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Modulation of heme function by genetic modification of the active site of horse heart myoglobin Hildebrand, Dean Patrick 1996

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M O D U L A T I O N O F H E M E F U N C T I O N B Y G E N E T I C M O D I F I C A T I O N O F T H E A C T I V E S I T E O F H O R S E H E A R T M Y O G L O B I N by Dean Patrick Hildebrand B.Sc, The University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF BIOCHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1996 ® Dean Patrick Hildebrand In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ""i&ooW .^-s^ r-y ° t r^olecxJor T^.QVQ The University of British Columbia Vancouver, Canada Date hNp^ o L i g ABSTRACT Site-directed mutagenesis has been used to construct variants of horse heart myoglobin to probe the structural contributions of specific amino acid residues in the active site of this protein. In particular four groups of variants were examined: (i) The proximal histidine (H93) ligand to the heme iron was substituted with tyrosine and cysteine as occurs in the coordination environments of catalase and cytochrome P-450, respectively, (ii) The distal histidine (H64) was substituted with valine and isoleucine to assess the effects of nonpolar residues on the coordination and ligand binding properties of myoglobin, (iii) Double variants, H64V/H93C and H64I/H93C, were also constructed to study the influence of these substitutions on the ability of the proximal cysteine ligand to ligate to the heme iron, (iv) Insight concerning the functional consequences of increasing the polarity of the distal heme binding pocket was sought through investigation of the V68H and V67A/V68S variants. The influence of mutations on the proximal and distal side of the heme were examined by spectroscopic and electrochemical methods. The electronic, EPR and NMR spectra of the H93 Y ferriMb derivative suggest that the axial ligation of the heme iron in this variant is pentacoordinate, with the heme iron coordinated by a phenolate group provided by the proximal Y93 residue. Unlike the H93Y variant, however, no experimental conditions were identified that allowed quantitative ligation of the proximal cysteine residue to the heme iron in the H93C variant. All of the spectroscopic evidence for the H64V/H93C and H64I/H93C variants, however, supports the assignment of thiolate as the ligand to the heme iron in the oxidized forms of these proteins. With the exception of an additional high-spin component in the electronic absorption spectrum, the V68H Mb variant exhibits absorption maxima similar to those of ferricytochrome b5. Electronic absorption, MCD and EPR spectroscopy of the double variant, V67A/V68S, confirm that the coordination environment of this protein is the same as that of wild-type Mb.The ii increase in active site polarity may account for the enhancement in the rate of coupled oxidation of heme exhibited by the variant upon aerobic exposure to ascorbate relative to that observed for wild-type myoglobin. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x ABBREVIATIONS xii INTRODUCTION A. History of myoglobin 1 B. Physiology of myoglobin 1 C. Structure of myoglobin 2 D. Comparative description of heme environments in various proteins 6 1. Myoglobin 6 2. Peroxidases 8 3. Catalase 10 4. Cytochrome P-450 13 5. Structural comparisons 15 6. Additional examples 16 E. Modulation of heme protein reduction potentials 16 F. Structural and functional studies of myoglobin through site-directed mutagenesis 19 1. Binding of 0 2 and CO to myoglobin 22 2. Characterization of distal heme pocket variants of myoglobin 25 3. Characterization of proximal ligand variants of myoglobin 26 G. Spectroscopic properties of heme proteins 27 iv 1. Oxidation and spin states of heme proteins 28 2. Electronic spectroscopy 32 3. Magnetic circular dichroism (MCD) spectroscopy 34 (i) MCD spectroscopy of ferrous heme centres 35 (ii) MCD spectroscopy of ferric heme centres 36 4. Electron paramagnetic resonance (EPR) spectroscopy 37 5. Axial ligand assignment by an EPR-MCD approach 39 H. Objectives of this work 40 EXPERIMENTAL PROCEDURES A. Site-directed mutagenesis and myoglobin expression 42 B. Purification of myoglobin 43 C. Spectroscopic characterization of variant myoglobins 44 1. Electronic absorption spectroscopy 44 2. Electron paramagnetic resonance (EPR) spectroscopy 45 3. Nuclear magnetic resonance (NMR) spectroscopy 45 4. Magnetic circular dichroism (MCD) spectroscopy 46 5. Fourier transform infrared (FTIR) spectroscopy 46 D. Electrochemical measurements 47 1. Spectroelectrochemical experiments 47 2. Photochemical experiments 48 E. Cyanogen bromide titrations 49 F. Reconstitution experiments 50 v G. Coupled oxidation of myoglobin 50 H. Biliverdin extraction and HPLC analysis 51 RESULTS I. Proximal ligand variants of myoglobin A. The H93Y variant 53 1. Electronic absorption spectroscopy 53 2. Electrochemistry 53 3. EPR spectroscopy 57 4. NMR spectroscopy 57 B. The H64V, H93C and H64V/H93C variants , 60 1. Electronic absorption spectroscopy 60 2. MCD spectroscopy 64 3. NMR spectroscopy 69 4. EPR spectroscopy 71 5. Electrochemistry 73 6. Cyanogen bromide titration 73 7. Reconstitution of H64I/H93C myoglobin 76 8. Unusual pH-dependence of H64V and H64I myoglobin 79 II. Variants designed to increase the polarity of the distal active site of myoglobin A. The V68H variant 82 1. Electronic absorption spectroscopy 82 2. MCD spectroscopy 84 vi 3. EPR spectroscopy 87 4. Electrochemistry 89 5. NMR spectroscopy 89 B. The V67A/V68S variant 92 1. Electronic absorption spectroscopy 92 2. Electrochemistry 95 3. Coupled oxidation of myoglobin 95 DISCUSSION 105 I. Proximal ligand variants A. The H93Y variant 106 1. Spectroscopic considerations 106 2. Structural considerations 108 3. FerrousH93Y myoglobin and catalase activity I l l B. The H64V, H93C and H64V/H93C variants 112 1. Comparison of horse and human H93C myoglobin 112 2. Trans effects on proximal coordination by Cys93 115 3. Nature of the Cys93-heme iron bond 115 4. Unusual pH-dependence of H64V and H64I myoglobin 116 II. Variants designed to increase the polarity of the distal heme binding site A. V68H myoglobin 120 1. Spectroscopic considerations and ligand binding 121 2. Structural considerations concerning the nature of the V68H bond 122 vii B. The V67A/V68S variant 125 1. Structural considerations 125 2. Coupled oxidation reaction of myoglobin 127 3. FTIR analysis of carbonyl myoglobin 129 4. Structural similarities between myoglobin and heme oxygenase 131 III. Conclusions 132 BIBLIOGRAPHY 134 viii LIST OF TABLES 1. Proximal and distal variants of myoglobin 20 2. Mutagenic oligonucleotides 42 3. Electronic absorption maxima and molar absorbances for wild-type and H93 Y myoglobin . 55 4. Electronic absorption maxima and molar absorbances for wild-type, H64V, H93C and H64V/H93C myoglobin 62 5. Electronic absorption maxima and molar absorbances for wild-type and V67A/V68S myoglobin 93 ix LIST OF FIGURES 1. Three-dimensional structure of horse heart myoglobin 4 2. Amino acid sequence of horse heart myoglobin 5 3. Active site structures of cytochrome c peroxidase and myoglobin 7 4. Active sites of bovine liver catalase and cytochrome P-450 12 5. Spin-states of ferric and ferrous heme iron 30 6. Electronic absorption spectra of the H93 Y Mb variant 54 7. Spectroelectrochemical titration of the H93 Y Mb variant 56 8. EPR spectra of the H93Y Mb variant 58 9. NMR spectrum of the H93Y Mb variant 59 10. Electronic absorption spectra of the H93C Mb variant 61 11. MCD spectra of oxidized wild-type, H93 C, and H64 V/H93 CMb 65 12. MCD spectra of reduced wild-type, H93C, and H64V/H93C Mb 66 13. MCD spectrum of oxidized H64V Mb 68 14. NMR spectra of wild-type, H64V, H93C, and H64V/H93C Mb 70 15. EPR spectra of wild-type, H64V, H93C, and H64V/H93C Mb 72 16. Spectroelectrochemical titration of the H64V/H93C Mb variant 74 17. Spectroelectrochemical titration of the H64V Mb variant 75 18. CNBr titration of myoglobin 77 19. Heme binding to the H64I/H93C Mb variant 78 20. pH titration of the H64V Mb variant 80 21. pH dependence of the EPR spectrum of the H64V Mb variant 81 22. Electronic absorption spectra of the V68H Mb variant 83 x 23. NIR-MCD of the V68H Mb variant 85 24. Visible MCD spectra of the V68H Mb variant 86 25. EPR spectrum of the V68H Mb variant 88 26. Spectroelectrochemical titration of the V68H Mb variant 90 27. NMR spectrum of the V68H Mb variant 91 28. Visible MCD spectrum of the V67A/V68S Mb variant 94 29. pH titration of the V67A/V68S Mb variant 96 30. Spectroelectrochemical titration of the V67A/V68S Mb variant 97 31. Electronic absorption spectra of myoglobin in the presence of ascorbate 98 32. Kinetics of the coupled oxidation reaction 99 33. Electronic absorption spectra of myoglobin in the presence of ascorbate 101 34. HPLC analysis of coupled oxidation reaction products 102 35. FTIR of the carbonyl derivative of myoglobin 104 36. Crystal structure of the H93Y Mb variant 109 37. Proposed reaction scheme for the coupled oxidation reaction of myoglobin 128 xi ABBREVIATIONS B L C bovine liver catalase CaCl 2 calcium chloride CaF 2 calcium fluoride CcP cytochrome c peroxidase cam camphor C H 3 O H methanol CNBr cyanogen bromide deoxyMb deoxymyoglobin D M E dimethylester DMSO dimethyl sulfoxide D N A deoxyribonucleic acid D T T dithiothreitol Em midpoint reduction potential E D T A ethylenediamine tetraacetic acid EPR electron paramagnetic resonance FTIR Fourier transform infrared Hb hemoglobin HC1 hydrochloric acid HPLC high pressure liquid chromatography Im imidazole KC1 potassium chloride Mb myoglobin xii Mb0 2 ; oxyMb oxymyoglobin MCD magnetic circular dichroism metMb metmyoglobin MgCl 2 magnesium chloride mV millivolt NaCI sodium chloride NaHC0 3 sodium bicarbonate NaPi sodium phosphate NH 4 H 2 P0 4 ammonium phosphate NIR-CT near infrared charge transfer NMR nuclear magnetic resonance nm nanometer OTTLE optically transparent thin-layered electrode ox oxidized [Fe(III)] QMAIS quantum mechanically admixed intermediate spin-state red reduced [Fe(II)] SCE saturated calomel electrode SHE standard hydrogen electrode T Tesla Tris tris(hydroxymethyl)aminoethane UV ultraviolet xm INTRODUCTION A. History of myoglobin Scientific interest in myoglobin was initiated almost two