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Structural basis for heme degradation in Staphylococcus aureus Ukpabi, Georgia Nonye 2012

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STRUCTURAL BASIS FOR HEME DEGRADATION IN STAPHYLOCOCCUS AUREUS  by  Georgia Nonye Ukpabi B. Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2012  © Georgia Nonye Ukpabi, 2012  ABSTRACT  IsdG and IsdI are heme degrading enzymes from the bacterium Staphylococcus aureus. Heme degrading enzymes use heme as a substrate and cofactor to degrade the porphyrin ring and release the central iron atom. Previous work on the structure of these enzymes showed metal coordination by a conserved His residue in the heme complex of the inactive IsdG-N7A variant and in the cobalt protoporphyrin IX complex of IsdI. In these structures, the porphyrin ring is highly distorted from planarity resulting in the β- and δ-meso carbons being displaced toward the distal side of the heme pocket and the α- and γ-meso carbons towards the His proximal side. This heme distortion is described as ruffling and is not present in the classical family of heme degrading enzymes called the heme oxygenases (HO) that bind heme in a planar manner. Thus, heme ruffling is proposed to be an important structural feature for the reaction mechanism of IsdG and IsdI. The role of heme ruffling in IsdI activity was examined in this study. For the first time, IsdI was cocrystallized in complex with its true substrate, heme, and the structure was solved to 1.50 Å resolution. The structure revealed extensive heme ruffling of 2.1 Å as determined by normal-coordinate analysis. IsdI was then engineered to adopt a flatter heme by mutating a conserved tryptophan residue, W66, in the heme pocket to the less bulky side chains of tyrosine, phenylalanine, leucine and alanine. Of the IsdI variants tested, only W66Y was amenable to X-ray structure solution. Heme ruffling in the variant was lessened to about 1.4 Å, demonstrating that W66 is an important contributor to heme distortion in IsdI. The activity of the W66Y variant was reduced to half that of wild-type suggesting a link between heme distortion and enzyme activity in IsdI.  ii  PREFACE  Part of the work presented in this thesis is drawn from published literature with corresponding publications listed. The work was made possible through collaborations, primarily with Dr. Eric Skaar’s lab at Vanderbilt University and Dr. A Grant Mauk’s lab at the University of British Columbia. Since the collaborations provide crucial support to the findings, some of the materials from the publications are integrated into thesis and the relative contributions to the work are outlined below.  Grigg, J. C., Ukpabi, G., Gaudin, C. F. M., and Murphy, M. E. P. (2010) Structural biology of heme binding in the Staphylococcus aureus Isd system. J. Inorg. Biochemistry 104, 341-348 This review article was written by members of Dr. Michael Murphy’s lab. I wrote and created figures for the Isd heme degrading enzyme section. J. Grigg wrote the abstract, introduction, biology, cell wall anchored surface receptors and membrane transport sections. C. Gaudin wrote the related Isd Systems and Summary sections. The manuscript was edited by Dr. Michael Murphy. Only the section on Isd heme degradation was adapted and expanded in Chapter 1 of the thesis.  Reniere, M. L., Ukpabi, G. N., Harry, S. R., Stec, D. F., Krull, R., Wright, D. W., Bachmann, B. O., Murphy, M. E., and Skaar, E. P. (2010) The IsdG-family of haem oxygenases degrades haem to a novel chromophore. Mol. Microbiol. 75, 1529-1538 The thesis contains work from this paper written with Dr. Eric Skaar’s lab. I produced recombinant IsdI, crystallized the protein, and determined structures of IsdI in complex with heme. M. Reniere expressed the protein, carried out activity assays and with the remaining authors carried out structure determination of the reaction product, as referenced in the discussion section of this thesis. I wrote the first draft of my respective sections of the methods iii  and results, which Dr. M Murphy edited. M. Reniere wrote the first draft of the manuscript and Dr. E. Skaar and Dr. M. Murphy edited. The text and figures describing data that I contributed was adapted and used in the thesis. This includes the methods, results and discussion on the crystal structure determination of IsdI with heme.  Ukpabi, G., Takayama, S. J., Mauk, A. G., Murphy, M. E. P. Inactivation of IsdI Heme Oxidation by an Active Site Substitution that Diminishes Heme Ruffling. In preparation. The thesis contains work from a manuscript in which Dr. S.J. Takayama and I shared coauthorship that will be submitted. I cloned, expressed, and carried out activity assays on the IsdI W66 variants. I crystallized the protein and determined structures of the W66Y variant. Dr. S.J. Takayama carried out experiments on electronic absorption spectra, 1H NMR spectra and cyclic voltammetry. I wrote the first draft of the abstract and introduction. Dr. S.J. Takayama and I wrote our respective experimental, results and discussion sections, and Dr. M. Murphy and Dr. A. Grant Mauk edited the manuscript. Only the sections of manuscript describing data I produced were adapted and used in the thesis.  Takayama, S. J., Ukpabi, G., Murphy, M. E. P., and Mauk, A. G. (2011) Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI. Proc. Natl. Acad. Sci. 108, 13071-13076 The final contribution is from a published manuscript in which Dr. S.J. Takayama and I shared co-authorship. I expressed and crystallized IsdI protein and determined the structure of IsdI bound to heme and cyanide. Dr. S.J. Takayama carried out experiments on electronic absorption spectra, NMR spectra, and cyclic voltammetry. Dr. S.J. Takayama wrote the first draft of the abstract and introduction. Dr. S.J. Takayama and I wrote our respective experimental, results, and discussion sections, and Dr. M. Murphy and Dr. A. Grant Mauk edited the manuscript. This paper is referenced in the discussion section of the thesis. iv  TABLE OF CONTENTS  ABSTRACT .................................................................................................................................... ii PREFACE ...................................................................................................................................... iii TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii LIST OF SYMBOLS AND ABBREVIATIONS .......................................................................... ix ACKNOWLEDGMENTS ............................................................................................................. xi CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1  Staphylococcus aureus ..................................................................................................... 1  1.1.1  Host iron availability .................................................................................................... 2  1.1.2  Heme transport in S. aureus ......................................................................................... 4  1.2  Heme-degrading enzymes ................................................................................................ 6  1.2.1  Class 1: Heme oxygenase family.................................................................................. 6  1.2.2  Class 2: IsdG-family ..................................................................................................... 8  1.2.3  IsdG-family homologues in other species .................................................................. 12  1.3  Crystallization of the IsdG-family enzymes................................................................... 14  1.4  Objective of the present study ........................................................................................ 16  CHAPTER 2. MATERIALS AND METHODS .......................................................................... 18 2.1  Materials ......................................................................................................................... 18  2.1.1  Chemical supplies and media ..................................................................................... 18  2.1.2  Bacterial strains and plasmids .................................................................................... 18  2.2  Protein preparation ......................................................................................................... 19  2.2.1  Site-directed mutagenesis of IsdI ............................................................................... 19  2.2.2  Overexpression and protein purification .................................................................... 20  2.2.3  Heme reconstitution .................................................................................................... 21  2.3  Functional characterization ............................................................................................ 22 v  2.3.1  Heme degradation assay ............................................................................................. 22  2.3.2  pH dependence of activity .......................................................................................... 22  2.3.3  Heme reduction........................................................................................................... 23  2.3.4  Effect of desferrioxamine ........................................................................................... 23  2.4  X-ray crystallography..................................................................................................... 23  2.4.1  IsdI-heme crystallization ............................................................................................ 23  2.4.2  IsdI-W66 variant crystallization ................................................................................. 24  2.4.3  Data collection and structure solution ........................................................................ 25  CHAPTER 3. RESULTS .............................................................................................................. 26 3.1  Protein preparation ......................................................................................................... 26  3.1.1  Protein expression and purification ............................................................................ 26  3.1.2  Protein reconstitution and characterization ................................................................ 26  3.2  Functional characterization ............................................................................................ 28  3.2.1  Activity of IsdI and the W66 variants ........................................................................ 28  3.2.2  pH dependence of activity .......................................................................................... 31  3.2.3  Heme reduction........................................................................................................... 32  3.2.4  Effect of desferrioxamine ........................................................................................... 33  3.3  X-ray crystallography..................................................................................................... 