist ic Implications Two distinct catalytic mechanisms have been reported for copper-containing nitrite reductases where catalysis proceeds via either an N-coordinate (Hulse et al. 1989), or O-coordinate (Chapters 3 and 4) (Adman et al. 1995, Murphy et al. 1997b) bound nitrite. Crystallographic and spectroscopic studies show a clear preference for nitrite to bind in a bidentate O-coordinate fashion to an oxidized copper of CuNiRs. Functional biomimetic models however show that nitrite binds preferentially in an N-coordinate fashion to a reduced copper (Halfen et al. 1996, Halfen et al. 1994). Although O-coordinate binding of nitrite has also been observed in these models, only N-coordinate binding is accompanied by 123 Chapter 5 -1257 AfNiR variants stoichiometric production of NO (Halfen et al. 1996, Monzani 2000). Modeling studies with the native nitrite-soaked AfNiR structure suggests that the topology of the active site, in particular the presence of the side-chain of Ile257, precludes nitrite binding in an N-coordinate fashion (Murphy et al. 1997b). Despite enlargement of the active site with mutations at position 257, no N-coordinate binding of nitrite is observed in the high-resolution crystal structures presented here. At most, a monodentate O-coordination is observed that correlates with a reduced specific activity. The monodentate O-coordination may provide insight into a catalytic intermediate as suggested previously from the nitrite-soaked H255N crystal structure (Chapter 4). Collectively, the structural and functional studies presented here are inconsistent with the proposed requirement for an N -coordinate nitrite as suggested by the biomimetic copper complexes. Furthermore, a bidentate, O-coordinate binding mode of nitrite is required for full catalytic activity. 5.3.4 Sulfite directed removal on NO Clearly, the ability of different amino acids at position 257 to alter significantly the activity of AfNiR and the binding mode of nitrite suggests that isoleucine 257 plays an intimate role in determining substrate specificity. Surprisingly, addition of sulfite to the nitrite reductase activity assay appears to rescue the impaired activity of the I257A, I257G, I257M and I257T AfNiR, while showing little or no effect with the native enzyme, or the I257L and I257V variants. An initial hypothesis was that sulfite was being reduced in the enlargened active sites of the variant enzyme to a form capable of chemically removing nitrite from solution. Several studies were carried out to test this hypothesis. No significant transfer of electrons, as monitored by the reoxidation of reduced pseudozurin, was measured 124 Chapter 5 -1257 AfNiR variants with I257A when incubated with sulfite as the sole substrate (Figure 5.3). Furthermore, no detectable removal of sulfite from solution was observed with the basic fuschin assay (Figure 5.4). Finally, no clear evidence of sulfite binding to the type II copper was observed in the sulfite-soaked I257A crystal structure. Collectively, these data suggest that sulfite is unlikely to be a direct substrate. The chemical reactivity between nitric oxide and sulfite has been shown in several studies to be rapid and lead to the formation of a variety of products (Harvey 1995, Zang 1993). A reaction stoichiometry of approximately 1.5 - 2.0 to 1 of nitric oxide to sulfite (Harvey 1995) has been measured from electrochemical studies consistent with the formation of the dinitrososulfite anion (Schroeter 1966). A more extensive study incorporating synthetic iron complexes to nucleate the reaction between nitric oxide and sulfite in solution also identified formation of the dinitrosulfite anion (Zang 1993). The reactivity of NO and sulfite has also shown to be biologically relevant, where the addition of sulfite is known to inhibit NO induced platelet aggregation. These observations led to the hypothesis that the physiological toxicity associated with sulfite is due to the rapid sequestration of NO thereby limiting the essential role as a neurotransmitter (Harvey 1995). Kinetic studies have shown that NO is a potent, micromolar inhibitor of AcNiR (Jackson et al. 1991). Following an initial burst of NO production as monitored with a gas chromatograph, the amount of NO produced by the enzymes leveled off rapidly and N 2 0 production was detected. Rapid removal of NO by either sparging or trapping with deoxyhemoglobin resulted in a significant increase in the amount of NO produced with no detectable N 2 0 production. Furthermore, nitrosylation of hydroxylamine by AcNiR has also been shown, which led to a proposed catalytic mechanism for CuNiRs involving a copper 125 Chapter 5 -1257 AfNiR variants nitrosyl intermediate (Hulse et al. 1989). Taken together, these observations and the data presented in this Chapter suggest that sulfite serves to trap NO minimizing rebinding of NO and accompanying inhibition of the enzyme. The increase in specific activity is confined to select AfNiR variants and may represent a difference in the NO release processes between the variants and the native enzyme. An alternate possibility is that these variants allow a closer approach of sulfite to the active site with nitric oxide bound. To pursue effectively the hypothesis describing the role of sulfite in the specific activity the AfNiR variants methods employing gas chromatography interfaced with a mass spectrometer to analyze gas production, especially NO, are currently being developed. 126 Chapter 6 - General discussion and outlook Chapter 6 - General discussion and outlook The experimental approach of combining mutagenesis, functional studies and high-resolution crystallography as presented here is unique in the field of CuNiR research. As such, this thesis provides a unique perspective that advances significantly the study of these denitrifying enzymes. In this Chapter, I discuss the data and conclusions presented in this thesis in the context of the current literature. 6.1 Summarizing the proposed catalytic mechanisms for CuNiRs The molecular mechanism of CuNiRs remains controversial. Figure 6.1 represents a detailed version of the O-coordinate nitrite binding mechanism as it appears in Chapter 4 with accompanying text boxes highlighting the major areas of controversy. In this mechanism, a protonated nitrite molecule displaces the ligand water and binds in a bidentate O-coordinate fashion to the oxidized type II copper center. Electron transfer from the type I copper is followed by the formation of a proposed transient complex in which a hydroxyl group and nitric oxide (NO) are bound simultaneously to a pentacoordinated copper. A final protonation event, possibly initiated indirectly through His255, results in the release of NO and regeneration of the water ligand to the type II copper. The catalytic mechanism for CuNiRs proposed by Suzuki and co-workers is largely similar to the O-coordinate mechanism presented here. Recent site-directed mutagenesis studies and steady state kinetics however, suggest two variations (Kataoka et al. 2000). One of these variations is that nitrite binds in the deprotonated form to an oxidized type II copper and the second is that His255 is responsible for direct donation of a proton to the reaction intermediates. 