centuries ago with the discovery of a red pigment associated with muscle tissue that appeared distinct from that found in the circulatory system. The first spectroscopic experiments concerning myoglobin appeared in the late nineteenth century and reported the electronic spectrum of this pigment was slightly different from that of hemoglobin (Kuhne, 1865; Morner, 1897; Kagen, 1973). Morner (1867) suggested the name myoglobin for the muscle pigment, to emphasize that it is distinct from, yet related to hemoglobin. As late as the early 20th century opinion was still divided concerning the distinction between myoglobin and hemoglobin. This uncertainty was based on the possibility that the slight spectral differences observed were actually artifacts of the extraction procedure. This issue was settled in the 1930s with spectrophotometry of intact muscle tissue and the crystallization of myoglobin from horse heart muscle. Adair's investigation of hemoglobin (1923) and Theorell's (1934) studies of myoglobin demonstrated the difference in molecular weight and iron content between the two globins. Theorell j performed the first oxygen saturation experiments that demonstrated the hyperbolic nature of oxygen binding to myoglobin, compared to the sigmoidal nature of hemoglobin binding curves (Barcroft & Roberts, 1909; Hill, 1910). Analogous experiments were reported by Hill (1936) for oxygen binding to the canine globins and thus firmly established the difference in oxygen binding affinities between myoglobin and hemoglobin. This important functional difference between these two proteins would later prove to be the key to understanding their physiological roles. B. Physiology of myoglobin Myoglobin is a small heme protein which functions in the reversible binding of dioxygen. Myoglobin is located within muscle cells and is found in species ranging from mollusc to man. The 1 distribution and amount of myoglobin present varies between muscle types, but generally appears in larger concentrations in muscles used for slower, repetitive contractions (Millikan, 1939). A good source, for example, is that of horse heart where myoglobin occurs in concentrations ranging from 2.5-6.0 mg/g wet weight (Kagen, 1973). The myoglobin content of skeletal muscle from deep-sea diving mammals such as sperm whales is -90 mg/g of tissue (Scholander, 1940). This extremely high concentration makes the musculature almost black in color (Kendrew et al., 1954). Originally, the role of myoglobin was assumed to be one of storage, but today it is clear that the function of long-term oxygen storage is more relevant in diving animals in which the blood supply of oxygen is severely limited. A more active role for myoglobin has been suggested in mechanisms that facilitate oxygen entry into the cell and diffusion from the plasma membrane to the mitochondria (Wittenberg, 1970; Wittenberg & Wittenberg, 1987; Jelicks & Wittenberg, 1995). Due to the low solubility of oxygen in solution, a facilitated diffusion mechanism may enhance the rate of oxygen flux to the mitochondrial regions (Wittenberg, 1970). Myoglobin must be maintained in the reduced form to be physiologically active, as this is the only state that permits oxygen binding. C. Structure of myoglobin Myoglobin is one of the most thoroughly characterized proteins, owing in part to the fact that metmyoglobin from sperm whale was the first protein for which a crystallographically determined three-dimensional structure was reported (Kendrew et al., 1958, 1960). Subsequent studies have expanded the number of refined structures to include met-, deoxy- (Takano, 1977) and oxy- (Phillips, 1978) sperm whale myoglobin, as well as met- (Evans & Brayer, 1988, 1990) horse heart myoglobin. The met- and deoxy- prefixes refer to the ferric and ferrous-unligated forms of the protein, respectively. Synthetic genes encoding recombinant myoglobins have been expressed in Escherichia coli (Varadarajan et al., 1985; Springer & Sligar, 1987; Guillemette et al., 1991) in quantities 2 sufficient for the crystallographic structural analysis of sperm whale (Phillips et al., 1990) and horse heart myoglobin (Maurus et al.). Structural comparisons between the recombinant and natural proteins reveal only minor changes in main chain atom and surface side chain positions, as well as variability in the processing of the N-terminal methionine. These structures provide the starting points for comprehensive structural and functional analyses of myoglobin variants produced by site-directed mutagenesis. The 1.9 A resolution structure of horse heart myoglobin (Evans & Brayer, 1990; Figure 1) reveals a protein tertiary structure very similar to that of the related protein from sperm whale, as would be expected from the high degree of sequence identity (89 %) between the two species (Figure 2). The polypeptide chain component consists of 153 amino acids (MW = 17,568), over 80 % of which occur in eight helical segments (designated A-H). Of these, seven are 3-613 (a-helix) helices and one is a 3 1 0 helix. The heme moiety, iron-protoporphyrin IX, acts as a cofactor for this class of protein and is buried within the active site fold of the globin, with only -18 % of the total heme surface exposed to external solvent (Stellwagan, 1978). This heme group is associated with the protein through van der Waals contacts within an apolar cleft formed between helices B, C and E to G. The a-meso position of the porphyrin is buried furthest into the binding cleft. Changes in overall folding are imparted by heme binding, as seen by the large difference in apparent molecular weights of apo and holomyoglobin on size exclusion columns. In addition, thermal denaturation studies reveal a significant decrease in melting point upon removal of heme (T1 / 2 ~ 70 °C and 80 °C at pH 7.0, respectively; personal observations). The central iron atom of the heme group is coordinated in a distorted octahedral geometry, with four ligands provided by the pyrrole nitrogens of the porphyrin ring and a fifth by the NE2 group of the proximal histidine (H93). The sixth coordination position is exchangeable. In metmyoglobin 3 Figure 1: Stereo drawing of the a-carbon backbone chain of horse heart myoglobin. Every tenth amino acid is labeled. 4 1 (a) G-L-S-D-G-E-T-Q-Q-V-L-N-V-T-G-K-V-E-A-D (b) V E L H A (c) L 21 (a) I-A-G-H-G-Q-E-V-L-I-R-L-F-T-G-H-P-E-T-L (b) V D-I K-S (c) F K 41 (a) E-K-F-D-K-F-K-H-L-K-T-E-A-E-M-K-A-S-E-D (b) R (c) S D 61 (a) L-K-K-H-G-T-V-V-L-T-A-L-G-G-I-L-K-K-K-G (b) V-T A (c) A-T 81 (a) H-H-E-A-E-L-K-P-L-A-Q-S-H-A-T-K-H-K-I-P (b) (c) I 101 (a) I-K-Y-L-E-F-I-S-D-A-I-I-H-V-L-H-S-K-H-P (b) E R (c) V C Q Q 121 (a) G-D-F-G-A-D-A-Q-G-A-M-T-K-A-L-E-L-F-R-N (b) N K (c) N K 141 (a) D-I-A-A-K-Y-K-E-L-G-F-Q-G (b) Y (c) M S-Q Figure 2: Amino acid sequence alignment of (a) horse (Dautrevaux et al., 1969), (b) sperm whale (Edmundson, 1965) and (c) human (Romero Herrera & Lehmann, 1971) myoglobin. Only the residues that differ from the horse sequence are listed. 5 this position is occupied by a water molecule (Wat 156), whereas in the reduced form this site is either unoccupied (deoxyMb) or bound by dioxygen (oxyMb). The water ligand present in metMb is stabilized by a hydrogen bond to the NE2 of the distal histidine (H64). In metmyoglobin the porphyrin ring is slightly distorted due to non-coplanarity of the pyrrole rings. The heme iron atom is found to reside essentially in the plane defined by the four pyrrole nitrogen atoms (Evans & Brayer, 1990). D. Comparative description of heme environments in various proteins Heme proteins as a class possess a wide range of biological functions that are as varied as dioxygen activation (eg. Mb, Hb) to electron transfer (eg. cytochromes). As a result the manner in which the environment provided by the active sites of these proteins dictate the chemical reactivity of the heme prosthetic group is of fundamental interest. With the use of available crystallographic information, several suggestions concerning this issue have been proposed (e.g. Poulos & Finzel, 1984; Dawson, 1988; Poulos, 1988; English, 1994). 1. Myoglobin The active sites of oxidized horse heart myoglobin and cytochrome c peroxidase (CcP) are compared in Figure 3. The heme group of myoglobin contains a high-spin, hexacoordinate iron with a proximal histidine (H93) axial ligand and a distal water ligand (Watl56). The imidazole ring of the distal histidine (H64) is perpendicular to the heme plane and is hydrogen bonded to Watl56. Note that the ligand binding site on the distal side of the heme is flanked on either side by two apolar residues, F43 and V68. Analysis of the active site structure shows no clear path linking it to the external solvent so that this region can be regarded as relatively hydrophobic. A network of hydrogen bonds exists on the proximal side of the heme that includes H93, S92, 189, H97 and heme propionate A. The hydroxyl group of S92 is hydrogen bonded to the carboxylate 6 Figure 3. Structural comparison of the active sites of yeast cytochrome c peroxidase (Finzel et al., 1984) and horse heart myoglobin (Evans & Brayer, 1990). Hydrogen bonding networks are shown with dotted lines. In the myoglobin structure, the hydrogen bond involving the side-chain carbonyl of L89 to H93 has been omitted for clarity. 7 of propionate A, the side-chain carbonyl of L89, as well as to ND1 of H93. The ND1 group of H93 is also hydrogen bonded to the main chain carbonyl of L89. The interactions between S92, L89 and H93 presumably help orient the proximal histidine. The other carboxyl oxygen of propionate A is hydrogen bonded to NE2 of H97. 