34  3.3.1  Crystallization of IsdI ................................................................................................. 34  3.3.2  Structure of IsdI-heme (oxidized and reduced) .......................................................... 35  3.3.3  Structure of IsdI-W66Y-heme .................................................................................... 38  CHAPTER 4. DISCUSSION ........................................................................................................ 44 4.1  Functional characterization of IsdI and the W66 variants.............................................. 44  4.2  Structural Study of IsdI and the W66Y variant .............................................................. 44  4.2.1  Crystallization ............................................................................................................. 44  4.2.2  Structure of oxidized and reduced IsdI-heme ............................................................. 46  4.2.3  Structure of IsdI-W66Y-heme .................................................................................... 47  4.3  IsdI heme degradation reaction ...................................................................................... 49  CHAPTER 5. CONCLUSIONS ................................................................................................... 53 REFERENCES ............................................................................................................................. 54 vi  LIST OF TABLES  Table 1-1. Crystallization conditions for various forms of IsdG-family proteins ........................ 15 Table 2-1. List of primer sequences used in construction of IsdI variants. .................................. 20 Table 3-1. Kinetic and Spectral Properties of IsdI and W66 variants .......................................... 30 Table 3-2. IsdI-heme data collection and refinement statistics..................................................... 36 Table 3-3. IsdI-W66Y-heme data collection and refinement statistics......................................... 39  vii  LIST OF FIGURES  Figure 1-1. Structure of heme. ........................................................................................................ 3 Figure 1-2. Schematic representation of the Isd system in S. aureus. ............................................ 5 Figure 1-3. Schematic of the heme oxygenase reaction mechanism. ............................................. 8 Figure 1-4. Structural comparison of S. aureus IsdI-CoPPIX and N. meningitidis HemO. ......... 10 Figure 1-5. Multiple sequence alignment of known IsdG-family homologues. ........................... 14 Figure 3-1. Visible electronic spectra of IsdG, IsdI, and the W66 variants and of pyridine hemochrome assays. ..................................................................................................................... 27 Figure 3-2. Heme degradation activity of IsdG, IsdI and the W66 variants. ................................ 29 Figure 3-3. pH dependence of heme degradation. ........................................................................ 31 Figure 3-4. Electronic spectra of reduced IsdI and IsdG. ............................................................. 32 Figure 3-5. Heme degradation in the presence of desferrioxamine. ............................................. 34 Figure 3-6. Structure of the active site of IsdI-heme. ................................................................... 37 Figure 3-7. Structure of the active site of the IsdI-W66Y variant. ............................................... 40 Figure 3-8. Superimpostion of IsdI and the W66Y variant. ......................................................... 42 Figure 4-1. Distortion of the porphyrin ring. ................................................................................ 48 Figure 4-2. The substrate and products of IsdG and IsdI heme degradation using ascorbate as the reductant in the presence of catalase. ............................................................................................ 50  viii  LIST OF SYMBOLS AND ABBREVIATIONS  λmax  Wavelength of absorbance maximum  Å  Ångstrom (1 Å = 0.1 nm)  ABC  Adenosine triphosphate binding cassette  Amp  Ampicillin  Ampr  Ampicillin resistance  B. anthracis  Bacillus anthracis  B. japonicum  Bradyrhizobium japonicum  B-factor  Crystallographic temperature factor  Bis-tris  Bis(2-hydroxy-ethyl)amino-tris(hydroxymethyl)methane  BSA  Bovine serum albumin  Buffer A  0.02 M Tris, pH 7.5, and 0.2 M NaCl  CV  Cyclic voltammetry  CLS  Canadian Light Source  CoPPIX  Protoporphyrin IX with a central Co3+ ion  DNA  Deoxyribonucleic acid  E. coli  Escherichia coli  Fur  Iron (Fe) uptake regulatory protein  H2O2  Hydrogen peroxide  Hb  Hemoglobin  H-bond  Hydrogen bond  heme  Protoporphyrin IX with a central Fe3+ ion  hemin  Hydrochloride form of heme, protoporphyrin IX with a central Fe3+ ion  His6-tagged  Six-histidine tag  HmuD  Heme-degrading enzyme D from Bradyrhizobium japonicum  HmuQ  Heme-degrading enzyme Q from Bradyrhizobium japonicum  HO  Heme oxygenase  HPLC  High-performance liquid chromatography  HRESIMS  High Resolution Electron Spray Ionization Mass Spectroscopy ix  IPTG  Isopropyl-β-D-thiogalactopyranoside  Isd  Iron-regulated surface determinant  kDa  Kilodaltons  LB  Luria Bertani  M. tuberculosis  Mycobacterium tuberculosis  MhuD  Mycobacterial heme utilization degrader  MRSA  Methicillin resistant Staphylococcus aureus  MW  Molecular weight  N. meningitidis  Neisseria meningitidis  NADPH  Nicotinamide adenine dinucleotide phosphate (reduced)  NEAT domain  NEAr Transporter domain  NMR  Nuclear magnetic resonance  NSD  Normal coordinate structural decomposition  R-factor  Crystallographic refinement factor (R-merge, R-work and R-free)  r.m.s.d.  Root mean square deviation  ROS  Reactive oxygen species  P. aeruginosa  Pseudomonas aeruginosa  PDB  Protein Data Bank  PEG  Polyethylene glycol  PPIX  Protoporphyrin IX  ppm  Parts per million  S. aureus  Staphylococcus aureus  SDS-PAGE  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  Soret  An intense peak in the blue region of the optical absorption spectrum of a hemoprotein.  TEV  Tobacco etch virus  Tris  Tris(hydroxymethyl)aminomethane  UV  Ultraviolet  v/v  Volume-to-volume ratio  WT  Wild-type  w/v  Weight-to-volume ratio x  ACKNOWLEDGMENTS  I would like to express my gratitude to my supervisor Dr. Michael Murphy for his invaluable intellectual contribution to this thesis as well as his continuous support and understanding. I also need to thank Dr. Woo Cheol Lee for his generous help and instruction in the laboratory during the initial stages of my project. This thesis could not have been completed without their guidance and constructive criticism. I acknowledge financial support for this work from the CIHR. I thank the CLS for synchrotron data collection. Acknowledgements are also due to all my colleagues in Dr. Murphy's group for their suggestions and comments on my research, and technical assistance. Finally, I would like to thank my family for their continued encouragement and support.  xi  CHAPTER 1. INTRODUCTION 1.1 STAPHYLOCOCCUS AUREUS Staphylococcus aureus is a Gram-positive, spherical bacterium and a member of the family Staphylococcaceae (1). S. aureus is a commensal of approximately 30% of healthy individuals and typically colonizes the nasal passages, skin and mucous membranes (2,3). Under opportunistic conditions, S. aureus can cause a wide range of infections, from mild skin abscesses to serious toxic shock syndrome. Methicillin resistant S. aureus (MRSA) is a leading cause of infection around the world (4). In the United States, medical data collected between 2005 through 2008 shows more than 21 000 cases of invasive MRSA infections (5). Strains of MRSA can be acquired in both the community and hospital settings, with hospital-acquired MRSA infections usually occurring in patients undergoing surgery or using indwelling catheters (6). In addition, many MRSA strains are resistant to other antimicrobial agents (4). The prevalence of multidrug resistant S. aureus stresses the need for new therapeutic targets against the organism. Iron is an essential nutrient that must be obtained by S. aureus during infection of the host. The host, however, attempts to limit the availability of free iron as a defense strategy against infection. In order to circumvent host restriction methods, S. aureus encodes iron transport systems that allow it to extract iron from its surroundings and take it up into the cell. An important iron source is in the form of heme. The major heme transport system in S. aureus is the iron-regulated surface determinant (Isd) system that was discovered in 2002 (7,8). The Isd system was found to be essential for S. aureus virulence in a murine model of infection (9). It is responsible for relaying heme inside the cell, where cytoplasmic enzymes degrade it to release  1  the central iron atom for use as a nutrient. The work described here focuses on uncovering the structural basis of how the Isd enzymes degrade heme.  1.1.1 HOST IRON AVAILABILITY Although the total amount of iron in the human body is quite abundant (4 g), the concentration of free iron ions is kept extremely low at 10-18 M (10). This is achieved through careful regulation and transport control of the metal by host proteins. Iron is absorbed from the intestine and transported throughout the body by iron-binding glycoproteins, such as transferrin and lactoferrin. Excess iron is stored as a mineral by the protein ferritin, providing an iron reserve within cells that limits the level of free iron ions in the blood (10). This system avoids the problem of iron precipitating as Fe(OH)3 at neutral pH in the presence of dioxygen. It also prevents the formation of damaging hydroxyl radicals from the Fenton reaction (i.e. the iron supported transfer of one electron to H2O2) (11). As a result, the level of free iron in the human body is not sufficient to support bacterial growth since iron is required by most pathogenic bacteria in the range of 0.4 – 4 µM (12). The bacteria must circumvent host restriction methods to obtain the nutrient. The majority of host iron is complexed in the cofactor heme (13,14). Heme is the active component of proteins such as hemoglobin, myoglobin, and cytochromes. The molecule is composed of four pyrrole groups linked together by meso-carbon bridges to form a tetrapyrrole ring (Figure 1-1). A single iron atom is chelated at the center of the ring by four pyrrole nitrogen atoms, accounting for four of the six possible ligand bonds the iron needs to complete octahedral coordination. The axial positions may be occupied by a protein residue, such as a histidine in the case of hemoglobin, or an inorganic ligand such as water or dioxygen. Heme also contains eight  2  substituent groups attached to its ring: four methyl groups, two vinyl groups, and two propionate groups.  Figure 1-1. Structure of heme. Heme consists of four pyrrole groups and a central iron atom. In total, the molecular weight of heme is approximately 616.5 Da. The meso-carbons are labeled α, β, δ and γ. The heme substituent positions are numbered: 2, 4-vinyl; 1,3,5,8-methyl; 6,7-propionate.  