127 Chapter 6 - General discussion and outlook Suzuki and co-workers propose that a deprotonated nitrite molecule serves as an acceptor in forming a hydrogen bond with the protonated side-chain of Asp98 (Kataoka et al 2000). His255 Averill and co-workers propose that nitrite binds initially in an In-coordinate fashion to a reduced type II copper (Hulse et al 1989). Wat H - d 7 1098 / / H H-— 9 % I J 2+ % - s Cu V Asp98 V H—6 H ' V H + Suzuki and co-workers suggest that during the reaction, the side-chain of His255 is reoriented such that it can donate directly the second proton required to release NO (Kataoka et al, 2000). Eady and co-workers propose that nitric oxide undergoes linkage isomerism from O to N-coordinate to allow the formation of a copper-nitrosyl intermediate (Dodd et al 1997). Figure 6.1 Summary catalytic mechanism for CuNiRs 128 Chapter 6 - General discussion and outlook From the nitrite-soaked D98N crystals structure presented in Chapter 4 and collaborative FT-IR studies of the D98N AfNiR variant (Zhang et al. 2000), significant data exists to indicate that nitrite binds in a protonated form and donates a proton in forming the hydrogen bond with the deprotonated side-chain of Asp98. Addressing the second point of controversy, attempts to model His255 in the native nitrite-soaked structure in an orientation such that a geometrically favorable hydrogen bond could be formed with the nitrite substrate was unsuccessful (Chapter 3) suggesting an indirect proton donation role for His255. The N-coordinate nitrite binding mechanism proposed by Averill and co-workers (Hulse et al. 1989) differs fundamentally from the mechanism presented here and is similar to that of the heme cd\ NiRs. In this CuNiR mechanism nitrite binds in an N-coordinate fashion to a reduced copper and catalysis proceeds via a copper-nitrosyl intermediate. The main controversial point lies in the coordination of nitrite and hence the nature of subsequently formed chemical intermediates. Although N-coordinate nitrite has been observed in biomimetic complexes (Halfen et al. 1996, Halfen et al. 1994, Monzani 2000), this binding mode has yet to be observed in the enzyme including the several nitrite-soaked structures presented in this thesis. Collectively, the data presented here, along with the current literature, suggest a significant role for the enzyme in directing a catalytically productive O-coordinate binding of nitrite in the active site. A recent variation on the N-coordinate mechanism proposed by Eady and co-workers (Dodd et al. 1997) suggests that, similar to the mechanism proposed here, nitrite binds initially in an O-coordinate fashion to the active site copper. During catalysis, however, nitric oxide undergoes linkage isomerization from O to N-coordinate to allow for the formation of a copper-nitrosyl complex as suggested by the Averill mechanism (Averill 129 Chapter 6 - General discussion and outlook 1994, Hulse et al. 1989) and observed with the cd\ NiRs. The studies described in this thesis do not contradict directly this variation of the mechanism, but several observations suggest a mechanism for CuNiRs that is distinct from that of the cd\ NiRs. 6.2 Distinct mechanisms for heme cd\ and CuNiRs: supportive evidence A variety of techniques have been used successfully to probe the catalytic mechanism of heme cd\ NiRs where the reduction of nitrite proceeds via the formation of heme nitrosyl intermediate. Despite the similarities suggested by the biomimetic models, there are many differences in the reaction chemistry between the heme cd\ and CuNiRs suggesting that reduction of nitrite by these two enzymes proceeds via distinct mechanisms. One of the major differences involves the interaction between the enzyme and the reaction product, nitric oxide. In both cases NO is a potent inhibitor, but only in CuNiRs does an accumulation of NO lead to the formation of N 2 0 (Jackson et al. 1991). A second major difference between the heme cd\ NiRs and CuNiRs involves the release mechanism for NO. The cd] NiRs enzymes rely on an active site Tyr residue to displace the bound NO whereas release of NO in CuNiRs appears to be largely governed by diffusion. A third noteworthy difference involves the correlation of the reduction potential between the metal co-factors within the cd\ and CuNiRs. Both enzymes coordinate two different heme or copper co-factors that are involved in either electron transfer (heme c and type I copper) or directly in catalysis (heme d\ and the type II copper). Similar studies on heme cd\ NiRs show that in the resting state of the enzyme, the d\ heme in the active site has a reduction potential nearly 100 mV greater than the c heme center and is therefore preferentially reduced. Pulse radiolysis studies show that in the absence of substrate, the 130 Chapter 6 - General discussion and outlook reduction potentials for the copper atoms in CuNiRs are closely matched (Suzuki et al. 1997) suggesting a requirement for nitrite to bind prior to electron transfer from the type I site. The preference for the active site copper to be in the oxidized state in the resting enzyme provides evidence that even the first catalytic step, the binding of nitrite differs from that of the heme cd\ NiRs. The preference for the type II copper to be reduced in the resting state as required by the mechanism of Hulse and Averill (Hulse et al. 1989) prior to nitrite binding also presents an interesting biological dilemma. Reduced copper-nitric oxide complexes are chemically more stable than the oxidized complex. As such, when the type II copper is in the oxidized state, NO release is facilitated. These observations are consistent with the cd\ NiRs where the reduction potentials of the heme groups are such that the active site heme d\ can be reduced by the heme c following release of NO. However, this is unlikely to occur in the CuNiRs where multiple studies have shown that in the resting state of the enzyme the type II copper is in the oxidized state (Howes et al. 1994, Murphy et al. 1997b, Strange et al. 1995, Strange et al. 1999). From a biological perspective, it would be advantageous for the enzyme if the type II copper was oxidized in the resting state, otherwise the product of the reaction, NO, would be a more potent inhibitor of the enzyme. The more effective inhibition of CuNiRs by NO, which is indirectly suggested by the Hulse et al (Hulse et al. 1989) and Dodd et al (Dodd et al. 1997) is biologically unproductive and could lead to a build-up of NO that would likely be lethal to the organism. 131 Chapter 6 - General discussion and outlook 6.3 Binding modes of nitrite: a mechanistic dilemma A chemical requirement for the formation of a copper-nitrosyl intermediate, as proposed by Averill and co-workers (Hulse et al. 1989), is that nitrite or nitric oxide must bind in an N-coordinate fashion to the type II copper. Several functional studies with biomimetic synthetic copper complexes support this mode of nitrite binding where stoichiometric production of NO has been monitored following the addition of acid (Beretta et al. 2000, Halfen et al. 1996, Halfen et al. 1994, Monzani et al. 2000). Crystallographic, E X A F S and ENDOR studies of several different biologically relevant CuNiRs show that nitrite binds preferentially via a bidentate O-coordination to an oxidized Cu(II) center (Howes et al. 1994, Murphy et al. 1997b, Strange et al. 1995, Strange et al. 1999). Additionally, the formation of a brown colour in the native AfNiR nitrite-soaked crystals suggests that the O-coordination of nitrite is catalytically competent (Murphy et al. 1997b). Biomimetic models have been observed with this latter type of coordination but no accompanying reduction of nitrite has been measured (Beretta et al. 2000, Casella et al. 1996, Monzani et al. 2000). Overall, ten different nitrite-soaked crystal