2. Peroxidases The peroxidase family of heme proteins, as typified by yeast cytochrome c peroxidase, catalyzes substrate oxidation with the concomitant reduction of hydrogen peroxide (Frew & Jones, 1984; English, 1994). A general reaction scheme for peroxidases can be described as: H202 + 2HA - 2H20 + 2A where A represents the substrate that is oxidized. The range of oxidizable substrates used by peroxidases varies significantly and determines the physiological function of the peroxidase. In the case of CcP, two molecules of ferrocytochrome c are oxidized per molecule of hydrogen peroxide reduced. Other important examples include the fungal peroxidases from Phanerochaete chrysosporium, lignin peroxidase and manganese peroxidase. Although the true substrate of lignin peroxidase is uncertain, manganese peroxidase catalyzes the oxidation of Mn(II) to Mn (III) by hydrogen peroxide and is believed to be involved in the oxidation of lignin. Myeloperoxidase, a mammalian peroxidase present in neutrophils, catalyzes the oxidation of chloride ions as an antimicrobial defense mechanism. Thus, reduction of hydrogen peroxide is variously associated with the antioxidant, biotransformation and defensive functions of heme peroxidases. Although the physiological functions of the various peroxidases differ considerably, their reactions with hydrogen peroxide are less diverse and involve heterolytic cleavage of the peroxide bond to produce a reactive intermediate. The initial step involves binding of a peroxide molecule to 8 the ferric ion followed by oxidation of the protein to yield an intermediate referred to as compound I. Thus, one electron comes from Fe(III) while the other comes from the porphyrin ring, to produce an oxyferryl species and a porphyrin cation radical (X-Fe(IV)=0) (Blumberg et al., 1968; Dolphin et al., 1971; Hewson & Hager, 1979). Compound I of CcP, however, is unusual in that the radical centre is located on the sidechain of W191 instead of the porphyrin ring (Sivaraja et al., 1989). Subsequent oxidation of the substrate(s) reduces compound I and returns the enzyme to its resting Fe(III) state. Yeast CcP is one of the best studied peroxidases because it was the first for which a high-resolution crystal structure was reported (Finzel et al, 1984). CcP consists of a single polypeptide containing 294 amino acids (34 kDa) that is folded into distinct N and C-terminal domains with the heme group located at the interface. Approximately 50% of the sequence is arranged into 10 a-helical segments and 12% exists as p-sheet structure. Although the heme is completely buried within the protein, with the closest edge being approximately 10 A from the molecular surface, a large channel is present (5 A deep and 10 A long) on the distal side of the heme that presumably allows access to solvent and/or substrate molecules. As Figure 3 illustrates, the heme iron of CcP is coordinated to a histidyl residue (H175) while the distal side is dominated by polar residues (R48, W51 and H52) and several well ordered water molecules. The heme iron is normally pentacoordinate (high-spin), although the ligation state has been shown to be pH and temperature sensitive (Yonetani & Anni, 1987). An extensive network of hydrogen bonds occurs in CcP that involves active site residues and ordered water molecules (Figure 3). The distal residues R48, W51 and H52 form a large ligation pocket for peroxide, and are hydrogen bonded to three water molecules, which are presumably displaced upon ligand entry. A position analogous to the distal histidine of myoglobin is occupied by 9 the H52 residue. Unlike myoglobin, the propionates of CcP are fully extended and are not exposed to the surface of the protein, being held in place by a network of hydrogen bonds. The proximal side of the heme also contains an extensive hydrogen bond network important for the catalytic function of CcP, involving D235, H175, W191 and a water molecule. The carboxylate of D235 is hydrogen bonded to ND1 of H175, the indole nitrogen of W191 and an internal water molecule, and is therefore critical for maintaining the proper orientation of these residues. Lignin peroxidase (LiP) (Poulos et al., 1993) and manganese peroxidase (MnP) (Sundaramoorthy et al., 1994) from Phanerochaete chrysosporium possess tertiary structures similar to that of CcP, despite sequence identities of <20 % between the fungal enzymes and CcP. The active site structures of LiP, MnP and CcP differ primarily in the substitution of phenylalanine residues in the fungal enzymes at positions analogous to W51 and W191 of CcP. A phenyalanine on the proximal side of the heme in LiP and MnP may contribute to the location of the cation radical on the porphyrin ring rather than on the protein. The presence of a tryptophan residue on the proximal side of the heme, however, does not necessarily result in an indole-containing radical as part of compound I. For example, ascorbate peroxidase (Patterson et al., 1995) contains a tryptophan residue in a position analogous to W191 of CcP, but unlike compound I of CcP, this enzyme contains a porphyrin radical. 3. Catalase Catalase is found in nearly all aerobic organisms and acts as an antioxidant by catalyzing the dismutation of hydrogen peroxide according to the following reaction: 2H202 - 02 + 2H20 The reaction of catalase with hydrogen peroxide proceeds by a mechanism similar to that of the 10 peroxidases. Catalase catalysis involves the heterolytic cleavage of the peroxide bond to form compound I (Fita & Rossmann, 1985). The reduction of compound I, however, is achieved by the oxidation of a second molecule of hydrogen peroxide rather than by oxidation of another substrate (Schonbaum & Chance, 1976). Like many of the peroxidases, catalase compound I is an oxyferryl intermediate with the cation radical contained on the porphyrin ring (Araiso et al., 1976). Crystal structures are available for Penicillium vitale (Vainshtein et al., 1986), Micrococcus lysodeikticus (Yusifov et al., 1989), HPII of Escherichia coli (Bravo et al., 1995) and bovine liver catalase (Murthy et al., 1981; Fita & Rossmann, 1985). Bovine liver catalase (BLC) is a tetramer of molecular weight 230 kDa with each of the identical, 509 residue subunits binding a single heme group. The core of each subunit is an eight stranded p-barrel comprised of two similar, four-stranded antiparallel P-sheets and represents the largest of the four domains in each subunit. The remaining three domains are constructed primarily of a-helices and coil. The heme group in catalase is completely buried within the protein, approximately 20 A from the molecular surface, to which it is connected by a relatively hydrophobic channel. Presumably, peroxides gain access to the active site via this channel (Fita & Rossmann, 1985). A closer look at the active site of bovine liver catalase (Figure 4) reveals a pentacoordinate heme, ligated by the phenolic hydroxyl group of Y357. There is no evidence of a water molecule in the sixth ligation position of the iron. Like the peroxidases, the propionate groups of BLC are part of a network of hydrogen bonds and have no access to the surface of the protein. The distal side of the heme is fairly hydrophobic containing a valine (V73) and two phenylalanines (F152 and F160) in close proximity to the iron. There is also an asparagine (N147) and a histidine (H74) that are similar to R48 and H52 of CcP. The distal histidine of catalase (H74), unlike CcP and myoglobin, lies almost parallel to the heme group. On the proximal side, Y357 is within hydrogen bonding distance 11 Phe-87 Cytochrome-P450 Catalase Figure 4: Structural comparison of the active sites of bovine liver catalase (Murthy et al., 1981) and cytochrome P-450 from Pseudomonasputida (Poulos et al., 1986). Hydrogen bonding networks are shown as dotted lines. Camphor (cam) is shown in the active site of P-450. 12 of R353, the presence of which may lower the p ^ of the phenolic hydroxyl group to allow phenolate binding to the iron. 4. Cytochrome P-450 The cytochromes P-450 are members of a large class of monooxygenases found in plants, animals and microorganisms (Ortiz de Montellano, 1986; Omura, 1993) that catalyze the hydroxylation of aliphatic and aromatic substrates in a wide range of biologically important processes. The net reaction catalyzed by this enzyme can be written as: RH + 02- ROH + H20 where R represents the substrate to be hydroxylated. The electrons for the reaction are supplied by electron transfer proteins, the identities of which depend on the source of the P-450. Because many of these enzymes are bound to microsomal membranes, their characterization has proven difficult. However, studies involving the soluble cytochrome P-450 from the bacterium Pseudomonasputida have provided a wealth of structural and mechanistic information. This bacterial P-450 (P-450cam) is part of the pathway responsible for the utilization of camphor as a carbon source and uses an iron-sulfur protein, putidaredoxin, as its source of electrons (Katigiri et al., 1968; Horiuchi et al., 1993). The cytochrome P-450 reaction scheme is more complex than that of the peroxidases, but similar intermediates (e.g. ferric peroxide bound and compound I intermediates) have been proposed, but not necessarily identified in the enzymic reaction (Imai et al., 1989). The oxygen-bound form is the last isolatable intermediate in the reaction cycle (Peterson et al., 1972). The fact that hydrogen peroxide can support the P-450 hydroxylation mechanism by circumventing the need for molecular oxygen and reducing equivalents suggests that, like the peroxidases, a ferric-bound peroxide intermediate is involved in the mechanism. Although cytochromes P-450 bind oxygen in a manner 13 similar to that of the globins (Eisenstein et al., 1977), the active site structure is conducive to oxygen activation. These enzymes catalyze the heterolytic cleavage of the dioxygen bond to form a reactive intermediate leading to substrate hydroxylation. Crystal structures are available for the bacterial cytochrome P-450cam in both the camphor-bound and camphor-free forms (Poulos et al., 1986; 1987). P-450cam is a monomer containing 414 residues arranged in -50 % helical structure and -25 % antiparallel P-sheet. Each monomer contains a single heme group located between two helical segments and is completely buried within the protein matrix. A channel to the molecular surface is located on the distal side of the heme, but it appears to be too small to allow substrate entry into the active site. A closer look at the active site of P-450^ (Figure 4) reveals that the ligation to the heme iron is provided by an axial cysteine residue (C357). In the presence of camphor, which binds in close proximity to the distal side of the heme, the iron is pentacoordinate due to the vacancy of the sixth coordination position. Hydrophobic contacts with F87, L244, V247 and V295 provide a suitable binding site for camphor and presumably force the correct orientation for the substrate to react with an adjacent, activated oxygen atom bound as the sixth ligand to the heme iron. In addition, a hydrogen bond is formed between the carbonyl oxygen of camphor and the hydroxyl of Y96. In the camphor-free form, the overall tertiary structure of P-450cam is preserved; however, the camphor binding site is occupied by a hydrogen-bonded network of five ordered water molecules. One of these waters acts as the sixth ligand to the iron, resulting in a hexacoordinate (low spin) heme. A threonyl residue (T252) is located adjacent to the ligand binding site and may stabilize bound oxygen through a hydrogen-bonding interaction. This residue may also act as part of a complex proton-relay system involving D251 and additional basic residues, which protonate the oxygen ligand prior to bond scission (Gerber & Sligar, 1994). Substitution of D252 with aliphatic residues greatly reduces the 14 monooxygenase activity of the enzyme (Imai et al., 1989). 