Heme is not readily available for bacteria to use as an iron source during infection because it is bound to host hemoproteins. Hemoglobin is the most abundant such protein accounting for up to 70 % of the iron content in the human body (14). Hemoglobin in circulating erythrocytes is involved in the important process of transporting oxygen throughout the body. Upon infection, S. aureus hemolysins are likely responsible for releasing hemoglobin from the erythrocytes (15). Any free heme released is potentially harmful to cells because heme is a hydrophobic molecule that is capable of damaging DNA, diffusing into the lipid layer of cell membranes and disrupting cellular integrity (16). In humans, the glycoproteins haptoglobin and  3  hemopexin bind free hemoglobin and heme, respectively, with high affinity (17,18). S. aureus is able to scavenge heme from these different sources using the cell wall associated proteins of its heme uptake system.  1.1.2 HEME TRANSPORT IN S. AUREUS The primary heme transport system in S. aureus is the iron-regulated surface determinant system (7,8). It is required for maximal growth of S. aureus on heme as the sole iron source, and allows the bacteria to effectively grow on heme, hemoglobin and hemoglobin-haptoglobin complexes (19-22). The proteins of the Isd system are encoded in five transcriptional units: isdA, isdB, isdCDEFsrtBisdG, isdH, and isdI that are under transcriptional regulation of the Fur repressor, resulting in maximal expression under low iron conditions (20). The system was originally linked to heme transport through gene disruption of isdA, isdF, srtA, and srtB, leading to decreased association of heme with S. aureus cells (20). Inactivation of isdH and isdB also led to decreased growth on hemoglobin as an iron source (22). The Isd system relays heme into the bacterial cell using proteins that are localized at the cell wall and cytoplasmic membrane (Figure 1-2). Heme transfer through the cell wall has been demonstrated to be a directional process through in vitro transfer experiments that showed heme is bound by the surface exposed proteins IsdB or IsdH, transferred to IsdA, then to IsdC and finally to the lipoprotein IsdE (23-25). Heme is bound through NEAT domains of these proteins as it is transported toward the cell membrane (26). Finally, the heme is shuttled through the lipid bilayer by the ABC transporter, IsdF. The function of IsdD is unknown.  4  Figure 1-2. Schematic representation of the Isd system in S. aureus. Heme transport by the nine Isd proteins. The cell wall anchored proteins are IsdB, IsdH, IsdA, and IsdC. IsdB and IsdH are shown binding hemoglobin and heme, respectively. IsdE and IsdF are the substrate binding proteins and permease components of the ABC transporter, respectively. The arrows show the directionality of heme transfer between the proteins of the system. Abbreviations: Isd components are labeled; Hb, hemoglobin; iron atoms are depicted as red circles.  In the cytoplasm, heme is degraded by the enzymes IsdG and IsdI (27). IsdG and IsdI are not typical heme degrading enzymes, as their protein sequences and structural fold are distinct from the classical heme oxygenases (HO) (28). They were originally implicated in growth on heme as the sole iron source in complementation study that showed S. aureus IsdI can restore growth of a Corynebacterium ulcerans HO deletion mutant (8). In addition, genetic disruption of S. aureus IsdG and IsdI led to impaired growth of S. aureus on iron-chelated minimal media that 5  was supplemented with heme (29). Although members of the IsdG-like family degrade heme, a structural comparison to HOs reveal differences in the active site that imply the two families do not share a similar reaction mechanism (30). IsdG and IsdI have been found to be structurally related to monooxygenases involved in the synthesis of antibiotics in Streptomyces (30). The following Section 1.2 highlights the similarities and differences between HOs and the IsdGfamily enzymes.  1.2  HEME-DEGRADING ENZYMES  1.2.1 CLASS 1: HEME OXYGENASE FAMILY The enzyme HO is responsible for oxidative degradation of heme in many organisms from mammals to bacteria. There are two main isoforms in mammals, HO-1 and HO-2, with typical molecular weights of 33 and 36 kDa, respectively. The fold of the enzyme is predominately α-helical, with one molecule of heme sandwiched between a distal and proximal helix, with the latter providing the histidine ligand that coordinates the heme-iron. Mammalian HO has a hydrophobic tail at the C-terminus that allows it to bind microsomal membranes (31). HO homologues have also been identified in Gram-positive and Gram-negative bacteria, including HemO from Neisseria meningitidis (32), HmuO from Corynebacterium diphtheriae (33), and pa-HO from Pseudomonas aeruginosa (34). The bacterial enzymes are soluble proteins of approximately 22 – 24 kDa that lack a hydrophobic tail, but otherwise share a high degree of structural similarity with mammalian HO-1 (33). HO degrades heme to biliverdin in a multistep reaction that requires a total input of three molecules of O2 and seven electrons (35). The final heme degradation product is predominantly biliverdin IXα through cleavage of the heme ring at the α-meso carbon. In the course of ring cleavage, the iron is released and the α-meso carbon atom is removed quantitatively as CO. The 6  only known exception is pa-HO, which has a preference for cleavage at the β- and δ-meso carbons through rotation of the heme in the active site compared to the orientation in typical HOs (36). In mammals, heme catabolism requires support from additional enzymes. Electrons for HO activity are supplied by NADPH through NADPH-cytochrome P450 reductase. The final biliverdin product is rapidly converted to bilirubin, following the action of biliverdin reductase, then conjugated with glucuronic acid and excreted (37). Homologues of mammalian NADPHcytochrome P450 reductase and biliverdin reductase have not been identified in pathogenic bacteria; however, the HO reaction can be supported in vitro by incubating the HO-heme complex with ascorbate as the electron donor, resulting in degradation of the heme to a biliverdin-iron complex that remains bound to the enzyme (38). The ferric iron chelator, desferrioxamine, can remove iron from the complex and release free biliverdin (38). The reaction mechanism of classical HOs has been elucidated in detail (see the review article by Unno et al (39)). A schematic representation of the reaction is shown in Figure 1-3. First, heme bound to HO is reduced, producing ferrous iron to which dioxygen binds to form oxyheme. Further reduction and protonation of oxyheme will result in a ferric hydroperoxo intermediate. The heme ring then undergoes hydroxylation at the α-meso carbon. The α-meso hydroxyheme in the deprotonated state has radical character and reacts with oxygen to produce verdoheme and CO. Verdoheme is subsequently converted to biliverdin and free iron in a step that requires reducing equivalents and O2. Product release involves sequential reduction of the ferric biliverdin complex to the ferrous state, release of the ferrous iron, and finally dissociation of biliverdin from the protein. The slowest step is biliverdin release.  7  Figure 1-3. Schematic of the heme oxygenase reaction mechanism.  1.2.2 CLASS 2: ISDG-FAMILY IsdG and IsdI have been identified as the heme-degrading components of the Isd system in S. aureus (8,28-30). IsdG and IsdI have been shown to bind heme, producing a Soret peak at 412 nm in the electronic spectra (27). Biochemical assays under aerobic conditions show either enzyme can release iron from heme in vitro in the presence of an electron donor such as ascorbate or NADPH cytochrome P450 reductase (27,28,30). The reaction does not proceed in the absence of dioxygen (28); however, it is not yet known whether the reaction proceeds by oxygen activation in a way similar to the HO reaction. IsdG and IsdI enzyme activity is also substantially hindered by reconstituting the enzymes with analogues of heme containing metals  8  other than Fe3+(28). The metalloporphyrins that have been tested include Co-, Ga-, Mn- and Zncontaining protoporphyrin IX (PPIX), which the enzymes are unable to degrade. A detailed investigation into the catalytic mechanism of IsdG and IsdI is lacking, and the final degradation product has not been unequivocally determined. IsdG and IsdI, along with their homologues from Bacillus anthracis and Bradyrhizobium japonicum, have been suggested to produce biliverdin from heme in a process similar to the HO (27,40,41). These conclusions were drawn from spectral evidence observed during the IsdG or IsdI reactions that appeared to be consistent with biliverdin formation, which is typically characterized spectroscopically by an increase in absorbance between 600 – 700 nm (32). High-performance liquid chromatography (HPLC) analysis of the reaction products also directed similar claims, as the reaction products were found to produce peaks that eluted at times similar to a biliverdin standard (27,41); however, direct structural investigation of the degradation product was lacking from the analysis. Thus, it is unclear whether the enzymes truly form a biliverdin end product similar to HO. The first structural insights into the function of IsdG and IsdI came from solving the apo crystal structures and mutational analysis of conserved residues in IsdG (30). The enzymes exist as homodimers that adopt a ferredoxin-like + sandwich fold with a -barrel at the dimer interface (Figure 1-4) (30). This is in contrast to the monomeric and predominantly α-helical fold of canonical HO (39). The structures of apo IsdG and IsdI contain clefts between the -barrel and -helixes that were proposed to accommodate the heme substrate.  9  Figure 1-4. Structural comparison of S. aureus IsdI-CoPPIX and N. meningitidis HemO. A, the dimeric fold of IsdI-CoPPIX, and, B, the monomeric α-helical fold of the HemO. C, the hydrophobic active site of IsdI, and, D, hydrophilic HO. IsdI-CoPPIX (PDB ID: 2ZDP); HemO (PDB ID: 1J77).  More recently, the structures of IsdI bound to CoPPIX and an inactive IsdG-N7A variant bound to heme were solved, providing the first look at substrate binding to the enzymes (28). The lack of structural homology between the heme binding sites of these enzymes and classical HO can be seen in the comparison of S. aureus IsdI-CoPPIX to N. meningitidis HemO in Figure 1-4. The IsdI homodimer (Figure 1-4A) contains two deep hydrophobic clefts where the heme moieties are bound to axial histidine ligands, while HemO (Figure 1-4B) binds a single heme molecule primarily between two α-helices.  