structures of eight AfNiR variants, with D98N and H255N in both the reduced and oxidized states, are presented in this thesis. The different modes of bound nitrite are summarized in Figure 6.2. Interestingly, despite the range of binding modes for nitrite, no N-coordinate nitrite binding was observed. Substitution of Ile257 with six different residues results in both monodentate and bidentate coordination of nitrite to the type II copper. Only the variants that coordinated a bidentate form of nitrite displayed high activity. Substitutions of Asp98 and His255 also resulted in different modes of nitrite binding, but drawing conclusions from these variant structures was 132 Chapter 6 — General discussion and outlook more difficult due to the added variable of the ionizable nature of the native side-chains. A monodentate coordination of nitrite is observed in the H255N nitrite-soaked structure but interestingly, the free oxygen of the bound nitrite is directed nearly 120 ° away from the analogous oxygen in the 1257 variant structures (Figure 6.2 B). Figure 6.2 The multiple conformations of the nitrite in the AfNiR variants presented in this thesis. Panel (A) shows the different binding modes of nitrite in the nitrite-soaked 1257 variant crystal structures. Panel (B) includes nitrite from the oxidized D98N (black atoms) and H255N (grey atoms) structures drawn in purple. With the exception of these purple nitrite molecules, nitrite molecules are coloured blue to red with increasing specific activity: Red, I257V[NC»2"]; orange, native AfNiR; light-orange, I257L[N02"]; yellow, I257M[N02"]; green, I257A[N0 2"]; cyan, I257G[N0 2 ']; blue, I257T[N02"]. A B Asp98 133 Chapter 6 - General discussion and outlook Despite a bidentate mode of nitrite binding in the D98N nitrite-soaked structure, the inability of nitrite to form a hydrogen bond with the non-ionizable side-chain of Asn98 resulted in a poorly active enzyme. Furthermore, in the reduced nitrite-soaked D98N and H255N crystal structures, the only significant structural observation in the active site is that nitrite appeared to bind at a reduced occupancy. The preference for nitrite to bind O-coordinate is also maintained in the nitrite-soaked crystal structure of the distantly related CuNiR (AniA) from Neisseria gonorrhoeae despite the crystals being grown at pH 10.5 (Boulanger 2001c). Taken together, these data suggest strongly that a bidentate mode of nitrite binding accompanied by a hydrogen bond to the residue at position 98 is required for productive catalysis. These data are inconsistent with the catalytic requirement for an N-coordinate nitrite, but do not provide further information on the proposed isomerization reaction (Dodd et al. 1997). 6.4 The essential role for Asp98 Several structural similarities exist between the coordination of the active site copper in CuNiRs and the zinc sites in superoxide dismutase (SOD), thermolysin, astacin and carbonic anhydrase (Murphy et al. 1997b, Murphy et al. 1995, Strange et al. 1995, Strange et al. 1999). The common structural motif of carbonic anhydrase, astacin and CuNiR is a metal site with three protein ligands, a fourth distal ligand water and a proton abstracting group (a carboxyl or hydroxyl group) forming a hydrogen bond to the ligand water (Strange et al. 1995). Asp98 in AfNiR is structurally analogous to the proton abstracting residues found in the zinc containing enzymes, suggesting a related role for this residue. The data presented in this thesis establishes a critical role for Asp98 in catalysis 134 Chapter 6 - General discussion and outlook (Chapter 3). Structural characterization of D98N AfNiR in the oxidized resting state indicates that Asn98 is poorly ordered (Chapter 3), with the binding of nitrite showing little effect in stabilizing the conformation of this residue (Chapter 4). By comparison, Asp98 is stabilized as shown by lower B-factors following nitrite binding in native AfNiR. FT-IR CO experiments of native AfNiR recorded over a pH range of 6.0 to 8.0 suggest that Asp98 is deprotonated thereby requiring nitrite to bind in the protonated form to the native enzyme (Zhang et al. 2000). These observations are consistent with five crystal structures of AcNiR solved at pH values between 5 and 6.8 that show minimal structural change in the active site (Adman et al. 1995). Recent steady state kinetics show an increase in K m for nitrite to the D98N and D98E AcNiR mutants of 200 fold and 15 fold, respectively (Kataoka et al. 2000), indicating that a negatively charged Asp98 may be required for high affinity binding of the substrate. Collectively, the available data indicate that the disorder observed for nitrite and Asn98 in the D98N[N0 2"] crystal structures (Chapter 4) is a result of the inability of the N52 atom of Asn98 to form a hydrogen bond with nitrite bound in the protonated form. Furthermore, the data presented here identifies the hydrogen bond between Asp98 and nitrite in the native structure as essential in anchoring nitrite in the active site for productive catalysis. This hydrogen bond is also likely to serve as a direct link through which protons are donated during catalysis. In spite of the crystallographic, spectroscopic and functional studies indicating an essential role for Asp98 (AfNiR) during catalysis, a recent study has led to the reevaluation of the proposed function of Asp98 (Prudencio et al. 2001). From this study, the functional impairment of the D92N variant of AxNiR as measured with chemical reductants is largely 135 Chapter 6 - General discussion and outlook reversed (~ 60%) when the proteaceous electron donor is used. The binding of azurin is proposed to result in a conformational change in AxNiR accompanied by a reorientation in the side-chain of Asn92 restoring the catalytic productivity. A major source of controversy regarding these studies is that, in the activity assays using reduced pseudoazurin as the source of electrons, a 100 fold molar excess of nitrite was used relative to the assay performed with the chemical reductants. The increased concentration of substrate present undoubtedly changes the dynamics of the reaction and could result in the increased activity. Despite the criticism, this observation deserves a more thorough characterization 6.4 Conclusions • Asp98 and His255 are essential for full catalytic activity of AfNiR (Chapter 3). o The hydrogen bond between Asp98 and nitrite in the native structure is essential in anchoring nitrite in the active site and for productive catalysis (Chapters 3 and 4 and Zhang et al (2000). o His255 likely directs the productive mode of nitrite binding through conservation of an active site hydrogen bond network including solvent molecules (Chapters 3, 4). • Non-engineered dinuclear type I copper sites are observed in nitrite-soaked reduced D98N and H255N structures (Chapter 4). o Presents a general model for evolution of dinuclear C U A sites. o May also represent a physiologically relevant change during catalysis potentially used in regulating the redox chemistry and overall activity of CuNiRs. • Ile257 directs the catalytically essential bidentate mode of nitrite binding (Chapter 5). o I257V variant coordinates a bidentate mode of nitrite binding and is more active than native enzyme, o I257T variant is the least active of the 1257 variants, nearly 100 fold less than I257V, and coordinates a monodentate form of nitrite. 