5. Structural comparisons Comparison of the active sites of structurally characterized heme enzymes provides insight into how structural features dictate the chemical reactivity of the heme iron centre. One obvious difference between these proteins is the nature of the proximal ligand provided to the heme iron. Although the globins and the peroxidases both contain a proximal histidine ligand, subtle differences in the nature of the hydrogen bond to ND1 exist between these two classes of heme protein. The anionic carboxylate group (D235) in CcP is a better hydrogen bond acceptor group than the neutral carbonyl (L89) and hydroxyl group (S92) of myoglobin. As a result the proximal histidine of CcP is considerably more negative and possesses greater imidazolate character than that of myoglobin. The proximal tyrosine of catalase and cysteine of cytochrome P-450 are deprotonated and occur as the phenolate and thiolate forms, respectively. Thus, a common feature among these heme enzymes is the substantial anionic nature of the proximal ligands. Such ligands increase the electron density at the iron which in turn stabilizes the higher oxidation states of the iron and may also facilitate the heterolytic cleavage of the peroxide bond. Some notable differences between these enzymes can also be identified on the distal side of the heme. Unlike myoglobin, CcP and catalase both contain polar residues (R48 and N147, respectively) in addition to the distal histidine. The added polarity of these residues may stabilize the separation of charge that develops during peroxide bond scission. Other than a threonine residue (T252), the distal side of cytochrome P-450 is hydrophobic, so the electron-donating power of the proximal thiolate may be sufficient for the same 0-0 bond cleavage. Another striking difference between myoglobin and the heme enzymes is their significant variation in size. CcP, catalase and P-450 are much larger than myoglobin, a characteristic that is 15 accompanied by the sequestering of the heme completely within the protein matrix. By protecting the heme groups from bulk solvent, the enzymes may minimize potential damage resulting from formation of the highly reactive intermediate, compound I, produced during the reaction cycle. 6. Additional examples The cytochromes are a class of heme proteins responsible for many essential electron transfer functions found in biology, and they have been shown to vary considerably in the nature of their heme iron coordination environment (Moore & Pettigrew, 1990). The axial ligands of the heme groups of cytochromes are provided by the protein, with variations of these ligands "tuning" the Fe(III)^Fe(II) reduction potential to suit the physiological role of the protein. The crystal structure of yeast iso-1-cytochrome c (Louie et al., 1988), for example, shows that the iron is coordinated by a histidine and a methionine, whereas cytochrome b5 contains bishistidine axial ligation (Mathews et al., 1972, 1979). Recently, a unique coordination environment was reported for cytochrome/(Martinez et al., 1994). Unexpectedly, the heme iron in this cytochrome was shown to be coordinated by a histidine residue and the a-amino group of the N-terminus. Unlike the previous examples, the heme groups of the cytochromes almost always possess a low-spin iron, and the heme prosthetic groups are partially exposed to solvent. One exception to this are the cytochromes c', which possess heme iron in an intermediate or spin admixed state (see below). E. Modulation of heme protein reduction potentials The relationship governing the reduction potential of an electroactive species is given by the Nernst equation: E = E° + ^ l n i ^ L m 16 where Am and Ared represent the concentration of oxidized and reduced species, respectively, E is the potential of the system, E° is the standard reduction potential, R is the gas constant, T is the temperature, n is the number of electrons transferred, and F is the Faraday constant. In biological systems, E is the solution potential relative to the standard hydrogen electrode (SHE) and E° becomes Em the midpoint reduction potential. The solution potential at which the concentrations of oxidized and reduced species are equal is defined as the midpoint potential. Because the conditions required for working with proteins are typically non-standard, the pH, ionic strength and temperature relevant to determination of the midpoint potential must be stated explicitly. The midpoint potential represents the relative stability of the oxidized and the reduced species and the tendency of the redox-active centre to donate (or accept) electrons. From the Nernst equation it is apparent that conditions that stabilize the oxidized species or destabilize the reduced species will decrease the midpoint potential. In the case of heme proteins, the net charge of the Fe(II) heme centre is 0, and the net charge of the Fe(III) centre is +1 because the net electrostatic charge of the metal-free porphyrin core is -2. Therefore, an environment of high dielectric constant stabilizes Fe(III) relative to Fe(II), while a low dielectric favors Fe(II) (Kassner, 1972, 1973). The magnitude of the influence of the apoprotein-heme interaction on the electrochemical behavior of the heme iron is demonstrated by the range of Em values observed for heme proteins. For example, cytochrome c2 from Rhodospirillum rubrum has been reported to exhibit a reduction potential of +320 mV vs. SHE (Schlauder & Kassner, 1979) while the heme iron of catalase is estimated to be <-500 mV vs. SHE (Williams, 1974). Although the effect of dielectric constant on the heme iron centre is probably the single most important determinant of heme protein electrochemical behavior (Moore et al., 1986), several other important structural factors have been shown to affect the reduction potentials of heme proteins. Among the most 17 important of these factors is the identity of the axial ligands (Moore & Williams, 1977). Quantitative characterization of the magnitude of individual structural characteristics to the electrochemical behavior of heme proteins is complicated by the difficulty in identifying naturally-occurring proteins that differ in just one relevant structural feature. For this reason, comparative studies of cytochromes or heme enzymes from various species are limited by the fact that most relevant proteins differ in several critical structural aspects. Nevertheless, such studies in combination with studies of model heme complexes have enjoyed some success in identifying the relatively large effects of axial ligands on the reduction potentials of heme proteins (e.g., Moore & Williams, 1977; Wilgus et al., 1978). Based on such analyses, axial ligation by a methionyl residue is expected to increase the reduction potential of a heme iron centre if other factors remain constant. Replacement of methionine with histidine or lysine is expected to result in progressively greater decreases in potential. More recently, semisynthetic forms of cytochrome c in which the normal M80 axial ligand to the heme iron has been replaced with a variety of non-naturally occurring amino acid residues have provided somewhat more satisfying experimental characterization of the magnitude of axial ligand contribution to the reduction potential of this protein (Wallace & Clark-Lewis, 1992). This latter study, however, is limited by the absence of rigorous characterization of active site structure induced by these modifications and by the use of equilibrium techniques for the determination of midpoint potentials. In such studies, use of direct electrochemical methods is preferable owing to the potential existence of, and contributions from, more than one conformer of the active site that may result from the destabilizing effect of replacing M80 (e.g., Barker & Mauk, 1992). In the case of heme enzymes, the anionic character of the proximal ligand to the heme iron decreases the reduction potential of these proteins further insofar as the increased electron density 18 of anionic ligands stabilizes the Fe(III) state. Cytochrome P-450, with a proximal cysteine residue, exhibits midpoint potentials of -170 and -270 mV vs. SHE in the absence and presence of camphor, respectively (Gunsalus et al., 1974). Although both CcP and myoglobin possess proximal histidine residues, the midpoint potential of the peroxidase (Em = -190 mV (Conroy et al., 1978)) is significantly lower than that of myoglobin (Em = 61 mV (Lim, 1990)) at least in part as the result of the greater strength of the hydrogen bond formed between D235 and ND1 of the proximal histidine residue relative to the hydrogen bond formed between H93, the backbone carbonyl (189), and the hydroxly group (S92) of myoglobin (Valentine et al., 1979; O'Brien & Sweigart, 1985). This interpretation is supported by the observation that the D235A variant of CcP exhibits a reduction potential that is 100 mV greater than that of the wild-type enzyme (Goodin et al., 1993). The lower potentials observed for CcP, P-450, and catalase presumably contribute to the stabilization of the higher oxidation states of the iron that are involved in the catalytic mechanism of these enzymes. F. Structural and functional studies of myoglobin through site-directed mutagenesis With the advent of generally applicable methods for use of site-directed mutagenesis in the study of protein function (Zoller & Smith, 1982, 1983), the difficulty in obtaining proteins that differ by one amino acid residue are largely eliminated, and the investigation of protein structure and function has entered a new era. The first metalloproteins that were studied in this manner were hemoglobin (Nagai et al., 1985), cytochrome c (Pielak et al., 1985), CcP (Goodin et al., 1986), and myoglobin (Braunstein et al., 1988; Springer et al., 1989). In the case of myoglobin, access to specifically modified proteins permitted detailed investigation of mechanistic models of ligand binding that had previously been resistant to such study. A representative summary of the extensive work concerning structural and functional characterization of active site variants of myoglobin from various species is provided in Table 1. 