10  The distal side of the heme pocket is substantially different between the IsdG-like and HO protein families. IsdG and IsdI lack the hydrogen bonding solvent network that is essential for HO activity (42), as seen by comparing IsdI to HemO in Figure 1-4C and D. In IsdI-CoPPIX, the metal center is six-coordinate with a distal chloride ligand that is stabilized by an H-bond to residue Asn6 (Figure 1-4C). The coordinating chloride ion is located between the metal and residue Ile53, where the long arm of the branched alkyl side chain is rotated away from the chloride ion to accommodate ligand binding. In the structure of IsdG-N7A, the heme-iron is fivecoordinate allowing the homologous Ile54 residue to rotate toward the distal iron site (28). In contrast, a distal water molecule is found in the structure of HemO (Figure 1-4D). Insights into the natural distal ligand of IsdG and IsdI may be gained by determining the structure of heme bound to the native enzymes. Perhaps the most interesting feature in the structures of IsdG and IsdI is the extreme conformational distortion of the porphyrin ring that occurs upon binding (28). Heme distortion can be assessed by normal-coordinate structural decomposition (NSD) (43). Shelnutt et al have quantified distortion of the heme macrocycle in terms of the six lowest frequency out-of-plane deformations: ruffling, saddling, doming, propellering, and waving in the x and y planes (43,44). The NSD method determines the contribution of each of these six deformations to give the best description of heme distortion found in the X-ray crystal structure, with the uncertainty arising from the atomic coordinates in the crystal structure. The output is given in terms of the displacement along each of these normal coordinates in Ångstroms (45). By the NSD method, the porphyrin ring distortion of IsdI and IsdG is best described as ruffled, which appears to be due to extensive steric interactions with hydrophobic residues in the binding cleft, particularly by Trp66, Val79, Asn6, and Phe22 in IsdI (equivalent residues are Trp67, Val80, Asn7, and Phe23  11  in IsdG). How the unusually high degree of ruffling contributes to IsdG and IsdI enzyme catalysis is unclear. Other hemoproteins have been found to exhibit nonplanar distortion of the porphyrin ring through structural analysis by the NSD method; however, it is not present to the extent observed for IsdG and IsdI. An example is the heme nitric oxide/oxygen sensing domain from Thermoanaerobacter tengcongensis that uses hydrophobic residues in the binding pocket to create saddling and ruffling distortions of ~1 Å each (46,47). Another example is mitochondrial cytochrome c, in which the protein has a ruffled heme group of up to 1 Å (43,44). Heme distortion in cytochrome c is conserved across species in the protein class and has been proposed to be important for electron transfer function (44,48). Shelnutt et al postulated that a bound porphyrin can only be non-planar when the protein exerts substantial force on the molecule, because in the absence of protein heme is planar (49). Thus, heme distortion that is conserved across a protein family is thought to be of importance to biological function.  1.2.3 ISDG-FAMILY HOMOLOGUES IN OTHER SPECIES Following the discovery of IsdG and IsdI in S. aureus, bioinformatic analysis identified homogolous heme degrading enzymes in other bacterial genomes. These include homologues in Bradyrhizobium japonicum, Bacillus anthracis, and Mycobacterium tuberculosis, all of which have been characterized experimentally (40,41,50). B. japonicum has two paralogous proteins, HmuD and HmuQ, with weak similarity to S. aureus IsdG, having pairwise sequence identity of 24% and 25%, respectively, over the length of the complete alignment. B. anthracis has a genomic region with similarity to the isd operon in S. aureus that contains an IsdG homologue with 35% sequence identity (40). The M. tuberculosis homologue MhuD, which has 22% sequence identity to IsdG, is the only other member of this family aside from IsdG and IsdI to 12  have its structure solved (50). Interestingly, MhuD was found to bind a diheme complex that it is unable to degrade (50), yet the significance of this is unknown. The aligned protein sequences of the IsdG homologues are shown in Figure 1-5, and the conserved residues are boxed. The majority of the conserved residues (Gly33, Phe34, Thr55, Trp57, Phe63, Trp66 and Ala75 in IsdI) are concentrated on one side of the heme pocket in a region around the β-meso carbon. On the opposite side of the pocket, a loop composed of residues 80 – 87 in IsdI (81 – 88 in IsdG) becomes ordered upon heme binding (28,30). Of the remaining conserved residues, His76 provides the axial ligand to the heme iron; Asn6 provides an H-bond to the distal iron ligand; and Gly13 is part of a small loop that hangs from one end of the β-barrel. Three of the conserved residues (Asn6, Trp66, and His76 in IsdI) have been implicated in catalysis of the IsdG family, as standard alanine mutations of the equivalent residues in IsdG resulted in abolishment of enzyme activity (30).  13  Figure 1-5. Multiple sequence alignment of known IsdG-family homologues. Columns of absolutely conserved residues are in boxes. The conserved residues required for function (Asn, Trp, and His) are highlighted by a star over the box. Created with Geneious Software (51).  1.3  CRYSTALLIZATION OF THE ISDG-FAMILY ENZYMES Prior structure work on S. aureus IsdG and IsdI focused on crystallization of the apo or  inactivated forms of the enzymes (28,30). IsdI had been crystallized bound to a substrate analogue CoPPIX that it is unable to degrade and the inactive variant of IsdG (N7A) was crystallized bound to heme (28). The native enzymes had not been crystallized bound to their true substrate, heme, which is crucial for a complete structural analysis of the reaction mechanism. The only other member of this family that had been crystallized was the 14  mycobacterium homologue, MhuD, which was crystallized in both the apo-form and as an inactive diheme complex (50). The crystallization conditions for the mentioned structures are shown in Table 1-1.  Table 1-1. Crystallization conditions for various forms of IsdG-family proteins Protein  Salt  Buffer  Precipitant  0.25 M sodium chloride  sodium cacodylate, pH 6.5  25% w/v PEG 4000  None  sodium citrate, pH 3.2  15% w/v PEG 3350  0.2 M ammonium acetate  Bis-tris, pH 6.5  30% w/v PEG 4000  IsdG-N7A-heme  None  Bis-tris, pH 5.3  24% w/v PEG 3350  Apo-MhuD  None  Bis-tris, pH 6.5  25% w/v PEG 3350  0.25 M sodium chloride, 10 mM triethylamine hydrochloride  Bis-tris, pH 5.0  20% w/v PEG 3350  Apo-IsdI IsdI-CoPPIX Apo-IsdG  MhuD-diheme  Based on these conditions, the IsdG-like enzymes appear to prefer crystallizing at acidic pH with polyethylene glycol (PEG) as the precipitant. PEG is a long-chain polymer that can compete with macromolecule solutes for aqueous solution (52). PEG is commercially available in a variety of molecular weights, from 1000 – 20 000 MW, and is a common precipitant in crystallization screens (53). It has been reported in the literature that peroxide and aldehyde formation occurs in PEGs, in a reaction that is accelerated by light, elevated temperature and the presence of oxygen, which can affect the stability and crystallization of biomolecules such as proteins (54-56).  15  1.4  OBJECTIVE OF THE PRESENT STUDY IsdG and IsdI are enzymes found in the pathogen S. aureus that release iron from heme.  The heme substrate is an aromatic compound that is planar in isolation; however, when bound to IsdG and IsdI, the porphyrin molecule is highly distorted from planarity. The porphyrin ring takes on a large ruffling deformation (NSD of ~2 Å), such that the β- and δ-meso-carbons that connect the pyrrole groups are displaced towards the distal side of the heme. The causes of heme ruffling and its implications on the reaction mechanism are not well understood. In IsdI, Trp66 is located at the proximal side of the heme pocket in contact with the β-meso-carbon. This residue is conserved within this family of enzymes, and I hypothesize that it plays an important role in heme ruffling and enzyme activity. The importance of Trp66 and its contribution to heme ruffling in IsdI are addressed in this study. The goal of this work is to gain insight into how IsdI induces ruffling of the heme macrocycle and how this affects enzyme activity. The approach involved attempting to flatten the heme ring by site directed mutagenesis of IsdI at residue Trp66. Of the engineered variants, W66Y was the most amenable to X-ray structure solution. This variant was characterized with respect to catalytic activity, heme ruffling, and electronic properties. In addition, for the first time IsdI was crystallized bound to its true substrate heme using low temperature conditions. Heme distortion in wild-type IsdI was compared to that of the W66Y variant, and the degree of ruffling was assessed by normal-coordinate analysis. A comparison of the heme groups in the native and variant structures revealed a substantial decrease in heme ruffling by the variant. In addition, the activity of the W66Y variant was reduced to half that of wild-type. Together the data suggest that Trp66 contributes to IsdI catalysis by sterically inducing a large degree of heme ruffling, contrasting with the heme environment of classical HO enzymes. The findings provide a better  16  understanding of how the IsdG-family enzymes degrade heme and contribute to heme catabolism in S. aureus.  17  CHAPTER 2. MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 CHEMICAL SUPPLIES AND MEDIA Chemicals were purchased from Fisher Scientific and Frontier Science, unless otherwise noted. The restriction endonuclease, BamH1, was obtained from New England Biolabs. DNA polymerases used include Phusion (New England Biolabs) and Plantinum Pfx (Invitrogen). IPTG was obtained from Fermentas. Primers were synthesized at Integrated DNA Technologies. Acrylamide and electrophoresis reagents were purchased from Sigma. Nickel Sepharose resin obtained from GE Healthcare Biosciences. Bacteria media components for LB broth were purchased from Sigma. The antibiotic ampicillin used at 100 µg/mL when appropriate. The HT Index screen used in crystallization trials was from Hampton Research. Crystallization reagents PEG 3350 and Bis-tris were purchased from Fluka; and magnesium chloride, ammonium acetate, and ammonium sulfate were purchased from Sigma. Dithionite and desferrioxamine were purchased from Sigma.  2.1.2 BACTERIAL STRAINS AND PLASMIDS The bacterial strain used for cloning and plasmid propagation was Escherichia coli DH5α. The strain used for protein expression was E. coli BL21(DE3). The E. coli cloning vectors pET15bisdG and pET15bisdI were used for recombinant protein expression, as described previously (27). The vectors contained an Ampr cassette that encoded a His6-tag and a TEV protease cleavage site at the N-terminus. There were extra residues between the TEV protease cleavage site and the N-terminus of the target protein. The tag remnants for IsdG included an additional twelve residues (GAHMGIQRPTST) at the N-terminus, while recombinant IsdI 18  contained an extra three residues (GAH). Bacterial stocks made from overnight cultures were stored at -80 °C in LB containing 10% glycerol.  