136 Chapter 6 - General discussion and outlook • Sulfite likely traps NO produced from I257A, I257G, I257T and I267V variants preventing rebinding of NO and enzyme inhibition. o Perhaps the less occluded active site of these variants relative to the native enzyme facilitates a direct interaction with the copper-nitrosyl inhibition product. • The enzyme directs strongly the bidentate mode of nitrite binding via the oxygen atoms to an oxidized type II copper (Chapters 3,4 and 5). 6.5 Outstanding questions and future directions 1) Do defined catalytic intermediates exist for CuNiR during the reduction of nitrite to nitric oxide? Typically, enzyme mechanisms proceed through a multi-step pathway involving sometimes several chemically defined intermediates. Traditionally, crystallography has provided a snapshot of an enzyme in a chemically stable form. The information obtained from these experiments, such as determining the mode of substrate binding and identifying potential structural and catalytic residues, can be invaluable in defining a molecular mechanism. However, the static nature of these experiments limits generally a thorough characterization of all but the most stable reaction states. Mutagenesis and transition state analogues have been used successfully to extend the application of crystallography but have the potential to bias the true biological systems. I suggest using time-resolved crystallography to characterize AfNiR in action. This technique has been used recently to characterize intriguing changes in conformation of the heme co-factor in a heme cd\ N iR (Nurizzo et al. 1999). Applying this type of approach is technically challenging and requires an ability to trap or stabilize chemical intermediates such that there is a homogeneous population throughout the molecules in the unit cell. One 137 Chapter 6 - General discussion and outlook method to observe catalytic processes on the required time scale for CuNiRs is to use "caged" reductants that are capable of releasing an electron upon photo-activation. If successful, these experiments could verify if the reaction processes such as the isomerization reaction suggested by Dodd et al (Dodd et al. 1997). 2) What is the binding mode of nitric oxide to the type II copper during catalysis? In the absence of the dynamic time-resolved crystallographic approach, NO bound structures may provide significant mechanistic information. To date, no nitric oxide copper complex has been observed structurally in CuNiR. The structural and functional data of the AfNiR variants presented in this thesis suggest strongly that the nitrite binds in an O-coordinate fashion to the type II copper. This observation has led to the revised isomerization mechanism, where during catalysis NO flips from O to N-coordinate to allow the formation of the proposed nitrosyl intermediate. I propose two different approaches to answer this question. First is to solve an NO bound crystal structure. Characterizing the mode of NO binding to the type II copper in CuNiRs would aid in understanding the formation and release of NO and the disproportionation reactions where NO rebinds to the enzyme and is converted to N2O. The initial suggestion that NO2" bound in an O-coordinate fashion was provided by ENDOR where no relevant coupling between the copper and nitrogen of 15N02~ was measured indicating that the nitrogen was too distal from the copper to be a ligand (Howes et al. 1994). In the second approach, I propose a similar experiment with 1 5 N O to determine the preference for O or N-coordinate for NO and hence the validity of the proposed isomerization reaction. 138 Chapter 6 - General discussion and outlook 3) Are the dinuclear sites in the reduced D98N and H255N[N02~] attainable in the native enzyme under physiological concentrations of copper? The dinuclear type I copper site observed in the D98N and H255N[NC»2~] structures provide a unique perspective regarding the evolution of traditional dinuclear C U A sites as these structures represent the first non-engineered expansion of a type I site to a dinuclear copper site similar to a C U A site. Efforts are currently being pursued to determine the biological relevance of this type I copper site expansion in the native AfNiR crystals under physiological concentrations of copper (<1 pM Q1Q2). The goal is to determine whether the altered copper coordination could potentially be used in regulating the redox chemistry and overall activity of copper-containing nitrite reductases. 4) What are the chemical products of sulfite and NO Several different activity assays have shown clearly that addition of sulfite rescues some of the catalytic activity of the I257A, I257G, I257T, and I257V AfNiR variants. The most likely explanation is that sulfite traps NO, which is a potent inhibitor of NiR, preventing it from rebinding to the enzyme. To characterize the products of the reaction between sulfite and NO an assay using a gas chromatograph interfaced with a mass spectrometer is being developed. In this assay, reduced pseudoazurin will be used as the electron donor to AfNiR variants in an anaerobic vial under an argon atmosphere. The gas phase will be injected into the gas chromatograph and the liquid phase directly into the mass spectrometer. 139 Bibliography Abraham, Z. H. , Lowe, D. J. & Smith, B. E. (1993). Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres. Biochem. J. 295, 587-593. Abraham, Z. FL, Smith, B. E., Howes, B. D., Lowe, D. J. & Eady, R. R. (1997). pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans. Biochemical Journal 324(Pt 2), 511-6. Adman, E. T., Godden, J. W. & Turley, S. (1995). The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with N02- bound and with Type II copper depleted. J. Biol. Chem. 270, 27458-27474. Adman, E. T. and Murphy, M.E.P. (2001). Copper-nitrite reductase. In Handbook of Metalloproteins (Messerschmidt, A. , Huber, R., Poulos, T. and Wieghardt, K. , ed.), Vol . 2, pp. 1381-1390. 2 vols. John Wiley & Sons Ltd., Sussex. Arciero, D. M . G., A. , Hendrich, M.P. and Hooper, A . B . (1998). Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 37, 523-529. Averill, B. A . (1996). Dissimilatory Nitrite and Nitric Oxide Reductases. Chem. Rev. 96, 2951-2964. Averill, B. A . (1994). Novel copper nitrosyl complexes: contribution to the understanding of dissimilatory, copper-containing nitrite reductases. Agnew. Chem. Int. Ed. Engl. 33, 2057-2058. Aylott, J. W., Richardson, D.J. and Russell, D.A. (1997). Optical biosensing of nitrate ions using a sol-gel immobilized nitrate reductase. Analyst. 122, 77-80. Beretta, M . , Bouwman, E., Casella, L. , Douziech, B., Driessen, W.L., Gutierrez-Soto, L. , Monzani, E. and Reedjik, J. (2000). Copper complexes of a new tridentate imidazole-containing ligand: spectroscopy, structures and nitrite reductase activity. The molecular structures of {Cu(biap)(N02)2} and {Cu(biap)Br2}. J. Inorg. Chim. Acta 310,41-50. 140 Berks, B. C , Ferguson, S.J., Moir, J.W.B. and Richardson, D.J. (1995). Enzymes and associated electron transport systems that catalyze the respiratory reduction of nitrogen oxides and oxyanions. Biochim. et Biophys. Acta 1231, 97-173. Blackburn, N . J., Barr, M . E., Woodruff, W. H. , van der Ooost, J. and de Vries, S. (1994). Metal-metal binding in biology: E X A F S evidence for a 2.5 A copper-copper bond in the CuA center of cytochrome C oxidase. Biochemistry 33, 10401-10407. Boulanger, M . J., Kukimoto, M . , Nishiyama, M . , Horinouchi, S. and Murphy, M . E. P. (2000). Catalytic roles for two water bridged residues (Asp98 and His255) in the active site of copper-containing nitrite reductase. J. Biol. Chem 275, 23957-23964. Boulanger, M.J. and Murphy, M.E.P. (2001a). Alternate Substrate Binding Modes to Two Mutant (D98N and H255N) Forms of Nitrite Reductase from Alcaligenes faecalis S-6: Structural Model of a Transient Catalytic Intermediate. Biochemistry. 