19 Table 1: Proximal and distal variants of myoglobin* Proximal variants Mutant Species Structure Selected References H93Y hh Y Hildebrand et al., 1995 H93Y SW N Egeberg et al., 1990; Morikis et al., 1990 H93Y hu N Adachietal., 1991, 1993 H93C hh N Hildebrand et al., 1995 H93C hu N Adachi et al., 1991, 1993 H93G SW Y DePillis et al., 1994; Banrick et al., 1994'" ; Decatur et al., 1995 Distal variants Mutant Species Structure Selected References H64Y hh Y Maurus et al., 1994"; Tang et al., 1994 H64Y SW Y Springer et al., 1989; Egeberg et al., 1990; Morikis et al., 1990; Braunstein et al., 1993; Brancaccio et al., 1994; Hargrove et al., 1994*; Li et al., 1994; Pin et al., 1994 H64A SW N Brantley et al., 1993; Hargrove et al., 1994; Li et al., 1994; Balasubramanian et al., 1994; Brancaccio et al., 1994; La Mar et al., 1994 H64A hu N Balasubramanian et al., 1993; Sakan et al., 1993; Lambright et al., 1993,1994; Brancaccio et al., 1994 H64G SW Y Braunstein et al., 1988,1993; Morikis et al., 1989,1990; Springer et al., 1989; Carver et al., 1990; Egeberg et al., 1990; Rohlfs et al., 1990; Rajarathnam et al., 1991,1992; Bormett et al., 1992; Brantley et al., 1993; Quillin et al., 1993'; Brancaccio et all, 1994; Carlson et al., 1994; La Mar et al., 1994; Li, 1994 H64G hu N Sakan et al., 1993; Brancaccio et al., 1994; H64V hh N Hildebrand etal., 1995 H64V SW Y Springer et al., 1989; Carver et al., 1990; Morikis et al., 1990; Rohlfs et al., 1990; Cutruzolla et al., 1991; Rajarathnam et al., 1991; Allocatelli et al., 1993; Brantley et al., 1993; Quillin et al., 1993'; Qin et al., 1993; Park et al., 1991; Rizzi et al., 1993; Rao et al., 1993; Braunstein et al., 1993; Brancaccio et al., 199.4; La Mar et al., 1994; Li et al., 1994 H64V po N Biram et al., 1993; Brantley et al., 1993; Cameron et al., 1993; Brancaccio et al., 1994 H64V hu N Ikedo-Saito et al., 1991,1992; Balasubramanian et al., 1993,1994; Sakan et al., 1993; Lambright et al., 1993,1994; Brancaccio et al., 1994 H64L SW Y Carver et al., 1990; Rohlfs et al., 1990; Brantley et al., 1993; Quillin et al., 1993"; Li et al., 1994; Balasubramanian et al., 1994; Brancaccio et al., 1994; La Mar et al., 1994 H64L hu N Ikedo-Saito et al., 1991,1992; Balasubramanian et al., 1993; Sakan et al., 1993; Lambright et al., 1993,1994; Brancaccio et al., 1994; Li et al., 1994 H64I hh N Bogumil et al., 1994, 1995; Hildebrand et al., 1995 H64I SW N Brancaccio et al., 1994; La Mar et al., 1994; Li et al., 1994 2o H64I hu N Sakan et al., 1993; Brancaccio et al., 1994 H64T hh Y Bogumil et al„ 1994,1995' H64T sw Y Springer et al., 1989; Brantley et al., 1993; Quillin et al., 1993"; Brancaccio et al., 1994; L i et al., 1994 H64Q sw Y Carver et al., 1990; Rohlfs et al., 1990; Rajarathnam et al., 1991; Brantley et al., 1993; Quillin et al., 1993'; Balasubramanian et al., 1994; Brancaccio et al., 1994; L a Mar et al., 1994; L i et al., 1994 H64Q hu N Ikedo-Saito et al., 1990,1991; Balasubramanian et al., 1993; Lambright et al., 1993,1994; Brancaccio et al., 1994; Sakan et al., 1993; Petrich et al., 1994 H64F sw N Springer et al., 1989; Carver et al., 1990; Morikis et al., 1990; Rohlfs et al., 1990; Park et al., 1991; Rajarathnam et al., 1991; Braunstein et al., 1993; Brancaccio et al., 1994; L i et al., 1994 H 6 4 M sw N Springer et al., 1989; Morikis et al., 1989,1990; Rohlfs et al., 1990; Braunstein et al., 1993; L i et al., 1994 H64R sw N Springer et al., 1989; Morikis et al., 1990; Rohlfs et al., 1990; Braunstein et al., 1993 H64R bo N Shimada e ta l , 1989 H64K hh N Bogumil et a l , 1994 H64K sw N Springer etal., 1989 H64D sw N Springer et al., 1989; Morikis et al., 1990; Rohlfs et al., 1990; Braunstein et al., 1993 H64C sw N Springer etal., 1989 H64W sw N L i et al., 1994 V 6 8 A sw N Egeberg et al., 1990; Hargrove et al., 1994; L i et al., 1994 V 6 8 A hu N Balasubramanian etal., 1993; Lambright etal., 1994 V68F sw N Egeberg et al., 1990; Ikeda-Saito et al., 1993; Hargrove et al., 1994; L i et al., 1994 V68L hu N Lambright et al., 1994; L i et al., 1994 V68I sw N Egeberg et al., 1990; Ikeda-Saito et al., 1993; L i et al., 1994 V68Q sw N L i et al., 1994 V68N hu N Varadarajan etal., 1989; Balasubramanian etal., 1993 V68S po N Smerdon etal., 1991; Cameron etal., 1993 V68T po Y Smerdon etal., 1991'; Cameron etal., 1993' V68D hu N Varadarajan et al., 1989 V68E hu N Varadarajan et al., 1989 V68H hu N Qin e ta l , 1994 V68H hh N Lloyd et al., 1995 (a)only single variants are listed. (b)(*) denotes structure containing reference of met form. Other abbreviations are: hh, horse heart; sw, sperm whale; hu, human; po, porcine; bo, bovine Mb. 1\ 1. Binding of 02 and CO to myoglobin The residues of myoglobin studied most intensely by mutagenesis are the distal histidine, H64, followed by V68. Both of these distal pocket residues are highly conserved among myoglobins from many species. Initial mutagenesis studies addressed the importance of these conserved residues on the properties of oxygen and carbon monoxide binding to reduced (deoxy) myoglobin. One informative means of evaluating the role of the protein environment in modulating the interaction of CO and 0 2 with the heme iron has been through investigation of model heme compounds. For example, the ratio of the equilibrium constants for binding of these two ligands (Kco/K02) is three orders of magnitude greater for heme free in solution than for heme at the active site of myoglobin (Springer et al., 1989). In other words, the active site of myoglobin decreases the relative affinity of the heme group for CO relative to dioxygen. This characteristic presumably reduces the potential toxicity of CO, a byproduct of metabolic heme degradation, to a tolerable level. Although ligand binding studies of model hemes have produced conflicting results concerning the relationship between CO affinity and CO orientation (e.g. Collman et al., 1976, 1983; Kim et al., 1989; Kim & Ibers, 1991), the lower affinity of CO for heme at the active site of myoglobin (and hemoglobin) has generally been attributed to the distortion of the coordinated CO ligand from the usual linear geometry to a bent orientation (Norvell et al., 1975; Collman et al., 1976). This unfavorable orientation results from the proximity of the distal histidine residue to the bound ligand. On the other hand, the favorable orientation of heme-bound dioxygen is bent and, therefore, is unperturbed by the proximity of the distal histidine. However, this "textbook" explanation of ligand discrimination in Mb has been questioned recently (see below). Structural evidence for hydrogen bond formation between coordinated dioxygen and NE2 of H64 (Hanson & Schoenborn, 1981; Phillips & Schoenborn, 1981) provides an additional basis for this environmental influence on relative 22 ligand binding affinities. A recent review by Springer (1994) provides a comprehensive summary of the kinetic and equilibrium studies reported for many of the myoglobin variants listed in Table 1. Among these studies, many concern characterization of variants in which H64 has been replaced with a variety of residues, in part to assess the role of the distal histidine residue in determining relative ligand binding affinities. The consensus from this work is that geometrical constraints by the distal histidine residue on the orientation of ligand binding is not sufficient to explain the relatively low affinity of myoglobin for CO. In particular, no correlation is observed between the size of the residue at position 64 and CO affinity. For example, substitution of H64 with either an alanyl or a phenylalanyl residue increases the affinity of the protein for CO three-fold. Further insight into contributory factors has been provided by crystallographic studies of wild-type deoxymyoglobin. Although the heme iron in deoxymyoglobin is pentacoordinate and the distal ligand binding site is vacant, a non-coordinated water molecule is present in the distal heme pocket that is hydrogen bonded to NE2 of H64 (Takano, 1977; Quillin et al., 1993). The proximity of this water molecule to the ligand binding site means that binding of ligands must involve displacement of this water molecule from the active site prior to ligand binding. This desolvation of the active site may represent the major kinetic barrier to ligand binding. Based on this assumption, replacement of H64 with hydrophobic residues affects ligand binding primarily through elimination of this critical water molecule from the active site of deoxymyoglobin rather than through any geometrical consequences of side chain volume on ligand binding (Quillin et al., 1993) Although replacement of H64 with hydrophobic residues also affects the binding of dioxygen to myoglobin, the effects of these substitutions are not the same as observed for CO binding. For example, the H64A variant exhibits increased affinity for CO but a 50-fold decrease in affinity for 23 dioxygen. In this case, the three-fold increase in the rate constant of dioxygen association observed in kinetic studies is more than offset by the 150-fold increase in the rate constant for dioxygen dissociation. Similar results have been observed upon replacing H64 with other aliphatic residues and are consistent with the stabilization of the bound dioxygen through hydrogen bonding interaction with H64 in the wild-type protein. This kinetic effect is much smaller in the case of CO presumably as the result of less stable hydrogen bonding interaction between CO and H64. Comparison of the three-dimensional structures of CO and oxymyoglobin reveals that a significant displacement of the imidazole of H64 occurs upon binding of CO to wild-type sperm whale myoglobin (Quillin et al., 1993). In contrast, the 2.7 A distance separating NE2 of H64 and the bound dioxygen ligand is ideal for hydrogen bonding (Quillin et al., 1993). Other residues in the distal heme pocket also contribute to the ligand binding properties of myoglobin. For example, the proximity of V68 to the distal coordination position can produce both steric and electrostatic consequences upon substitution with other amino acid residues. In the case of the V68T and V68S variants, the affinity for CO is reduced 5-fold while the affinity for dioxygen is reduced 17-fold. For both ligands, the rate constant for association decreases through stabilization of the non-coordinated water molecule that is hydrogen bonded to H64 in deoxymyoglobin. Significantly, the only variant in which H64 or V68 has been replaced that has been reported to retain the ability to form a stable dioxygen adduct is V68N (Springer et al., 1994). With this variant, it appears that dioxygen coordination can be stabilized through formation of an additional hydrogen bond with N68. Overall, characterization of the ligand binding properties of distal heme pocket variants of myoglobin has led to the conclusion that the major determinant for the relatively low Kco/KQ2 ratio exhibited by wild-type myoglobin is the stabilization of bound dioxygen rather than destabilization of CO binding. 