2.2 PROTEIN PREPARATION 2.2.1 SITE-DIRECTED MUTAGENESIS OF ISDI Site-directed mutagenesis was used to generate active site variants of IsdI at position 66. The plasmid pET15bisdI was used as the template after linearization by BamHI digest. The megaprimer method was performed involving two sets of PCR (57,58). First, a region of the isdI coding sequence was amplified between the mutagenic forward primer and the T7 terminator primer (see primer sequences in Table 2-1). The amplified DNA was used as a megaprimer for the second step of whole plasmid PCR, which involved using the original plasmid, pET15bisdI, as the template to create the new mutagenic plasmid. The plasmid was transformed into E. coli DH5α by electroporation and plated on LB-Amp and a single colony was chosen for plasmid propagation. The Promega Wizard kit was used to purify the plasmids. Insertion of the mutation was confirmed by sequencing at Beckman Coulter Genomics.  19  Table 2-1. List of primer sequences used in construction of IsdI variants.1 Primer Sequences Original Sequence: IsdI-W66 (WT)  5’-gaagatagctttaataattggttgaattccgatgtattt-3’  Forward (mutagenic) primers: IsdI-W66F  5’-gaagatagctttaataattttttgaattccgatgtattt-3’  IsdI-W66Y  5’-gaagatagctttaataattatttgaattccgatgtattt-3’  IsdI-W66L  5’-gaagatagctttaataatttgttgaattccgatgtattt-3’  IsdI-W66A  5’-gaagatagctttaataatgcgttgaattccgatgtattt-3’  Reverse primer: T7 terminator 1  5’-gctagttattgctcagcgg-3’  Underscored bases represent the mutations.  2.2.2 OVEREXPRESSION AND PROTEIN PURIFICATION IsdG, IsdI, and the IsdI-W66 variants were purified from E. coli BL21(DE3) as previously described (27). Briefly, the pET15b vector encoding the His6-tagged protein was transformed into Escherichia coli BL21 (DE3) cells by electroporation. Cells were grown in LB media containing ampicillin at 37 °C until reaching an OD600 ~ 0.75. Protein expression was induced in the presence of 0.25 mM IPTG and cells were grown overnight at 25 °C with shaking at 200 rpm. Cells were harvested by centrifugation at 4000 rpm for 10 minutes and then resuspended in 20 mM Tris, pH 7.5, and 200 mM NaCl (Buffer A). Cell lysates were produced by homogenization (Avestin) and applied to a 5 mL Ni-Sepharose column (GE Healthcare). The His6-tagged protein was washed with 10 column volumes of Buffer A containing 25 mM imidazole, and eluted with Buffer A containing 500 mM imidazole. The tag was removed by 20  adding tobacco etch virus (TEV) protease at a ratio of about 10 : 1 (His-tagged protein to TEV). After the reaction, TEV was removed by Ni-Sepharose affinity chromatography and the cleaved IsdG or IsdI protein was obtained in the flow through. The cleaved proteins were dialyzed into Buffer A, using a molecular weight cutoff of 6000 – 8000 Da. Typically, 100 – 150 mg of protein was obtained per L of culture. Purity was assessed by SDS-PAGE showing a single band that was greater than 95% pure.  2.2.3 HEME RECONSTITUTION IsdG, IsdI, and the IsdI-W66 variants were reconstituted with heme. First, the protein concentration was measured at an absorbance of 280 nm with an extinction coefficient of 16 960 M-1cm-1 for IsdG and IsdI; 12 950 M-1cm-1 for W66Y; and 11 460 M-1cm-1 for W66F, W66L, and W66A. Next, hemin was dissolved in 0.1 M sodium hydroxide to a final concentration of 20 mg/mL, and diluted to 2 mg/mL with 0.1 M Tris buffer, pH 7.5. Hemin was then added to the protein at equimolar plus 10 % excess amounts per monomer. After reconstitution on ice for 1 h, the sample was centrifuged at 14 000 rpm for 2 minutes and then applied to a G-25 Sephadex column (GE Healthcare) to remove the unbound heme. The final heme bound protein was dialyzed into Buffer A. The protein was concentrated in an Amicon ultra centrifugal filter unit (6000 - 8000 MWCO, Millipore), and the final protein concentration was determined by the Bradford method using BSA (Sigma Aldrich) as the standard. Successful heme reconstitutions were confirmed by UV-visible spectroscopy analysis revealing a Soret peak. Heme content was measured using the pyridine hemochrome assay (59) as follows. A solution containing 400 µL of protein in Buffer A, 70 µL of 1 M NaOH and 130 µL pyridine was placed in a cuvette. Solid sodium dithionite was then added, and the spectrum of the reduced pyridine hemochrome was recorded. The absorption at the characteristic peak at 418 nm was 21  used to calculate the concentration of the pyridine hemochrome using the extinction coefficient of 191.5 mM-1cm-1 (59).  2.3 FUNCTIONAL CHARACTERIZATION 2.3.1 HEME DEGRADATION ASSAY The activity of heme reconstituted IsdG, IsdI and the W66 variants were determined spectroscopically by the change in absorbance over time in the Soret region of the spectrum at room temperature. The assay cuvette contained heme reconstituted protein (10 μM) in Buffer A, to which sodium ascorbate was added at 1 mM to initiate the reaction. Electronic spectra were recorded from 300 to 700 nm with a Varian Cary-50 UV-visible spectrophotometer every 10 min for a total of 90 min. Heme degradation is characterized by a decrease in absorbance of the Soret over time and, thus, the reaction was followed by the change in absorbance at 396 nm over time. Rate constants were determined with GraphPad Prism software (60), by fitting the first order equation: A = Aoe-kt (A, absorbance; Ao, initial absorbance; k, rate constant; t, time).  2.3.2  PH DEPENDENCE OF ACTIVITY  The pH dependence of the activity was measured across a pH range of 6.0 to 8.5. The reaction was monitored by the decrease in absorbance at the wild-type Soret peak (412 nm). The assays were performed with 10 µM of heme reconstituted IsdG, IsdI, or IsdI-W66Y that had been reconstituted with heme. The buffer contained 0.1 M sodium phosphate between pH 6.0 – 8.5, 0.2 M NaCl and 0.1µM catalase. Catalase was present to remove exogenous peroxides that may cause non-enzymatic heme degradation through coupled oxidation (61). Heme degradation was initiated by adding the reductant sodium ascorbate to 1 mM. Pseudo-first order rate  22  constants were derived by fitting the decrease in the absorbance at 412 nm to a first order rate equation (see Section 2.3.1 Heme degradation assay).  2.3.3 HEME REDUCTION Reduced IsdI-heme and IsdG-heme were prepared by adding an excess of sodium dithionite (50 mM) to a solution of 20 mg/mL protein in an Mbraun Labmaster anaerobic glove box maintained at ~ 2 ppm of O2. The protein was then applied to a G-25 desalting column equilibrated with Buffer A to remove excess dithionite. Protein samples were sealed in an airtight cuvette with a path length of 1 cm before being removed from the glove box to record the UV-Visible spectra.  2.3.4 EFFECT OF DESFERRIOXAMINE The product of IsdG- and IsdI-mediated heme degradation was monitored spectroscopically upon addition of excess desferrioxamine to bind the iron released during the reaction. The reaction cuvette contained 19 µM of IsdG-heme or IsdI-heme and 250 µM desferrioxamine mesylate in 1 mL of Buffer A. The reaction was initiated by the addition of 1 mM sodium ascorbate as the reductant, and spectra were recorded at 10 minutes intervals for 110 minutes, at room temperature.  2.4 X-RAY CRYSTALLOGRAPHY 2.4.1 ISDI-HEME CRYSTALLIZATION Crystals of the IsdI-heme complex were prepared by mixing equal volumes of reservoir solution and protein (15 mg/mL in Buffer A) in sitting drop vapour diffusion plates. Red crystals of IsdI-heme grew when crystallization experiments were conducted at a lowered temperature of 23  4 °C (277 K) and 12 °C (285 K). Crystals were obtained in a reservoir solution of 0.1 M Bis-tris at pH 5.5, 0.2 M MgCl2 and 25 % w/v PEG 3350. This reservoir solution was mixed 1:1 with the protein solution. Prior to data collection, crystals were soaked in a cryoprotectant solution containing the crystallization reservoir and 10% ethylene glycol before being flash frozen in liquid nitrogen. Reduced crystals of IsdI-heme were obtained by soaking the ferric crystals in 50 mM sodium dithionite cryoprotectant solution for 10 minutes before freezing. Red crystals of the native IsdG-heme complex also grew at 4 °C when mixed with a reservoir solution of 0.2 M ammonium sulfate, 0.1 M HEPES at pH 7.5 and 25% w/v PEG 3350. However, these small rod-shaped crystals were not pursued due to poorer resolution of the diffraction (> 3 Å). Further optimization of these crystals may be necessary to improve the diffraction quality.  2.4.2 ISDI-W66 VARIANT CRYSTALLIZATION The final protein solution of W66Y-heme was concentrated to approximately 17 mg/mL in Buffer A and subjected to crystallization trials with the Hampton HT Index screen. Crystals grew when the protein solution was mixed in a 1:1 ratio with reservoir solution containing 0.1 M Bis-tris pH 5.0, 0.2 M ammonium acetate, and 25% w/v PEG 3350. The drops were set up at 4 °C in sitting drop vapour diffusion plates and thin plate-shaped crystals were deep red in colour, and formed in 4 - 7 days. The crystals were looped and dipped in cryoprotectant solution containing the crystallization reservoir supplemented to 20% ethylene glycol prior to data collection.  24  2.4.3 DATA COLLECTION AND STRUCTURE SOLUTION Diffraction data was collected under a cryostream at 100 K on beamline 08ID-1 at the Canadian Light Source (Saskatoon, SK). Data were processed and scaled with MOSFLM (62) and SCALA (63), respectively. Initial phases were obtained with the program MolRep (64), using a search model of the protein moiety from the IsdI-CoPPIX structure (PDB ID: 2ZDP). Programs in the CCP4 suite (65) were used to solve and refine the structures. Heme and water molecules were added to the structures during model building using the visualization software COOT (66) and the structures were refined with the program Refmac5 (67). The final structures were validated with the program PROCHECK (68) and figures were prepared with the program PyMOL (69). All chemical structural formulas were drawn using Symyx Draw 3.2 (45). The amount of heme ruffling in the final structures was analyzed using the normal-coordinate structural decomposition (NSD) program (43). The final structures were superimposed and the root-mean-square-deviation (rmsd) calculated using the program SuperPose (70).  25  CHAPTER 3. RESULTS 3.1 PROTEIN PREPARATION 3.1.1 PROTEIN EXPRESSION AND PURIFICATION Wild-type IsdG and IsdI proteins were expressed and purified as outlined in Section 2.2. Site-directed mutagenesis was used to generate variants of IsdI in which tryptophan at position 66 was replaced with a tyrosine (W66Y), phenylalanine (W66F), leucine (W66L), and alanine (W66A). The mutant forms of the gene were overexpressed in E. coli BL21 (DE3) to produce the corresponding protein using conditions similar to wild-type. All proteins were purified to greater than 95% homogeneity by SDS-PAGE analysis.  3.1.2 PROTEIN RECONSTITUTION AND CHARACTERIZATION Following protein expression, the His6-tags were removed and the proteins were reconstituted with hemin. UV-visible spectra were collected for each protein (Figure 3-1). The spectra of the variants are similar to that of wild-type IsdI except for W66L and W66A that have a broader Soret band. Finally, the pyridine hemochrome assay was used to determine the heme content of the reconstituted proteins (59). The spectra of pyridine hemochrome derived from each of the proteins displayed peaks at 418, 524, and 557 nm confirming that proteins bind heme (Figure 3-1). The protein concentration was determined and the molar ratio of heme to protein monomer was assessed: IsdG, 1.0; IsdI, 0.9; W66Y, 1.1; W66F, 0.7; W66L, 0.7; and W66A, 0.7. The results indicate that wild-type IsdG, IsdI, and W66Y bind heme at approximately 1:1 stoichiometry. The W66F, W66L and W66A variants of IsdI bound less heme, with approximately 70% of the protein loaded with heme, which is consistent with the visible spectra (Figure 3-1E-F). 