40(31), 9132-41. Boulanger, M.J . and Murphy, E.P.M. (2001b). Characterization of substrate specificity in nitrite reductase from Alcaligenes faecalis S-6: Screening novel enzymatic activities. Prot. Sci.(To be Submitted). Boulanger, M . J. and Murphy, E.P.M. (2001c). Crystal Structure of the Soluble Domain of the Major Anaerobically Induced Outer membrane Protein (AniA) from Pathogenic Neisseria: A New Class of Copper-Containing Nitrite Reductases. J. Mol. Biol. In press. Bradford, M . M . (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Braker, G., Fesefeldt, A . & Witzel, K. P. (1998). Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Applied & Environmental Microbiology 64(10), 3769-75. Brittain, T., Blackmore, R., Greenwood, C. & Thompson, A. J. (1992). Bacterial nitrite -reducing enzymes. Eur. J. Biochem. 209, 793-802. Brown, K. , Dijnovic-Carugo, K. , Haltia, T., Cabrito, I., Saraste, M . , Moura, J.J.G., Tegoni, I and Cambillau, M . (2000). Revisiting the catalytic Cuz cluster of N20 reductase: evidence of a bridging inorganic sulphur. J. Biol. Chem. 275, 41133-41136. 141 Brown, K. , Tegoni, M . , Prudencio, M . , Pereira, A . S., Besson, S., Moura, J. J., Moura, I. and Cambillau, C. (2000). A novel type of catalytic copper cluster in nitrous oxide reductase. Nat. Struct. Biol. 7, 191-195. Brunger, A . T. (1997). Free R Value: Cross-Validation in Crystallography. Methods Enzymol. 277, 366-404. Brunger, A . T., Adams, P. D., Clore, G. M . , Delano, W. L. , Gros, P., R.W., G.-K., Jiang, J. S., Kuszewski, J., Nilges, N . , Pannu, N . S., Rice, L . M . , Simonson, T. and G.L., W. (1998). Crystallography and N M R system (CNS): A new software system for macromolecular structure determination. Acta Cryst. D 5 4 , 905 - 921. Cardinale, J. A . and Clark, V . L . (2000). Expression of AniA, the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, provides protection against killing by normal human sera. Infect. Immun. 68(7), 4368-4369. Carr, G. J., Page, M.D. and Ferguson, S.J. (1989). The energy-conserving nitric-oxide reductase system in Paracoccus dentrificans. Eur. J. Biochem. 179, 683-692. Carr, G. J. and Ferguson, S.J. (1990). The nitric oxide reductase of Paracoccus denitrificans. Biochem. J. 269, 423-430. Casella, L. , Carugo, O., Gullotti, M . , Doldi, S. and Frassoni, M . (1996). Synthesis, structure and reactivity of model complexes of copper nitrite reductase. Inorg. Chem. 35, 1101-1113. Chang, C. K. (1986). The porphyrindione structure of heme dl.J. Biol. Chem. 261, 8593-8596. Clark, V . L. , Knapp, J. S., Thompson, S. & Klimpel, K. W. (1988). Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb. Path. 5(5), 381-90. Collaborative Computational Project, N . (1994). The CCP4 suite, programs for protein crystallography. Acta Cryst. D 5 0 , 760-763. Costa, C , Moura, J. J., Moura, I., Wang, Y . & Huynh, B. H . (1996). Redox properties of cytochrome c nitrite reductase from Desulfovibrio desulfuricans A T C C 27774. J. Biol. Chem. 271(38), 23191-6. 142 Coyne, M . S., Arunakumari, A. , Averill, B .A. and Tiedje, J .M. (1989). Immunological identification and distribution of dissimilatory heme cdl and nonheme copper nitrite reductases in denitrifyng bacteria. Appl. Environ. Microbiol. 55, 2924-2931. Coyne, M . S., Arunakumari, A. , Pankratz, H.S., and Tiedje, J .M. (1990). Localization of the cytochrome cdl and copper nitrite reductase in denitrifiying bacteria. J. Bacteriol. 172(5), 2558-62. Cramm, R., Siddiqui, B. and Friedrich, J. (1997). J. Bacteriol. 179, 6769-6777. Cutruzzola, F. (1999). Bacterial nitric oxide synthesis. Biochim. et Biophys. Acta 1411(2-3), 231-49. Cutruzzola, F., Arese, M . , Grasso, S., Bellelli, A . & Brunori, M . (1997). Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis. FEBS Letters 412(2), 365-9. Cuypers, H. and Zumft, W.G. (1993). Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene. J. Bacteriol. 175, 7236-7246. DeBoer, A . P. N . , Reijnders, W.N.M. Kuene, J.G. Stouthmer, A . H . and van Spanning, R.J.M. (1994). Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans. Antonie van Leeuwenhoek 66, 111-127. DeBoer, A . P. N . , van der Oost, J., Westerhoff, H.V., Stouthmer, A . H . and van Spanning, R.J.M. (1996). Mutational analysis of the nor gene cluster which encodes nitric oxide reductase from Paracoccus denitrificans. Eur. J. Biochem. 242, 592-600. Denariaz, G., Payne, W.J. and LeGall J. (1991). The denitrifying nitrite reductase of Bacillus halodenitrificans. Biochim. Biophys. Acta 1056, 225-232. Dermastia, M . T., T. and Hollocher, T.C. (1991). Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characterisitcs. J. Biol. Chem. 266(17), 10899-905. Dias, J. M . , Tahn, M.E. , Humm, A. , Huber, R, Bourenkov, G.P. Bartunik, H.D. Bursakov, S., Calvete, J, Caldeira, J. and Carneiro, C. (1999). Crystal structure of a periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC27774 at 1.75 A resolution by M A D : a molybdopterin enzyme with a single Fe4S4 cluster. Structure 7, 65-79. 143 Dodd, F. E., Hasnain, S.S., Abraham, Z.H., Eady, R.R and Smith, B.E. (1997). Structures of a blue-copper nitrite reductase and its substrate bound complex. Acta Cryst. D53, 406-418. Dodd, F. E., Van Beeumen, J., Eady, R. R. & Hasnain, S. S. (1998). X-ray structure of a blue-copper nitrite reductase in two crystal forms. The nature of the copper sites, mode of substrate binding and recognition by redox partner. J. Mol. Biol. 282(2), 369-82. Einsle, O., Messerschmidt, A. , Stach, P., Bourenkov, G.P., Bartunik, H.D., Huber, R. and Kroneck, P. (1999). Structure of a cytochrome c nitrite reductase. Nature 400, 476-480. Einsle, O., Stach, P., Messerschmidt, A. , Simon, J., Kroger, A. , Huber, R. and Kroneck, P .M.H. (2000). Cytochrome c nitrite reductase from Wollinella succinogenes. J. Biol. Chem. 35, 39608-39616. Felsenstein, J. (1996). Inferring phylogenics from protein sequences by parsimony, distance and likelihood methods. Methods Enzymol. 266, 418-426. Fenderson, F. F., Kumar, S., Liu, M . - Y . , Payne, W. J. and LeGall, J. (1991). Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes. Biochemistry 30, 7180-7185. Ferguson, S. J. (1994). Denitrification and its control. Antonie van Leeuwenhoek 66, 89-110. Ferguson, S. J. (1998). Nitrogen cycle enzymology. Curr. Opin. Chem. Biol. 2, 182-193. Fisher, D. E. and Fisher, M.J. (2001). The Nitrogen Bomb. In Discover, pp. 48-57. Fulop, V. , Moir, J. W., Ferguson, S. J. and Hajdu, J. (1995). The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cdl . Cell 81(3), 369-77. George, S. J., Allen, J.W., Ferguson, S.J. and Thorneley, R.N. (2000). Time-resolved infrared spectroscopy reveals a stable ferric heme NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cdl nitrite reductase with nitrite. J. Biol. Chem. 275(43), 33231-7. Glockner, A . B., Jungst, A . and Zumft, W.G. (1993). Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cdl free background (NirS-) of Pseudomonas stutzeri. Arch. Microbiol. 160, 18-26. 