24 2. Characterization of distal heme pocket variants of myoglobin Replacement of the distal histidine residue of sperm whale or horse heart myoglobin frequently results in unusual coordination environments for the heme iron, some of which appear to be relevant to the active sites of myoglobins from other species. For example, the crystallographically defined structures of the H64V and H64I variants of sperm whale metmyoglobin have revealed that these variants are pentacoordinate in the oxidized state (Quillen et al., 1993). These variants are reminiscent of the active site of the myoglobin from the invertebrate Aplysia limacina, which possesses a valine residue at the sequence position corresponding to H64. The three-dimensional structure of Aplysia myoglobin confirms that the heme iron of this protein is pentacoordinate at neutral pH (Bolegnesi et al., 1989). These findings confirm the importance of the stabilizing influence provided by the hydrogen bond formed by the distal histidine residue and the distally coordinated water ligand in the active site of the hexacoordinate wild-type myoglobins. In contrast, crystallographic characterization of the V64G and V64Q variants of sperm whale metmyoglobin establish that the distally coordinated water molecule is retained in each case (Quillen et al., 1993). In the case of the V64Q variant, the Q64 side chain can form a hydrogen bond with the coordinated water molecule and suggests that a similar interaction may exist in the active site of myoglobin from the Asian elephant. The amino acid sequence of elephant myoglobin is known to possess a glutamine residue at position 64 (Romero-Herrera, 1981). Although such an interaction is impossible with the V64G variant, the absence of a side chain at position 64 results in the availability of sufficient space for an additional water molecule in the position normally occupied by NE2 of H64. Hydrogen bonding between this new water molecule and the coordinated water stabilizes the hexacoordinate heme iron derivative. Some reported myoglobin variants produced by site-directed mutagenesis mimic structural 25 characteristics of naturally occurring human hemoglobin variants with unusual coordination environments. One such example is the H64Y variant of sperm whale (Egeberg et al., 1990; Pin et al., 1994) and horse heart (Maurus et al., 1994; Tang et al., 1994) myoglobin in which the phenolic side chain of Y64 occupies the distal coordination position as observed in HbM Boston (Ha64Y; Pulsinelli et al., 1973) and HbM Saskatoon (HP64Y; Nagai et al., 1983, 1989). The unique spectroscopic properties of coordination of the distal Y64 residue has resulted in use of a related double variant (V68F/H64Y) as a convenient means of determining the rate of heme dissociation from heme proteins (Hargrove et al., 1994). Addition of the heme-free variant (apo-V68F/H64Y; the V68F substitution increases the thermal stability of the apoprotein to an acceptable level) in excess to a protein that possesses a non-covalently bound heme group results in transfer of the heme to the apo-Mb variant. The reaction proceeds in this direction because the stability of heme binding to the variant is stabilized significantly by coordination of the Y64 residue. The reaction can be monitored conveniently owing to the unique spectroscopic properties of Y64 coordination. Experiments of this type can provide informative insight into the structural characteristics that stabilize heme binding to myoglobin and other heme proteins. 3. Characterization of the proximal ligand variants of myoglobin. Although studies of variants involving replacement of either the proximal or distal histidyl residues have been reported in the literature, far more work has been directed at understanding the role of the distal H64 residue. This fact is at least partially attributable to the difficulty in preparing H93 variants since these are invariably expressed in E. coli as a mixture of holo and apo-proteins (Egeberg et al., 1990; Adachi et al., 1991, 1993; Hildebrand et al., 1995). The fact that the only H93 variants reported are H93 Y and H93C suggests that substitution of H93 with other residues capable of providing a proximal heme iron ligand leads to secondary effects. Complications of such 26 substitutions are likely effects of distance or geometry that prevent proximal coordination or even the inability to bind heme or allow for proper protein folding. An alternative, novel approach to the study of proximal ligand effects in myoglobin was introduced recently by Barrick (DePillis et al., 1994; Barrick, 1994). The principle of this approach is that substitution of H93 with a glycyl residue should create a space in the proximal heme pocket for the binding of ligands of appropriate size from bulk solvent. This variant is expressed and purified in essentially the same manner as the wild-type protein, except that all procedures are performed in the presence of imidazole. The resulting protein has spectroscopic properties similar to those of wild-type metmyoglobin, and the crystallographically determined three-dimensional structure confirms that imidazole from solution is proximally coordinated to the heme iron (Barrick, 1994). The lack of covalent attachment of the imidazole ligand to the proximal F helix results in rotation of this group to minimize steric interaction between the ligand and the pyrrole nitrogen atoms. Thus, it appears that covalent tethering of the proximal imidazole ligand to the F helix in wild-type myoglobin forces the imidazole group into a specific orientation. The imidazole-bound variant binds CO and 02, and the electronic spectra of these derivatives are essentially identical to those of wild-type myoglobin. The unique property of this variant, however, is its ability to exchange the proximally-bound imidazole ligand for a wide range of ligands and thereby serve as a scaffold for efficient evaluation of derivatives with a wide range of proximal ligands to the heme iron. G. Spectroscopic properties of heme proteins. Spectroscopic studies of heme proteins provide important information concerning active site properties that are complementary to the structural information provided by crystallographic studies. Methods generally employed in such work include electronic absorption spectroscopy (Smith & Williams, 1970), electron paramagnetic resonance (EPR) spectroscopy (Palmer, 1985), visible and 27 near-infrared (NIR) magnetic circular dichroism (MCD) spectroscopy (Dawson & Dooley, 1983), nuclear magnetic resonance (NMR) spectroscopy (Goff, 1983), Mossbauer spectroscopy (Debrunner, 1983), and resonance Raman spectroscopy (Spiro & Czernuszewicz, 1995). One advantage of such techniques is that they can be used under a wide range of solution conditions and temperature to assess the influence of such factors on the coordination environment and electronic properties of the heme iron centre in a manner that would not be feasible by crystallographic methods alone. Spectroscopic characterization of heme protein variants is a powerful and convenient avenue of investigation because the protoheme IX prosthetic group serves as a spectroscopic reporter group that is sensitive to the surrounding protein environment. Since 1965, perhaps the most frequently sought goal of spectroscopic studies of heme proteins has been the identification of axial ligands responsible for heme iron coordination at the active sites of structurally uncharacterized proteins. However, initial efforts at identification of axial ligands, often on the basis of EPR spectroscopy alone, were subsequently found to be in error. Based on this history, a healthy skepticism arose concerning such work and remains to some extent today. As a result, spectroscopic methods for characterization of metalloprotein centres are often referred to as "sporting techniques" because the quarry frequently eludes capture. In fairness, much of the early inadequacy of these methods undoubtedly resulted from the lack of structurally characterized proteins and small molecule model systems that could provide reference or calibration standards for correlation of spectroscopic properties with their structural origins. As reviewed in detail below, contemporary methods have achieved a relatively high degree of reliability in determination of axial ligands of heme proteins through the use of a combination of spectroscopic techniques. J. Oxidation and spin states of heme proteins. Before considering the spectroscopic techniques that have been used to study myoglobin 28 variants in the current work, it is helpful to consider briefly the basic electronic properties of heme iron centres that are relevant to these techniques to help explain the basis for these techniques and to gain some insight into the type of information that they can provide. The electronic configuration of neutral iron, Fe(0), is represented as [Ar]3d 64s2. The two most common oxidation states are the ferrous, Fe(II) and ferric, Fe(III) states, which