26  Figure 3-1. Visible electronic spectra of IsdG, IsdI, and the W66 variants and of pyridine hemochrome assays. Thin lines represent the heme reconstituted electronic spectra of 10 µM IsdG (A), IsdI (B), W66Y (C), W66F (D), W66L (E) and W66A (F). The thick lines represent the reduced pyridine hemochrome spectra derived from the respective protein and diluted by a third. 27  3.2 FUNCTIONAL CHARACTERIZATION 3.2.1 ACTIVITY OF ISDI AND THE W66 VARIANTS The ability of IsdI, IsdG and the IsdI-W66 variants to degrade heme was assayed by UVvisible spectroscopy. The proteins were reconstituted with heme and then incubated with ascorbate to initiate the reaction. IsdG and IsdI rapidly degraded heme as shown by the disappearance of the Soret region of the absorbance spectrum (Figure 3-2A and B). Heme degradation was accompanied by a colour change of the solution from red to faint yellow. The activity differed among the IsdI variants, whose spectra are shown in Figure 3-2C-F. The rate of heme degradation for all proteins is compared in a kinetic time course of the decay at 396 nm in Figure 3-2G. Finally, kinetic constants were determined assuming heme degradation follows a one phase exponential decay equation (Table 3-1).  28  Figure 3-2. Heme degradation activity of IsdG, IsdI and the W66 variants. Heme degradation by IsdI and the W66 variants. Enzymatic degradation of the heme was analyzed by UV-visible spectroscopy for IsdG WT (A), IsdI WT (B), W66Y (C), W66F (D), W66L (E) and W66A (F). Assays were performed with 10 µM of protein in 0.02 M Tris buffer, pH 7.5, 0.2 M NaCl and 0.1 µM catalase. Ascorbate was added to 1 mM to initiate the reaction. The change in absorbance was recorded every 10 min for 90 minutes and the decrease at 396 nm was fit to a one phase exponential decay equation that was normalized for comparison (G). Arrows show the direction of the Soret change. 29  Table 3-1. Kinetic and Spectral Properties of IsdI and W66 variants Protein (10 µM)  Soret (nm)  Extinction Coefficient at Soret (mM-1cm-1)  Rate constant at 396 nm, k (min-1)  IsdG WT  412  73.0  0.055 ± 0.001  IsdI WT  412  71.1  0.065 ± 0.003  W66Y  401  56.4  0.030 ± 0.002  W66F  405  60.6  0.024 ± 0.002  W66L  408  56.2  0.0023 ± 0.0007  W66A  396  62.0  0.002 ± 0.002  The activity of the variants was significantly impaired compared to wild-type IsdI with an overall trend of decreasing activity with increasing severity of the mutation to smaller side chains in the order of Tyr, Trp, Leu to Ala. Initial spectra of the W66Y and W66F variants showed well-defined Soret peaks that became slightly blue-shifted as the Soret decayed. The rate constants for these variants were about half that of WT. In contrast, the W66L and W66A variants displayed slight to nearly undetectable heme degrading activity, as indicated by the negligible decrease in the absorbance for these two variants. The spectrum of W66A, in particular, appears more like free heme, forming a broad peak near 396 nm with a large shoulder at shorter wavelengths. The Soret of W66A became more defined in the presence of ascorbate with the loss of the shoulder that occurs to the left of the peak.  30  3.2.2  PH DEPENDENCE OF ACTIVITY  The activity versus pH profiles were measured for heme reconstituted IsdG, IsdI and the IsdI-W66Y variant over the pH range of 6.0 – 8.5 in sodium phosphate buffer. The profiles for IsdG and IsdI were similar with a pH optimum of 8.0 (Figure 3-3). At the acidic pH of 6.0, neither enzyme showed significant activity. Activity of the W66Y variant was also affected by changes in pH. Under the experimental conditions tested, the variant is functional above pH 7, with greatest activity at more alkaline pH.  Figure 3-3. pH dependence of heme degradation. The pH-dependence of IsdG, IsdI and W66Y activity is shown as the rate constant, k (min-1), plotted against pH. Activity was expressed as the pseudo-first order rate constant that was measured by the change in absorbance at 412 nm over time. The protein (10 µM) was reacted with ascorbate (1mM) in buffer containing 0.1 M phosphate, 0.2 M NaCl, and 0.1 µM catalase.  31  3.2.3 HEME REDUCTION IsdI-heme and IsdG-heme were reduced with sodium dithionite under anaerobic conditions. The excess sodium dithionite was subsequently removed with a desalting column after 15 minutes of incubation. There was an observable colour change of the solution from dark to bright red as the protein was reduced, and the Soret of the UV-visible spectrum became redshifted from 412 to 427 nm (Figure 3-4). Upon reduction, both proteins showed second absorption maxima at 557 nm. No spectral changes were observed when 50 mM sodium ascorbate was used to reduce the protein under these conditions (data not shown).  Figure 3-4. Electronic spectra of reduced IsdI and IsdG. The spectrum of oxidized (thin line) and dithionite reduced (thick line) IsdI (A) and IsdG (B) under anaerobic conditions. The peak maxima are labeled. The solution contained 12.5 µM IsdIheme or 10 µM IsdG-heme in 20 mM Tris-HCl, pH 7.5, and 0.2 M NaCl.  32  3.2.4 EFFECT OF DESFERRIOXAMINE IsdG and IsdI activity was assayed in the presence of the ferric iron chelator desferrioxamine to bind iron released during the reaction. In the case of HO, desferrioxamine removes ferric iron from the biliverdin-iron complex during the final step of the reaction with ascorbate (38). Therefore, IsdG-heme and IsdI-heme were incubated in the presence of excess desferrioxamine and ascorbate, and changes to the absorbance spectrum were observed. A new spectral feature appeared as the reaction progressed, as characterized by an increase between 450 – 480 nm that remained stable for the duration of the reaction (Figure 3-5A and B). This feature was observed in the absence of desferrioxamine; however, it only appeared transiently during the first 10 minutes of the reaction then decayed. In addition, there was a colour change of the solution from red to brownish-yellow. The yellow colour of the product is in stark contrast with the blue-green colour of the HO product, biliverdin, which absorbs in the region between 600 – 700 nm (32). It is more similar to bilirubin, which has a maximum absorbance around 440 nm (71).  33  Figure 3-5. Heme degradation in the presence of desferrioxamine. The reaction was carried out with 19 µM of IsdG (A), or IsdI (B), 1 mM ascorbate, and 250 µM desferrioxamine. The buffer contained 20 mM Tris-HCl, pH 7.5 and 0.2 M NaCl. Spectra were recorded at 10 min intervals for 110 min.  3.3 X-RAY CRYSTALLOGRAPHY 3.3.1 CRYSTALLIZATION OF ISDI Initial attempts to crystallize the IsdI-heme complex were unsuccessful. Rather than retaining the red colour of the enzyme-heme complex, the protein solution became faint yellow in colour, similar to the product formed at the end of the reaction as described in Sections 3.2 and 3.2.4. This suggested that turnover of the heme was occurring in the crystallization solution even though no reducing equivalents had been added to initiate the reaction. Initially, the crystallization trials for the protein were set up at room temperature (295 K). To reduce destruction of the bound heme during crystallization, the next set of crystal trials were conducted at the lower temperatures of 4° C (277 K) and 12° C (285 K). Deep red coloured crystals grew for both IsdI and IsdG at these temperatures when using PEG 3350 as the precipitant. The IsdI34  heme complex produced the highest quality diffracting crystals, and was therefore used for structure solution.  3.3.2 STRUCTURE OF ISDI-HEME (OXIDIZED AND REDUCED) The structure of the IsdI-heme complex was solved to 1.50 Å resolution with one IsdI homodimer in the asymmetric unit. Data collection and refinement statistics are shown in Table 3-2. The fold of the IsdI-heme complex is similar to that observed in the IsdI-CoPPIX structure, and the porphyrin ring is also highly ruffled. The NSD heme deviation from planarity is comparable that in IsdI-CoPPIX at 2.1 Å versus 2.3 Å, respectively. Surprisingly, elongated density was observed for a sixth iron ligand at the distal side of the heme. It was best modeled as a coordinating dioxygen species at full occupancy with an average Fe-O bond length of 2.05 Å (Figure 3-6A-C). The dioxygen species has a low B-factor of 14 Å2 and forms an H-bond to the Asn6 ligand at a distance of 3.2 Å. The presence of an oxygen species suggests that the hemeiron was photoreduced to Fe(II) in the X-ray beam during data collection. Further reduction of the oxygen species is suggested by the decreased Fe-O-O angle (108°). To obtain crystals of reduced IsdI-heme, dithionite was added to the crystals since it was found that dithionite could rapidly reduce the metal center, producing a visible colour change (see Section 3.2.3). Upon addition of dithionite, the colour change of the crystals from dark to bright red was easily identified. The crystals maintained their integrity during the soak and diffracted to a high resolution of 1.88 Å. The dithionite reduced crystals yield a structure without a density for a sixth iron ligand (Figure 3-6D), likely due to the consumption of oxygen in the crystal by the reductant. No other significant differences were noted between the dithionite reduced and native structures.  35  Table 3-2. IsdI-heme data collection and refinement statistics IsdI-heme (oxidized)  IsdI-heme (reduced)  48.11 - 1.50  58.03 - 1.88  P212121  P212121  a=58.24, b=66.78, c=69.43  a=58.03, b=67.93, c=70.67  Unique reflections  43991 (6351)  25682 (3674)  Completeness (%)  9s9.8 (100.0)  99.8 (100.0)  Average I/σI  13.1 (3.3)  12.2 (4.1)  Redundancy  6.8 (5.6)  7.0 (6.9)  Rmerge  0.077 (0.481)  0.094 (0.487)  Rwork (Rfree)  0.181 (0.215)  0.202 (0.242)  2256  2116  Protein  1884  1845  Solvent  282  185  Heme  86  86  21.7  29.6  Protein  19.7  28.8  Solvent  36.6  39.2  Heme  16.1  26.3  Dioxygen  14.4  -  Bond length (Å)  0.009  0.014  Bond angles (°)  1.118  1.348  Data Collection Resolution range (Å) Space group Unit cell dimension (Å)  R-factors  No. Atoms  Overall B-factor (Å2)  R.m.s. deviation  36  Figure 3-6. Structure of the active site of IsdI-heme. The structure of IsdI (green) bound to heme (grey) reveals density on the distal side modeled as a dioxygen species (green mesh) liganded to the heme-iron. A, 2Fo – Fc map contoured at 1σ. B, a Fo – Fc omit map for oxygen is contoured at 3σ. The heme α-, β- and δ-meso carbons are labelled. The γ-meso carbon is buried in the protein. C, the same figure as in (B), but looking down perpendicular to the heme from the distal side. The heme α-, β-, γ- and δ-meso carbons are labeled. D, crystals of IsdI (cyan) bound to heme (grey) were reduced for 10 min in 50 mM (excess) dithionite. The 2Fo – Fc map (light grey) is contoured at 1σ. Heme and selected amino acids are depicted in sticks and are labeled. Nitrogen, oxygen and iron atoms are coloured blue, red and orange, respectively.  