144 Godden, J. W., Turley, S., Teller, D. C., Adman, E. T., Liu, M . Y . , Payne, W. J. and LeGall, J. (1991). The 2.3 Angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes. Science 253, 438-442. Halfen, J. A. , Mahapatra, S., Wilkinson, E. C., Gengenbach, A. J., Young, V . G., Que, L. and Tolman, W. B. (1996). Synthetic modeling of nitrite binding and activation by reduced copper proteins. Characterization of copper(I)-nitrite complexes that evolve nitric oxide. J. Am. Chem. Soc. 118, 763-776. Halfen, J. A . and Tolman, W. B. (1994). Synthetic model of the substrate adduct to the reduced active site of copper nitrite reductase. J. Am. Chem. Soc. 116, 5475-5476. Han, J., Loehr, T .M. Lu, Y . , Sleverstone-Valentine, J., Averill, B .A. and Sander-Loehr, J. (1993). Resonance raman excitation profiles indicate multiple Cys --> Cu charge transfer transitions in type I copper proteins. J. Am. Chem. Soc. 115, 4256-4263. Harvey, S. B. and Nelssestuen, G.L. (1995). Reaction of nitric oxide and its derivatives with sulfites : a possible role in sulfite toxicity. Biochim. Biophys. Acta 1267, 41-44. Heiss, B., Frunzke, K . and Zumft, W.G. (1989). Formation of the N - N bond from nitric oxide by a membrane-bound cytochrome be complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri. J. Bacteriol. 171, 3288-97. Higgins, D. G., Thompson, J.D. and Gibson, T.J. (1996). Using C L U S T A L for multiple sequence alignments. Methods Enzymol. 266, 383-409. Hirasawa-soga, M . , Tamura, G. and Horie, S. (1983). Spectrophotometric and electron spin resonance studies on the substrate interactions of ferredoxin linked nitrite reductase from spinach. J. Biochem. (Tokyo) 94, 1833-1840. Hochstein, L . I. and Tomlinson, G.A. (1989). The enzymes associated with denitrification. Annu. Rev. Microbiol 42, 231-261. Hoehn, G. T. and Clark, V . L. (1992a). Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae. Infect. Immun. 60(11), 4695-703. Hoehn, G. T. and Clark, V . L . (1992b). The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein. Infect. Immun. 60(11), 4704-8. 145 Hoffmann, T., Frankenberg, N . , Marino, M . and Jahn, D. (1998). Ammonification in Bacillus subtilis utilizing dissimilatory nitrite reductase is dependent on resDE. J. Bacteriol. 180(1), 186-9. Holmes, A . J., Costello, A. , Lidstrom, M.E. and Murrell, J.C. (1995). Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbial. Lett. 132, 203-208. Howes, B. D., Abraham, Z. H. L. , Lowe, D. J., Bruiser, T., Eady, R. R. and Smith, B. E. (1994). EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015). Biochemistry 33, 3171-3177. Hulse, C. L. and Averill, B. A . (1989). Evidence for a copper-nitrosyl intermediate in denitrification by the copper-containing nitrite reductase of Achromobacter cycloclastes. J. Am. Chem. Soc. I l l , 2322-2323. Inatomi, K . and Hochstein., L.I. (1996). The purification and properties of a copper nitrite reductase from Haloferax denitrificans. Curr. Microbiol. 32, 72-76. Inoue, T., Gotowda, M . , Deligeer, Kataoka, K. , Yamaguchi, K. , Suzuki, S., Watanabe, H. , Gohow, M . & Kai, Y . (1998). Type 1 Cu structure of blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 at 2.05 A resolution: comparison of blue and green nitrite reductases. J. Biochem. 124(5), 876-9. IUPAC-IUB. (1970). Abbreviations and symbols for the description of the conformation of polypeptide chains. J. Biol. Chem. 245(24), 6489-6497. IUPAC-IUB. (1965). Combined commission on biochemical nomenclature abbreviations and symbols for chemical names of special interest in biological chemistry. Revised tentative rules. J. Biol. Chem. 241(3), 528-533. IUPAC-IUB. (1968). Commission on biochemical nomenclature a one letter notation for amino acid sequences tentative rules. J. Biol. Chem. 243(13), 3557-3559. Iwasaki, Y . , Takeuchi, T., Tamiya, E., Karube, I., Nishiyama, M . , Horinouchi, S., Beppu, T., Kadoi, H. , Uchiyama, S., Suzuki, S. and Suzuki, M . (1992). Electrocatalysis of nitrite reductase from Alcaligenes faecalis S-6 mediated by native redox partner. Electroanal. 4, 771-776. 146 Jackson, M . A. , Tiedje, J. M . and Averill, B. A . (1991). Evidence for an NO rebound mechanism for production of N20 from nitrite by the copper-containing nitrite reductase from Achromobacter cycloclastes. FEBS Lett. 291, 41-44. Jones, T. A. , Zou, J.-Y., Cowan, S. W. and Kjeldgaard, M . (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst. A47, 110-119. Jordan, P. A. , Thomson, A.J . Ralph, E.T. and Green, J. (1997). FNR is a direct oxygen sensor having a biphaisc response curve. FEBS. Lett. 416(3), 349-52. Kakutani, T., Watanabe, H. , Arima, K. and Beppu, T. (1981a). A blue protein as an inactivating factor for nitrite reductase from Alcaligenes faecalis strain S-6. J. Biochem. 89, 463-472. Kakutani, T., Watanabe, H. , Arima, K. and Beppu, T. (1981b). Purification and properties of copper-containing nitrite reductase from a denitrifying bacterium Alcaligenes faecalis strain S-6. J. Biochem. 89, 453-461. Kastrau, D. H. , Heiss, B., Kroneck, P .M. and Zumft, W.G. (1994). Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome be complex. Phospholipid requirement, electron paramagnetic resonance and redox properties. Eur. J. Biochem. 222(2), 293-303. Kataoka, K. , Furusawa, H. , Takagi, K. , Yamaguchi, K. and Suzuki, S. (2000). Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase. J. Biochem. 127, 345-350. Kim, C. H. and Hollocher, T.C. (1983). 15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. J. Biol. Chem. 258, 4861-4863. Kim, C. H. and Hollocher, T.C. (1984). Catalysis of nitrosyl transfer reaction by a dissimilatroy nitrite reductase (cytochrome c,dl). J. Biol. Chem. 259, 2092-2099. Knowles, R. (1982). Denitrification. Microbiol. Rev. 46, 43-70. Kobayashi, M . and Shoun, H. (1995). The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus oxysporum. J. Biol. Chem 270, 4146-4151. 147 Kohzuma, T., Shidara, S., Yamaguchi, K. , Nakamura, N . and Suzuki, S. (1993). Direct electrochemistry of copper-containing nitrite reductase from Achromobacter xylosoxidans NCIB 11015. Chem. Lett., 2029-2032. Korner, H. a. M . , F. (1992). Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri. Arch. Microbiol. 157(3), 218-222. Kukimoto, M . , Nishiyama, M . , Murphy, M . E. P., Turley, S., Adman, E. T., Horinouchi, S. and Beppu, T. (1994). X-ray structure and site-directed mutagenesis of a nitrite reductase from Alcaligenes faecalis S-6: roles of two copper atoms in nitrite reduction. Biochemistry 33, 5246-5252. Kukimoto, M . , Nishiyama, M . , Tanokura, M . , Adman, E. T. and Horinouchi, S. (1996). Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6. J. Biol. Chem. 271, 13680-13683. Kukimoto, M . , Nishiyama, M . , Ohnuki, T., Turley, S., Adman, E.T., Horinouchi, S. and Beppu, T. (1994). Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6. Biochemistry 33, 5246-52. Lacroix, L. B. , Shadle, S.E., Wang, Y . N . , Averill, B.A. , Hedman, B., Hodgson, K.O. and Solomon, E.I. (1996). Electronic structure of the perturbed blue copper site in nitrite reductase; spectroscopic properties, bonding and implications for the entatic / rack state. J. Am. Chem. Soc.(188), 7755-7768. Laskowski, R. A. , MacArthur, M . W., Moss, D. S. and Thornton, J. M . (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291. Leinweber, F. and Monty, K . (1987). Sulfite determination: fuschin method. Methods Enzymol. 143, 15-17. Libby, E. and Averill, B. A . (1992). Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase. Biochem. Biophys. Res. Comm. 187, 1529-1535. Lin, J. T. and Stewart, V . (1998). Nitrate assimilation by bacteria. Adv. Microb. Physiol. 39, 1-30. 148 Masuko, M . , Iwasaki, H. , Sakurai, S. and Nakahara, A. (1984). Characterization of nitrite reductase from a denitrifier, Alacaligenes sp. NCIB 11015. A novel copper protein. J. Biochem. 