have electronic structures of [Ar]3d 64s 0 and [Ar]3J 4s ,° respectively. As discussed previously, some heme enzymes can also form a ferryl, Fe(IV) ([Ar]3<i44s °) intermediate, but this state is less stable and less frequently studied. The distribution of electrons within the five 3c/-orbitals of a heme iron centre is determined by the geometry and bonding interactions of the coordinating ligands. The spectroscopic techniques used in the current study, in turn, provide information about the electronic structure of the heme iron from which information concerning the coordination environment of the heme iron centre can be inferred. A summary of the possible heme spin-state and stereochemical relationships observed in heme proteins is provided in Figure 5; along with an indication of heme proteins that exhibit these electronic states. Depending on the strength of the ligand field, a variety of spin states can be observed for both ferric and ferrous hemes (Scheidt & Reed, 1981). Coordination of the iron by the porphyrin ring and addition of one or two inequivalent axial ligands produces a system with tetragonal symmetry. The electronic configuration of ferric iron exhibits a range of states that can be arranged in the following order as the ligand field strength of the axial ligands decreases: low-spin (S = 1/2) > high-spin (S = 5/2) > intermediate-spin (S = 3/2). An additional spin-state, first described by Maltempo (1974), results from the coupling of the high and intermediate-spin states. Although this new spin state is frequently referred to as a quantum mechanically admixed spin state (QMAIS), this state is magnetically pure and should not be confused with a spin equilibrium, which represents 29 S=l / 2 S=5/2 S=3/2,5/2 S=3/2 z2 xz'yz 1h.+-^ - T T C.N. 6 example cyt. 6 5 , c - r -++  5 , 6 P-450 , C c P , Mb-H 20 4 -+* + 5 cyt. c' 5 , 6 z2 xz, _yz xy S=0 S=2 S = l = 4- — 4 -•4 -4 4- 4- +4-"^ •fr -fr -fr C.N. 6 5 4 example cyt. b5, c Mb Mb-CO Figure 5: Schematic diagram illustrating the different spin-states possible for ferric (top) and ferrous (bottom) heme. The <i-orbital energy levels are represented as straight lines and the electrons as arrows. Examples of natural heme proteins are listed underneath their respective spin-states along with the coordination number ( C N ) . 30 a mixture of two pure spin states. For example, compounds with axial ligands of moderate field strength produce a heme iron system that is close to the high-spin/low-spin crossover point. A sample of such a system is a mixture of high and low-spin species that are in thermal equilibrium. The hydroxide-bound form of myoglobin is an example of a system that exhibits such behavior at room temperature (Beetlestone & George, 1964; George et al., 1964). The extent of mixing in a quantum mechanical admixture also depends on the ligand-induced splitting of the <i-orbitals and a continuum can arise between the high and intermediate-spin states. If the energy of the x^-y2 orbital is increased sufficiently, the electron that normally occupies this orbital will partially occupy x^-y2 and xy simultaneously. If the energy of the x^-y2 orbital is raised further, an intermediate spin-state results. At present, no heme proteins are known that exhibit this spin behavior. In fact, the QMAIS state itself is relatively rare and has been observed only for some species of pentacoordinate cytochrome c. (Maltempo, 1974) and some model heme compounds (Reed et al., 1979). The majority of heme proteins exist at room temperature in either purely high-spin or low-spin states or as thermal equilibria of high-spin and low-spin states. Generally, the nature and number of axial ligands determine the spin-state and stereochemistry of Fe(III)-porphyrin centres. For example, all low-spin heme proteins are hexacoordinate. In the case of one such protein, cytochrome b5, the heme iron is coordinated by two strong field histidine ligands (Mathews et al., 1972; Argos & Mathews, 1975; Mathews, 1980). Replacing one of these ligands with a weaker ligand or replacing one of them with an amino acid residue incapable of providing an axial ligand results in formation of a high-spin system (Beck von Bodman et al., 1986; Sligar et al., 1987). The electronic configuration of the ferrous ion also exhibits various states that are observed in the following order as the strength of the axial ligands decreases: low-spin (S=0) > high-spin (S=2) 31 > intermediate spin (S=l). The effects of ligand field strength on the stereochemistry and spin-states of the ferrous ion are similar to those observed for the ferric ion. Hexacoordinate ferrous cytochromes possess low-spin heme iron centres as do the carbonyl derivatives of myoglobin and hemoglobin. Deoxymyoglobin and deoxyhemoglobin are examples of high-spin ferrous heme proteins. With ferrous heme systems, the intermediate spin-state has been observed only for certain model heme compounds (Collman et al., 1975). 2. Electronic spectroscopy Electronic spectroscopy provides information concerning the transitions between energy levels of different electronic states. The complex electronic spectra observed for heme proteins can provide insight into both the spin and oxidation states of the heme iron centre. The origins of the transitions in a typical heme protein spectrum can be classified as protein centred, heme centred (porphyrin or iron) or charge transfer. Protein centred transitions occur in the ultraviolet (UV) region. The far-UV region (-190-230 nm) and the near-UV region (-280 nm) are associated with peptide bond transitions and aromatic residues, respectively. The presence of the heme chromophore in these proteins results in electronic transitions in the visible region of the spectrum. The intensity and shape of the visible absorption bands are different for various heme proteins, but a typical spectrum consists of an intense (e = 100-170,000 M" 1 cm"1) band at -400-420 nm (the Soret, B, or y-band), and two absorption bands of lesser intensity (e = 10-15,000 M" 1 cm"1) with maxima between 500 and 600 nm (Qv and Q 0 or P and a bands)(see Makinen & Churg, 1983 and references therein). All heme proteins and model compounds exhibit these characteristically intense B and Q transitions that are assigned to n-n* transitions of the porphyrin macrocycle (Longuet-Higgens, et al., 1950). Gouterman (1961) employed a four-orbit model to describe the Q and B transitions as originating between the two highest occupied and two lowest unoccupied molecular (n) orbitals of 32 the porphyrin. A distinct three band pattern corresponding to the B and Q transitions is characteristic of the diamagnetic (low-spin) ferrous systems (e.g., cytochromes c and b5 and MbCO). This pattern is also observed for low-spin ferric systems, but the a and P maxima are less pronounced (e.g., ferricytochromes c and bs). Although these electronic spectra are primarily attributable to the porphyrin macrocycle, they are also quite sensitive to the electronic configuration of the iron centre. This relationship is demonstrated, for example, by the linear correlation between the position of the Soret band maxima and the magnetic susceptibility of a series of metMb derivatives (Makinen & Churg, 1983 and references therein). Conversion of metmyoglobin from a high-spin species to a low-spin species induced by ligand binding results in a concomitant red-shift of the Soret band maximum. The origin of the intense porphyrin (n-Tt*) transitions of heme complexes have been the subject of a number of theoretical studies. Identification and assignment of iron-centred (d-d) transitions are more difficult. In many cases, these d-d transitions are forbidden and, therefore, presumably absent or of quite low intensity. Even when these transitions do occur, they are frequently obscured by the far more intense porphyrin electronic transitions. Similarly, the charge transfer (CT) bands that result from the transfer of electrons between the iron and the porphyrin or axial ligand orbitals are also less well understood. However, the contributions of CT transitions to the spectra of some heme complexes have been established. For example, CT bands are particularly prevalent in high-spin ferric porphyrin derivatives and lead to additional complexity in the visible spectra of these systems (vide infra). The spectra of high-spin ferric porphyrins (e.g., metMb(H20)) are more complex than those of typical metalloporphyrins. The a and P bands are only partially discernable for such systems and additional features at -500 nm and -600-640 nm are present that are assigned to porphyrin-Fe(III) CT transitions (Williams, 1956). These absorption bands are sensitive to the type of axial ligation and 33 not observed when strong field ligands (e.g., cyanide) bind to the heme iron. As mentioned previously, moderately strong field ligands (e.g., as in MbOH) result in a thermal spin equilibrium between high and low-spin iron (Beetlestone & George, 1964). The spectra of such complexes appear as superpositions of the high and low-spin spectra so that both the Q bands and the high-spin CT bands are discernable. The electronic spectra of these systems are dependent on temperature because the thermal spin equilibrium favors the low-spin species at lower temperature which, in turn, results in disappearance of the CT bands (Feis et al., 1994). 