37  The orientation of heme binding was determined during structure solution. In the case of the IsdI-heme structures, binding of heme could presumably occur in two orientations that differ by 180° rotation about the α- and γ-meso heme axis. These two orientations can be differentiated by the positions of the vinyl and methyl heme substituents. The heme bound to IsdI, predominately adopts a single orientation, rather than binding in a mixture of two alternate orientations. The single orientation can be explained by specific contacts between heme and the surrounding protein environment. When the heme is modeled in the opposite orientation, difference electron density becomes apparent at the vinyl and methyl positions. In particular, a large positive peak is now located at the tip of the methyl group that had previously been the site of the 4-vinyl. On the other side of the heme pocket, steric contacts provided by Ile92, Val79 and Leu81 appear to restrict the presence of a vinyl group. Instead, a much better fit to the electron density map was attained with 1-methyl at this position. This orientation may be required for proper centering of the heme in the active site for catalysis.  3.3.3 STRUCTURE OF ISDI-W66Y-HEME The crystal structure of the W66Y variant of IsdI was solved to 1.90 Å resolution and the crystallographic statistics are shown in Table 3-3. The asymmetric unit of the crystal contains a homodimer consisting of the same α+β-barrel fold as wild-type IsdI-heme (PDB ID: 3LGN), and the structures superimpose with an rmsd of 0.68 Å for the Cα atoms of both chains. The heme pocket of chain A is shown in Figure 3-7A. The electron density surrounding the heme group (Figure 3-7B) reveals the heme-iron is pentacoordinate with no distal ligand. For the W66Y variant, the large unoccupied space remaining in the pocket after mutating Trp66 results in the heme molecule binding in either flipped orientation about its α,γ-axis of the meso carbons.  38  Table 3-3. IsdI-W66Y-heme data collection and refinement statistics IsdI-W66Y-heme Data collection Resolution range (Å) Space group Unit cell dimension (Å)  50.12 -1.90 P 212121 a=59.49, b=68.80, c=73.17  Unique reflections  24334 (3481)  Completeness (%)  100.0 (100.0)  Average I/σI  10.4 (4.2)  Redundancy  6.6 (6.7)  R-factors Rmerge  0.135 (0.430)  Rwork (Rfree)  0.193 (0.230)  No. Atoms  2208  Protein  1863  Solvent  259  Heme  86  Overall B-factor (Å2)  15.1  Protein  14.2  Solvent  22.3  Heme  10.8  R.m.s.deviation Bond length (Å)  0.010  Bond angles (°)  1.32  39  Figure 3-7. Structure of the active site of the IsdI-W66Y variant. A, the active site of IsdI-W66Y (gray) bound to heme. B, the 2Fo – Fc electron density map contoured at 1σ. Heme and selected amino acids are depicted in sticks and are labeled. Nitrogen, oxygen and iron atoms are coloured blue, red and orange, respectively.  40  The superposition of heme bound wildtype IsdI and W66Y is shown in Figure 3-8, with panel A showing the similarity of the overall fold and panel B comparing the active sites. The Tyr residue in the variant appears to mimic the position and function of the Trp residue in the wild-type structure, as shown in Figure 3-8B. The Tyr residue in the variant is angled toward the back of the heme pocket where it is involved in an H-bond network. This network uses a water molecule to bridge the Tyr Oη atom to a heme propionate group (Figure 3-8C). The indolic nitrogen of tryptophan is involved in this interaction with an equivalent water molecule in the wild-type structure. Nonetheless, the smaller Tyr residue relative to Trp creates space in the proximal side of the heme pocket that results in a flatter heme. In wild-type, the distance between Trp and the heme β meso-carbon is 3.5 Å. This distance is longer in the Tyr variant at 4.0 Å, suggesting a weaker steric interaction with the heme.  41  Figure 3-8. Superimpostion of IsdI and the W66Y variant. Structural comparison of IsdI WT and W66Y. A, The Cα trace of the structures with IsdI WT (blue, chain A; green, chain B) and W66Y (black, chain A; gray, chain B). The models superimpose to minimize root mean square deviation of 0.68 for the Cαs of each chain. The Nand C-termini are shown. B, superposition of the active sites of wild type (green) and the W66Y variant (grey) of IsdI. Heme and selected amino acids are depicted in sticks and are labeled. Nitrogen, oxygen and iron atoms are coloured blue, red and orange, respectively. C, the solvent bridge H-bond between residue 66 and the heme propionate group.  42  In the W66Y variant structure, the loss of the bulky Trp side chain results in conformational changes of other key residues in the active site (Figure 3-8B). At the distal side, Ile53 undergoes a rotation of its Cδ1 atom to a distance of 3.4 Å from the iron. The side chain of Asn6 shifts away from the iron center such that it is unable to stabilize binding of a distal iron ligand through H-bonding, as observed in the wild-type IsdI-heme structure. There is also an ordered water molecule on the distal side of the active site forming an H-bond to Thr55 (2.65 Å). The amount of heme distortion in the structures of wild-type IsdI and W66Y was analyzed by normal-coordinate analysis (44). NSD revealed the heme is significantly less ruffled in the variant than wild-type IsdI. The variant has ruffling displacements of 1.3 Å and 1.4 Å in chains A and B, respectively, compared to 2.1 Å in the wild-type IsdI structure, a difference of 0.7 Å.  43  CHAPTER 4. DISCUSSION 4.1 FUNCTIONAL CHARACTERIZATION OF ISDI AND THE W66 VARIANTS Trp66 is a hydrophobic residue on the proximal side of the heme binding pocket of IsdI that has been suggested to contribute to porphyrin distortion (28). This residue is in direct steric contact with the β-meso carbon and is conserved across the IsdG-like family (28,30). In this study, variants of Trp66 were demonstrated to be severely impaired in heme degrading activity. The more conservative IsdI variants, W66Y and W66F, retained about half the activity of the native enzyme. The other two variants, W66L and W66A, exhibited an altered heme binding environment, as determined by visible absorbance spectroscopy and heme content analysis, and suffered a near complete loss of activity. This conserved Trp residue is needed for full activity of IsdG, as shown with the loss in heme degrading activity of the equivalent variant, W67A, of IsdG (30). Although all four IsdI variants bound and retained heme after gel filtration, the pyridine hemochrome assay revealed heme content was about 70 % for the W66F, W66L and W66A variants, implying the Trp residue is likely important for substrate binding as well. To better understand the reasons for the loss in activity, the variant W66Y and wild-type IsdI were subject to structure solution by X-ray crystallography.  4.2 STRUCTURAL STUDY OF ISDI AND THE W66Y VARIANT 4.2.1 CRYSTALLIZATION A structural approach was used to examine the heme binding environment of IsdI. This first required obtaining crystals of IsdI-heme. The IsdI-heme complex was initially found to be a challenge to crystallize as standard crystallization methods were unsuccessful in producing crystals of the complex. The Hampton HT Index screen was used to test a pH range of 3 – 9, as 44  well as different salts and precipitant concentrations to force molecules out of solution and into crystals. The screen contains a variety of PEGs, a type of precipitant used in crystallization, which was successfully used to form crystals of apo and inhibited forms of IsdI at low pH (see Table 1-1) (28,30). However, no crystals of the IsdI-heme complex were obtained using this screen at room temperature. Instead, a colour change of the crystallization solutions occurred from red to pale yellow similar to the colour change observed during the heme degradation assay described in Section 3.2, suggesting enzymatic turnover of the heme was occurring in the crystallization solution. This was surprising given that no reducing equivalents had been added and at low pH the enzyme appears to be inactive (Figure 3-3). A method for limiting reactivity of heme during crystallization was sought. Originally, crystallization trials for the IsdI-heme complex were set up at room temperature (22 °C) without success. In an attempt to prevent degradation of the heme, the next step involved crystalizing the protein at 4 °C and 12 °C. The lower temperature was sufficient to obtain high resolution diffracting crystals of the IsdI-heme complex. The deep red coloured crystals grew in the shape of thin plates in the presence of PEG 3350 as the precipitant. Crystals of the W66Y variant with heme also formed readily at the lower temperatures. It is likely that PEG, a long-chain polymer and common precipitant, was contributing to heme reactivity in the crystallization solution. PEG is known to cause problems in crystallization when it decomposes into its breakdown products of aldehydes and peroxides, and produces crystal hits that are irreproducible (72). Peroxide formation in PEGs has been reported to be accelerated by light, elevated temperature and the presence of oxygen, and can affect the stability of biomolecules in solution (54-56). Future experiments could look into the temperature  45  sensitivity of the IsdI reaction, as well as the rate of heme decomposition in PEG to understand any underlying contributions to heme reactivity during crystallization.  4.2.2 STRUCTURE OF OXIDIZED AND REDUCED ISDI-HEME In the IsdI-heme structure, the presence of a dioxygen species implies that the heme-iron was photoreduced to Fe(II) in the X-ray beam during data collection. This oxygen species is not seen in the dithionite reduced IsdI-heme structure, possibly due to the removal of dioxygen from the crystal environment by dithionite (73). Photoreduction on the beamline is a serious issue when collecting X-ray data on metalloproteins, even though the diffraction data is collected at cryogenic temperatures. During data collection at a wavelength of 1 Å, about 10% of interacting photons are elastically scattered to give diffraction, the rest result in damage to the material in the crystal mainly through the photoelectric effect (74). The oxidation state and the spatial configuration of the atoms in the metal site can be affected by photoreduction on the beamline (75). A way to monitor changes in the metal center during data collection is through recording spectra of the protein in the crystal using microspectrophotometry (75). This has been used to characterize the oxidation state of other hemoproteins. For example, microspectrophotometry on single crystals of cytochrome ba3 oxidase from Thermus thermophilus was used to show that crystals of the oxidized enzyme are completely reduced in an intense synchrotron X-ray in nearly 1 min (76). Future work should involve characterization of the oxidation state of the iron during the reaction and validation of the formation of the oxygen species during data collection using a similar approach. The oxygen species modeled in the IsdI heme-binding pocket are aligned parallel to the β,δ-axis of the porphyrin ring (Figure 3-6C). This is in contrast to the HO family enzymes where oxygen is bent towards the α-meso carbon. The orientation of the dioxygen is restricted by Phe22 46  and Asn6, the latter which is also an H-bond donor to the oxygen species. In HO, regiospecificity is also achieved by steric limitations of the dioxygen ligand such that only the α-meso carbon is accessible for hydroxylation. In the two IsdI heme pockets of the asymmetric unit, the oxygen species are in opposite orientations such that either the β- or δ-meso carbon is aligned for possible hydroxylation. Though the heme is bound to IsdI in predominantly one orientation, the crystallographic data suggests that oxidation at both meso-carbons is possible. Due to the alignment of the oxygen species with respect to the heme ring in the active site, it was hypothesized that the product of heme degradation by IsdG-family enzymes exhibits unique regiospecificity compared with the canonical HO-1 like enzymes.  