96(2), 447-54. Mellies, J., Jose, J. and Meyer, T. F. (1997). The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase. Molecular & General Genetics 256(5), 525-32. Melville, S. B. and Gunslus, R.P. (1990). Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli. J. Biol. Chem. 265(31), 18733-6. Michalski, W. P. & Nicholas, D. J. D. (1985). Molecular characterization of a copper containing nitrite reductase from Rhodopseudomonas sphaeroides forma sp. denitrificans. Biochim. et Biophys. Acta 828, 130-137. Moir, J. W., Crossman, L.C. , Spiro, S. and Richardson, D.J. (1996). The purification of ammonia monooxygenase from Paracoccus dentrificans. FEBS. Lett. 387(1), 71-4. Monzani, E., Anthony, G.J., Koolhas, A. , Spandre, A. , Legieri, E., Casella, L. , Gullotti, M . , Nardin, G., Randaccio, L., Fontani, M . , Zanello, P. and Reedjik, J. (2000). Binding of nitrite and its reductive activation to nitric oxide at biomimetic copper centers. J. Biol. Inorg. Chem. 5, 251-261. Murphy, M . E., Lindley, P. F. and Adman, E. T. (1997a). Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions. Prot. Sci. 6, 761-70. Murphy, M . E., Turley, S. and Adman, E. T. (1997b). Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. Mechanistic implications. J. Biol. Chem. 272, 28455-28460. Murphy, M . E. P., Turley, S., Kukimoto, M . , Nishiyama, M . , Horinouchi, S., Sasaki, PL, Tanokura, M . and Adman, E. T. (1995). Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. Biochemistry 34, 12107-12117. Murphy, M . E. P., Turley, S. and Adman, E.T. (1998). On the mechanism of nitrite reductase: Complex between pseudoazurin and nitrite reductase from A. cycloclastes. In Biological Electron Transfer Chains: Genetics, Composition and Mode of 149 Operation (E., G. W. C. a. V. , ed.), pp. 115-128. Kluwer Academic Publishers, The Netherlands. Navaza, J. and Saludjian, P. (1997). AMoRe: An Automated Molecular Replacement Program Package. Methods Enzymol. 276, 581-594. Neese, F., Kappl, R., Hutterman, J., Zumft, W.G. and Kroneck, P .M.H. (1998). d. Biol. Inorg. Chem 3, 53. Nicholls, A. , Sharp, K . and Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11(4), 281-96. Nishiyama, M . , Suzuki, J., Kukimoto, M . , Ohnuki, T., Horinouchi, S. and Beppu, T. (1993). Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli. J. Gen. Micro. 139, 725-733. Nishiyama, M . , Suzuki, J., Ohnuki, T., Chang, H.C., Horinouchi, S., Turley, S., Adman. E.T. and Beppu, T. (1992). Site-directed mutagenesis of pseudoazurin from Alcaligenes faecalis S-6; Pro80Ala mutant exhibit marked increase in reduction potential. Prot. Eng. 5(2), 177-84. Nurizzo, D., Cutruzzola, F., Arese, M . , Bourgeois, D., Brunori, M . , Cambillau, C. and Tegoni, M . (1999). Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase, d. Biol. Chem. 274(21), 14997-5004. Olesen, K. , Veselov, A. , Zhao, Y . , Wang, Y . , Danner, B., Scholes, C. P. & Shapleigh, J. P. (1998). Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3. Biochemistry 37, 6086-6094. Otwinowski, Z. and Minor, W. (1997). Processing of x-Ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326. Park, S. Y . , Shimizu, H. , Adachi, S., Nagawa, A. , Tanaka, I., Nakahara, K. , Shoun, H. , Obayashi, E., Nakamura, H. , Lizuka, T. and Shiro, Y . (1997). Crystal structure from nitric oxide reductase from dentrifiying fungus Fusarium oxypsorum. Nat. Struct. Biol. 4, 827-832. Payne, W. J. (1981). Dentrification. John Wiley & Sons, New York. 150 Prudencio, M , Eady, R. and Sawyers, G. (2001). Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction. Biochem. J. 353, 259-266. Prudencio, M . , Pereira. A.S., Tavares, P., Besson, S., Cabrito, I., Brown, K. , Samyn, B., Devreese, B., VanBeeuman, J. and Rusnak, F. (2000). Purification and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica. Biochem. 39, 3899-3907. Raveli, R. B. G., Sweet, R .M. , Skinner, J .M., Duisenberg, A . J .M. and Kroon, J. (1997). STRATEGY: A program to optimize the starting spindle angle and scan range for X -ray data collection. J. Appl. Cryst. 30, 551-554. Richardson, D. J., Wehrfritz, J.M., Keech, A. , Crossman, L.C. , Roldan, M.D. , Sears, H.J., Butler, C S . Reilly, A . , Moir, J.W. and Berks, B.C. (1998). The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification. Biochem. Soc. Trans. 26, 401-408. Ryden, L . (1984). Structure and evolution of small blue proteins. In Copper Proteins and Copper Enzymes F7(Lontie, R., ed.), pp. 183-214. CRC Press, Boca Raton, Florida. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Nolan, C , Ed.), 3. 3 vols, Cold spring Harbour Laboratory Press. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). D N A sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. Schroder, I., Darie, S. and Gunsalus, R.P. (1993). Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor. J. Biol. Chem. 268(2), 771-4. Schroeter, L . C. (1966). In sulfur dioxide; Application in foods, beverages and pharmaceuticals. Pergamon, Oxford, 84-85.. Shapleigh, J. P. and Payne, W.J. (1985). Differentiation of cdl cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbial. Lett. 26, 275-279. Silvestrini, M . C , Falcinelli, S. Ciabatti, I., Cutruzolla, F. and Brunori, M . (1992). Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein. Biochem. J. 285, 661-666. 151 Silvestrini, M . C , Falcinelli, S. Ciabatti, I., Cutruzolla, F. and Brunori, M . (1994). Pseudomonas aeruginosa nitrite reductase (or cytochrome oxidase). An overview. Biochimie 76, 641-654. Smith, G. B. and Tiedje, J .M. (1992). Isolation and characterization of a nitrite reductase gene and its use as a probe for dentrifying bacteria. Appl. Environ. Microbiol. 58, 376-384. Solomon, E. I., Hare, J.W. and Gray, H.B. (1976). Spectroscopic studies and a structural model for blue copper centers in proteins. Proc. Natl. Acad. Sci. 73(5), 1389-93. Spiro, S. and Guest, J.R. (1990). FNR and its role in oxygen regulated gene expression in Escherichia coli. FEMS Microbial. Rev. 75, 399-428. Strange, R. W., Dodd, F. E., Abraham, Z. H. L. , Grossman, J. G., Briiser, T., Eady, R. R., Smith, B. E. & Hasnain, S. S. (1995). The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase. Nat. Struct. Biol. 2, 287-292. Strange, R. W., Murphy, L . M . , Dodd, F. E., Abraham, Z. H. , Eady, R. R., Smith, B. E. & Hasnain, S. S. (1999). Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase, d. Mol. Biol 287, 1001-1009. Suzuki, E., Horikoshi, N . & Kohzuma, T. (1999). Cloning, sequencing, and transcriptional studies of the gene encoding copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015. Biochem. Biophys. Res. Comm. 255, 427-31. Suzuki, S., Deligeer, Yamaguchi, K. , Kataoka, K., Kobayashi, K. , Tagawa, S., Kozhuma, T., Shidara, S. and Iwasaki, H. (1997). Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases. J. Biol. Inorg. Chem. 2, 265-274. Suzuki, S. K. , K. and Yamaguchi, K. (2000). Metal coordination and mechanism of multicopper nitrite reductase. Acc. Chem. Res. 33, 728-35. Tiedje, J. M . (1988). In Biology of anaerobic microorganisms. (Zehnder, A. J. B., Ed.), John Wiley & Sons. Tosques, I. E., Kwiatkowski, A . V. , Shi, J. and Shapleigh, J. P. (1997). Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3. d. Bacteriol. 179(4), 1090-5. 152 Tsukihara, T., Aoyama, H. , Yamashita, Y . , Tomizaki, R., Yamagauchi, H. , Shinzawa-Itoh, K. , Nakashima, R., Yaono, R. and Yoshikawa, S. (1995). Structures of metal site of oxidized bovine heart cytochrome c oxidase at 2.8 A . Science 269, 1069-1074. Van Spanning, R. J., De Boer, A . P., Reijnders, W. N . , Westerhoff, H. V . , Stouthamer, A . H. and Van Der Oost, J. (1997). FnrP and N N R of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation. Mol. Microbiol. 23(5), 893-907. Veselov, A. , Olesen, K. , Sienkiewicz, A. , Shapleigh, J. P. and Scholes, C. P. (1998). Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase. Biochemistry 37, 6095-6105. Wang, Y . and Averill, B .A. (1996). Direct observation by FT-IR spectroscopy of the ferrous heme - NO+ intermediate in the reduction of nitrite by a dissimilatory heme cdl nitrite reductase. J. Am. Chem. Soc. 118, 3972-3973. Watmough, N . J., Butland, G., Cheesman, M . R., Moir, J. W., Richardson, D. J. & Spiro, S. (1999). Nitric oxide in bacteria: synthesis and consumption. Biochim. et Biophys. Acta 1411(2-3), 456-74. Weeg-Aerssens, E., Wu, W., Ye, R.W., Tiedje, J .M. and Chang, C.K. (1991). Purification of cytochrome cdl nitrite reductase from Pseudomonas stutzeri J M 300 and reconstitution with native heme d l . J. Biol. Chem. 266, 7496-7502. Williams, P. A. , Fulop, V. , Garman, E.F., Saunders, N.F., Ferguson, S.J. and Hajdu J. (1997). Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme. Nature 389, 406-412. Wilmanns, M . , Lappalainen, P., Kelly, M . , Sauer-Eriksson, E. & Saraste, M . (1995). Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase with an engineered dinuclear copper center. Proc. Nat. Acad. Sci., U.S.A. 92, 11955-11959. Wilson, E. K. , Bellelli, A . , Cutruzzola, F., Zumft, W.G., Guttierrez, A . and Scrutton, N.S. (2001). Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(l) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation. Biohemical J. 355, 39-43. 153 Wolf, E. and Kim, P.S. (1999). Combinatorial codons: A computer program to approximate amino acid probabilities with biased nucleotide usage. Prot. Sci. 8, 680-688. Wu, Q., Storrier, G. D., Pariente, F., Wang, Y . , Shapleigh, J. P. and Abruna, H. D. (1997). A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized fdm of a pyrrole-derived bipyridinium. Analyt. Chem. 69(23), 4856-63. Ye, R. W., Fries, M.R., Bezborodnikov, S.G. Averill, B .A. and Tiedje, J.M. (1993). Characterization of the strucutral gene encoding a copper-containing nitrite reductase and homology of this gene of other denitrifiers. Appl. Environ. Microbiol. 59, 250-254. Ye, R. W., Haas, D., Ka, J. O., Krishnapillai, V. , Zimmermann, A. , Baird, C. and Tiedje, J. M . (1995). Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J. Bacteriol. 177(12), 3606-9. Yeates, T. O. (1997). Detecting and overcoming crystal twinning. Methods Enzymol. 276, 344-358. Zahn, J. A. , Arcerio, D .M. , Hooper, A . B . and DiSpirito, A . A . (1996). Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea. FEBS. Lett. 397, 35-38. Zang, K. E., R. (1993). Reaction of nitric oxide with sulfur(IV) oxides in the presence of iron(II) complexes in aqueous solution. d. Chem. Soc. Dalton Trans., 111-118. Zhang, H. , Boulanger, M . J., Mauk, A. G. & Murphy, M . E. P. (2000). Carbon monoxide binding to copper-containing nitrite reductase from Alcaligenes faecalis. J. Phys. Chem. B 104(46), 10738-10742. Zumft, W. G. (1997). Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 5333-616. Zumft, W. G., Dreusch, A. , Lochelt, S., Cuypers, H. , Friedrich, B. & Schneider, B. (1992). Derived amino acid sequences of the nosZ gene (respiratory N20 reductase) from Alcaligenes eutrophus, Pseudomonas aeruginoase and Pseudomonas stutzeri reveal potenital copper binding residues. Implications for the CuA site of N20 reductase and cytochrome C oxidase. Eur. J. biochem 208, 31-40. 154 Zumft, W. G., Gotzman, D.J. and Kroneck, P.M.H. (1987a). Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens. Eur. J. Biochem. 168, 301-307. Zumft, W. G., Gotzman, D.J. and Kroneck, P.M.H. (1987b). Type I, blue copper proteins constitute a respiratory nirite-reducing system in Pseudomonas aureofaciens. Eur. J. Biochem. 168, 301-307. 155 Appendix - Publications arising from graduate work 2001 Hein J.Wijma, Martin J. Boulanger, Annamaria Molon, Maria Fittipaldi, Martina Huber, Michael E.P. Murphy, Martin Ph. Verbeet and Gerard W. Canters (2001). Replacement of the Type-1 Site Ligand Histidinel45 by Glycine/Alanine in a green Nitrite Reductase creates a Centre that binds Exogenous Ligands in its Cu" State. Submitted to Biochemistry. 2001 Boulanger, M.J. and Murphy, M.E.P. (2001). Crystal Structure of the Soluble Domain of the Major Anaerobically Induced Outer membrane Protein (AniA) from Pathogenic Neisseria: A New Class of Copper-Containing Nitrite Reductases. 56 pages. In press J. Mol. Biol. 2001 Boulanger, M.J. and Murphy, M.E.P. (2001). Alternate Substrate Binding Modes to Two Mutant (D98N and H255N) Forms of Nitrite Reductase from Alcaligenes faecalis S-6: Structural Model of a Transient Catalytic Intermediate. Biochemistry. 40 (31), 9132-9141. 2001 Van Gastel, M . , Boulanger, M.J., Canters, G.W. Huber, M . , Murphy, M.E.P., Verbeet, M.Ph. and Groenen, E.J.J. (2000). A single-crystal electron paramagnetic resonance study at 95 GHz of the type 1 copper site of the green Nitrite reductase of Alcaligenes faecalis. J. Phys.Chem. B. 105, 2236-2243. 2000 Boulanger, M.J., Kukimoto, M . , Nishiyama, Horinouchi, S. and Murphy, M.E.P. (2000). Catalytic roles for two water bridged residues (Asp98 and His255) in the active site of copper-containing nitrite reductase. J. Biol. Chem. 275, 23957-23964. 2000 Zhang, H. , Boulanger, M.J., Mauk, A . G . and Murphy, M.E.P. (2000). Carbon Monoxide Binding to Copper-Containing Nitrite Reductase from Alcaligenes faecalis. J. Phys. Chem. B. 104 (46), 10738-10742. 156 Published Abstracts; 2001 Murphy, M.E.P. and Boulanger, M.J. (2001). Multiple Substrate Binding Modes Observed by X-ray Crystallography in Mutant Forms of Copper-Containing Nitrite Reductase. Annual meeting of the Imaging Society, Tokyo, Japan. 2001 Boulanger, M.J. and Murphy, M.E.P. (2001). Crystal Structure of the Soluble Domain of the Major Anaerobically Induced Outer membrane Protein (AniA) from Pathogenic Neisseria. Presented at the Fourth European symposium of the protein society, April 18-20. Paris, France. 2000 Boulanger, M.J. and Murphy, M.E.P. (2000). Mechanistic implications of substrate soaked crystal structures of two mutant (D98N and H255N) forms of nitrite reductase from Alacligenes faecalis S-6. Presented at the 6 t h Northwest Crystallography Workshop, July 7 - 9 . Eugene, Oregon. 2000 Van Gastel, M . , Huber, M . , Verbeet, M . , Canters, G.W., Boulanger, M.J., Murphy, E.P.M. and Groenen, E.E.J. (2000). Single-Crystal High-Field EPR on the Type I Copper Site in Nitrite Reductase: G-tensor and Electronic Structure of a Center for Electron Transfer. Presented at the International Biophysics and Biochemistry Conference. New Orleans, Louisiana. 2000 Verbeet, M . , Jeuken, L.C. , Wijma, H.J., Fittipaldi, M . , Boulanger, M.J., Huber, M . , Murphy, E.P.M. and Canters, G.W. (2000). Engineering Type I Copper Centers in Redox Enzymes for Hot Wiring. Presented at the NIH Metals in Medicine Conference. Seattle, WA. 157 1999 Boulanger, M . J . and Murphy, M.E.P. (1999). Structural analysis of active site mutants of Nitrite reductase; the role of twinning. Presented at the 14 th West Coast Protein Crystallography Workshop, March 14-17. Pacific grove, California. 1999 Boulanger, M .J . , Resell, F.I., Mauk, G.A. and Murphy, E.P.M. (1999). Probing the Structural and Spectral Properties of the Copper Centers in Nitrite Reductase. Presented at the Innovations in Molecular Biophysics Conference. Vancouver, Canada. 158 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0103826/manifest

Comment

Related Items