4.2.3 STRUCTURE OF ISDI-W66Y-HEME Of the four IsdI variants generated, only W66Y-heme was amenable to crystallization. The other variants, W66F, W66L and W66A, which had lower heme content (Figure 3-1), probably exist in as a mixture of heme bound and unbound species in solution, which would not support crystallization. The W66Y variant, however, had high heme content and produced a high resolution crystal structure of 1.90 Å. The W66Y variant had less heme distortion than wild-type. The smaller ring structure of the tyrosine appears to allow the heme to relax into a more planar conformation. In Figure 4-1, heme distortion is broken down in terms of normal coordindates for IsdI and various hemoproteins. It can be seen that heme ruffling is the major contributor to heme distortion in the IsdG-like family enzymes (IsdG-N7A, IsdI-W66Y, and IsdI-heme) in comparison to HOs (pa-HO and HemO with ~ 0.3 Å ruffling) and the relatively planar oxy-myoglobin. In the W66Y structure, the two heme groups were flatter, decreasing from 2.1 Å ruffling in the wild-type IsdI structure to 1.3 Å and 1.4 Å in the heme groups of chains A and B, respectively. These values are closer to 47  mitochondrial cytochromes c (1 Å ruffling), whose heme group is covalently linked to amino acid residues of the protein (44). Additionally, heme seating in W66Y is flipped 180° in comparison to wild-type IsdI (Figure 3-8A), as the flatter heme appears to be more permissive to the flipped orientation of heme binding due to the more spacious heme pocket.  oxy-myo  propellering waving(y)  cyt c  waving(x) HemO  doming saddling  Pa-HO ruffling IsdG-N7A  IsdI-W66Y  IsdI-heme -4  -2  0  2  Distortion (Å)  Figure 4-1. Distortion of the porphyrin ring. The NSD out-of-plane distortion (Å) of the porphyrin ring in the structures of oxy-myoglobin (oxy-myo, 1A6M), horse heart cytochrome c (cyt c, 1HRC), Escherichia coli HO (ChuS, 2HQ2), Pseudomonas aeruginosa HO (Pa-HO, 1SK7), Neisseria meningitidis (HemO, 1J77), IsdG-N7A (2ZDO), IsdI-W66Y, IsdI-heme (3LGN).  48  Another interesting feature in the W66Y structure is the H-bonding network that links the tyrosine side chain to a propionate group through a water molecule (Figure 3-8C). The tyrosine appears to mimic the same interaction of the indolic nitrogen of tryptophan in the wild-type structure. This suggests high affinity heme binding in IsdI may be achieved through anchoring the propionate groups inside the protein, which is seen in other hemoproteins such as hemopexin and cytochromes (77-80).  4.3 ISDI HEME DEGRADATION REACTION Concurrently with this study, HRESIMS and NMR were used to characterize the structure of the in vitro IsdG- and IsdI-mediated heme degradation products (81). The enzymes degrade heme to two oxo-bilirubin isomers, collectively named staphylobilins (Figure 4-2), which were HPLC-purified before structure analysis by NMR. The chromophores are similar to bilirubin in that they are yellow pigments that absorb maximally at ~462 nm (bilirubin absorbs maximally around 440 nm), yet differ by a carbonyl group at the α- or γ-meso carbon positions. Consistent with these findings, an increase in absorbance between 450 – 480 nm was seen during the heme degradation reaction that was more pronounced in the presence of desferrioxamine. This spectral feature could provide a way to monitor reaction progression up until iron release, as the presence of desferrioxamine appears to stabilize the product or intermediate formed. However, further structural characterization of the product formed in the presence of desferrioxamine is still required.  49  Figure 4-2. The substrate and products of IsdG and IsdI heme degradation using ascorbate as the reductant in the presence of catalase. The structure of the staphylobilins revealed information about where the heme ring is cleaved by IsdI. The heme macrocycle is cleaved at the β- or δ-meso carbons, producing the linear tetrapyrroles 5-oxo-δ-bilirubin or 15-oxo-β-bilirubin, respectively (81). This supports the idea that heme ruffling is important for controlling regioselectivity of heme cleavage during the reaction by exposing the β- and δ-meso carbons to the distal side of the heme for oxidative degradation of the heme macrocycle. The staphylobilin structures also reveal that the meso carbon at the site of cleavage is removed during the reaction; however, it is unknown whether CO is released during the reaction similar to the step of α-meso carbon removal during the HO mechanism. Heme ring cleavage at the β- and δ-meso carbon is also in agreement with the alignment of molecular oxygen in two orientations along the β,δ-meso carbon axis of the heme in the crystal structure of heme-bound IsdI. It results in exposure of the β- and δ-meso carbons to the distal face of the heme, while the remaining meso carbons are directed towards the proximal side  50  of the heme face, and thus protected from possible oxygen attack. This is in contrast to HO, where regioselectivity of the enzyme results in heme ring cleavage at the α-meso carbon. In the case of human HO-1, α-cleavage occurs with either heme seating (82). The only known exception is in the case of pa-HO from P. aeruginosa (34). The crystal structure of pa-HO revealed novel δ-regioselectivity of heme oxygenation is due to a ~100° rotation of heme seating within the active site. This places the δ-meso carbon in the position of the α-meso carbon of other HOs. The structure of pa-HO maintains the same overall fold as other bacterial and mammalian HO, including a conserved network of H-bonded solvent molecules important for dioxygen activation. The main interaction in pa-HO that stabilizes the unique heme orientation is a salt bridge between Lys132 and the heme 7-propionate, as well as hydrophobic contacts involving Leu29, Val33, and Phe189 with the heme methyl and vinyl groups (34). Although the IsdI reaction requires dioxygen, it is still unknown how the enzyme activates it to degrade the heme ring to the staphylobilin products. In this study, the IsdI reaction was initiated with ascorbate as the electron donor under aerobic conditions; however, ascorbate was unable to reduce the heme-iron in the absence of dioxygen. It was shown that the ferric IsdIheme complex can be reduced with dithionite in deoxygenated conditions, producing a shift in the Soret from 412 to 427 nm (Figure 3-4), while the same experiment with ascorbate does not result in a spectral shift (data not shown). In accordance with these findings, the reduction midpoint potential of IsdI obtained anaerobically (-88 mV) is more negative than that of ascorbate, while the reduction potential of the catalytic reduction peak seen aerobically is similar to that of ascorbate (83). The distal residue Asn6 may play a role in the initial stages of the reaction with dioxygen. The IsdI-heme structure revealed Asn6 is responsible for stabilizing ligand binding by providing  51  an H-bond to the bound dioxygen species. Furthermore, the equivalent Asn residue in IsdG has been directly implicated in activity, as a standard alanine mutation of the residue renders the enzyme inactive (30). In the IsdI-W66Y-heme structure, there is no distal ligand. The extra room in the heme pocket created from the decrease in heme ruffling allows for Asn6 to rotate away from the heme-iron where it is more difficult to form a hydrogen bond to a distal ligand. This suggests that flattening of the heme ring results in an environment that is not conducive for Asn6 to interact with the distal ligand, possibly contributing to the impaired activity of the W66Y variant. In summary, the IsdI reaction appears to be initiated by reduction of the heme-iron and is likely followed by dioxygen binding distally to the iron, an interaction that is stabilized by an Hbond to Asn6. Finally, the heme ring is cleaved at the β- or δ-meso carbons, which are putatively exposed for oxidative attack by heme ruffling. The reaction produces two staphylobilin isomers through a potential mechanism of dioxygen activation that still needs to be addressed.  52  CHAPTER 5. CONCLUSIONS The structural basis for heme ruffling in IsdI was investigated in this study by comparing the structure of wild-type IsdI to the ruffling variant, W66Y. Ruffling in the variant was lessened from 2.1 Å to 1.4 Å as determined by normal-coordinate analysis, demonstrating that Trp66 is an important contributor to heme distortion in IsdI. The activity of this variant was reduced to half of wild-type revealing a link between heme distortion and enzyme activity in IsdI. Collectively, the results from this structural investigation of IsdI support a role for Trp66 in heme ruffling and activity. The results provide insights into how heme acquired by the Isd system is degraded to staphylobilins, linear tetrapyrroles that differ by β- and δ-meso carbon cleavage of the macrocycle (81). This appears to be achieved through exposure of the β- and δ-meso carbons of the heme to the distal side of the heme pocket for regioselective oxidative degradation. In contrast, the typical product of the classical HO reaction is cleaved at the α-meso carbon to produce biliverdin IXα. The formation of staphylobilins in nearly equal yields appears to result from the ability of the enzyme to attack opposite sides of the heme ring, rather than binding the heme substrate in two alternative orientations. IsdI is a useful model for future studies of the IsdG-family enzymes due to its ease of mutagenesis and crystallization when the experiments are conducted at low temperatures. Future work on the reaction mechanism should involve characterization of intermediates involved in the reaction pathway, which may require further mutagenesis and spectroscopic analysis. Elucidation of the details of the reaction mechanism occurring between initial heme binding and product formation will also contribute to furthering our understanding of how these enzymes degrade heme and contribute to S. aureus pathogenesis. 53  REFERENCES  1.  Rogers, K. L., Fey, P. D., and Rupp, M. E. (2009) Epidemiology of Coagulase-Negative Staphylococci and Infections Caused by These Organisms in Staphylococci in Human Disease, Wiley-Blackwell. pp 310-332  2.  Kluytmans, J., van Belkum, A., and Verbrugh, H. (1997) Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10, 505-520  3.  Gorwitz, R. J., Kruszon-Moran, D., McAllister, S. K., McQuillan, G., McDougal, L. K., Fosheim, G. E., Jensen, B. J., Killgore, G., Tenover, F. 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