3. Magnetic circular dichroism (MCD) spectroscopy The origins of circular dichroism (CD) and magnetic circular dichroism (MCD) are based on the differential absorption of left and right circularly polarized light (Ae = e, - er). Although CD can be observed only for asymmetric chromophores, the presence of the external magnetic field applied in the MCD experiment induces optical activity by lifting the electronic orbital and spin degeneracies. Experimentally, CD and MCD spectroscopy differ only in that the sample is placed in a magnetic field for acquisition of an MCD spectrum; the intensity of the resulting MCD spectrum is a linear function of the applied magnetic field. The splitting of electronic states induced by the magnetic field is referred to as Zeeman splitting and can occur in either the ground state or the excited state, depending on the system in question (Schatz & McCaffery, 1969). The designations, A and C terms have been given to transitions that involve field-induced splitting of excited and ground states, respectively. The consequence of ground state splitting is a spectrum that is highly temperature dependent owing to the effects of the Boltzmann distribution. Additional splitting of electronic levels can occur through field-induced mixing of states to give rise to B terms. Information concerning the type of splitting can be obtained from the shapes and temperature dependences of the spectroscopic features (Stephens, 1976). An A term, for example, is characterized by a derivative band shape, 34 whereas a C term is generally absorbance shaped and highly temperature dependent. The B and C terms can have similar band shapes, but the former are usually independent of temperature. The same porphyrin n-7r* transitions that dominate the electronic spectra of metalloporphyrins are also responsible for the transitions observed in the MCD spectra of such systems. As discussed for electronic spectra, the additional complexities of d-d and CT-bands resulting from introduction of a metal to the porphyrin macrocycle are also features of MCD spectra. It is not surprising, therefore, that both spectroscopic techniques provide information concerning the oxidation and spin states of the iron center, the type of porphyrin ring, and the nature of the axial ligation. In contrast to electronic spectroscopy, however, the presence of a metal centre with a paramagnetic ground state results in MCD spectra that are highly temperature dependent as the result of mixing of the porphyrin and fi?-orbital states. Extensive MCD analyses of the various ligated forms and spin states observed for heme proteins and model heme compounds of known structure provide reference information that permit inferences to be drawn concerning the axial ligation and electronic properties of heme centres of unknown structure (Hatano & Nozawa, 1978; Cheesman et al., 1991). Correlations of this type are of particular use in the study of new heme proteins of undetermined structure or in the investigation of new axial ligation environments created through site-directed mutagenesis. (i) MCD spectroscopy of ferrous heme centres The use of MCD spectroscopy to differentiate low-spin (S=0) from high-spin (S=2) ferrous heme derivatives is relatively straightforward. In the case of the diamagnetic, low-spin ferrous heme systems, the MCD spectra are independent of temperature below -100 K. For examples such as ferrocytochrome c, the B and Q bands of the porphyrin are dominated by A term intensity. In contrast, paramagnetic, high-spin ferrous systems exhibit MCD intensity in the B and Q band regions 35 that is highly dependent on temperature. The MCD spectra of diamagnetic iron porphyrin centres exhibit relatively little variation with the nature of the axial ligands to the heme iron, so MCD spectroscopy of such systems is of less value in identification of the coordination environment of such systems. For example, the MCD spectra of model heme compounds that possess thiol/CO and imidazole/CO axial ligation are virtually superimposable (Collman, 1975). (ii) MCD spectroscopy of ferric heme centres Development of MCD spectroscopy as a technique for the identification of heme protein axial ligation has proven to be particularly successful for ferric heme protein derivatives. The MCD spectra of high-spin ferriheme proteins are complex owing to the presence of CT bands that alter the typical B and Q-band pattern. The contribution of the ferric iron centre to the MCD spectrum of ferriheme proteins is unusually sensitive to the electronic environment imposed on the metal by the axial ligands. For example, MCD spectra of various derivatives of ferrimyoglobin exhibit MCD band shapes that are sensitive to the chemical identity of the sixth ligand even for systems of the same spin state (Vickery et al., 1976). In addition, the intensity of the MCD transition in the Soret region is linearly correlated with the amount of low-spin species present with the intensity increasing as ligand field strength increases. The paramagnetic spin states of high and low-spin ferric heme increases the C-term intensity and renders the MCD spectra of such systems dependent on temperature. A unique feature of low-spin ferric heme centres is the low energy, near-infrared CT band (Day et al., 1967). This NIR-CT band (E c x) is attributable to a transition from the highest filled porphyrin (n) to ferric dXPd orbital level, the position of which is sensitive to the energy of the acceptor iron J-orbitals and, hence, to the identity of the axial ligands. The energy of this transition occurs in a region of the spectrum that is unaffected by the B and Q bands of the porphyrin macrocycle. Gadsby and Thomson (1990) have recorded the positions of the NIR-CT bands for a 36 wide range of low-spin ferric heme proteins and model compounds as a means of correlating E C T with the known iron coordination states. The absorption maximum observed for most systems of this type occurs at wavelengths >1200 nm. The absorption maxima observed for naturally-occurring heme proteins ranges from those observed for bacterioferritin (2200 nm; bis-methionine axial ligation), cytochrome c (1750 nm; histidine/methionine axial ligation), and cytochrome bs (1600 nm; bis-histidine axial ligation) (Cheesman et al., 1991). Thomson & Gadsby (1990) have suggested further that the intensity of the NIR-CT band in these systems may provide information about the orientation of the axial ligands. In many cases, the absorption maximum of the NIR-CT band is sufficient for assignment of axial ligation in proteins of this type. Nevertheless, some combinations of axial ligands cannot be distinguished from each other by NIR-MCD spectroscopy alone. In these cases, EPR data are required in addition to the information obtained from NIR-MCD to achieve unequivocal definition of the coordination environment (vide infra). 4. Electron paramagnetic resonance (EPR) spectroscopy EPR spectroscopy is a technique used for the detection of unpaired electrons in, for example, free radical or paramagnetic molecules (Abragam & Bleaney, 1970; Swartz et al. 1972; Wertz & Bolton, 1986). This magnetic resonance technique measures transitions between electronic Zeeman states. EPR is analogous to MCD in as much as the presence of an external magnetic field induces normally degenerate energy levels to become non-degenerate. Thus, the energy separation between different spin angular momenta levels is induced by the presence of an external magnetic field. The resonance condition is described by the following equation: AE = /?v = gpH (2) where AE is the difference in energy of the two spin states, h is Planck's constant, vis the (constant) operating frequency of the spectrometer, P is the electron Bohr magneton, and H is the resonance 37 magnetic field. The quantity measured in an EPR experiment is the dimensionless g-factor, which relates the resonance frequency and the external magnetic field. The designations gx, gy, and gz correspond to theg-values obtained when the magnetic field is parallel to each of the structural axes. This orientational dependence (anisotropy) determines the shape of the EPR spectrum and, along with actual g-value, provides insight into the local environment of the unpaired electrons (Palmer, 1985). For instances where gx = gy = g0 the systems is said to be isotropic, a situation that is rare for transition metals. Systems of this type generally exhibit either axial or rhombic anisotropy for which two or three unique g-values occur, respectively. For proteins with heme iron centres, the paramagnetic Fe(III) derivatives are most amenable to EPR spectroscopy. For low-spin, ferriheme centres, the type of symmetry (axial or rhombic) is determined by the relative energy levels of the dxz, dyz and d^ iron orbitals (Figure 5), which, in turn, are related to axial ligation. For an axially symmetric heme iron centre, the dxz and dyz orbitals are destabilized equally relative to d^. An additional (rhombic) distortion leads to an energy separation of d^ and d^. The degree of axial or rhombic distortion can be calculated directly from the ^-values (Palmer, 1985). For low-spin ferric heme centres (e.g., cytochromes c, cytochrome b5), the range of ^-values observed is between -3.8 and 0.5. From EPR measurements of a large number of low-spin ferric heme proteins, Blumberg and Peisach (1971) constructed so-called "Truth Diagrams" that relate the degree of rhombicity to the axial ligand field strength. For the assignment of coordination environments in many types of heme-containing systems, such analyses have significant predictive values owing to the clustering of ^ -values in domains that represent certain combinations of axial ligands. Situations have been identified subsequently, however, for which ambiguities in ^-values preclude unequivocal assignment of axial ligands solely on the basis of EPR spectroscopy. For heme iron centres possessing more than a single unpaired electron (e.g., high-spin 38 metmyoglobin, S=5/2), interactions between the c/-orbital electrons and the ligand field determine the energies of the spin substates. Analogous axial and rhombic perturbations described for low-spin heme centres also affect the EPR spectra of high-spin systems. Metmyoglobin is an example of an axial heme iron centre with g = 6.0 and = 2.0. Any rhombicity introduced by the ligand field in such centres results in splitting ofg± into^x andgy. Cytochrome P-450cam with the substrate camphor bound, for example, is highly rhombic and exhibits g-values of ~7.8, 3.9 and 1.8. In cases where rhombicity is quite small, g± is simply broadened and cannot be resolved into its components. The range of ^ -values observed for high-spin ferriheme proteins is -8-1.8 (Palmer, 1985). 5. Axial ligand assignment by an EPR-MCD approach Although examples can be found for which axial ligands have been assigned by the use of either EPR or MCD spectroscopy alone, a combination of the two methods is generally regarded as the optimal means for spectroscopic identification of axial ligands of low-spin ferriheme centres (Gadsby & Thomson, 1990). The interrelationship between these two techniques relates to the involvement of the Fe(III) d-orbitals in each method. As previously mentioned, EPR spectroscopy depends on the relative energy levels of the cL^, d^ and dyz orbitals, whereas NIR-MCD spectroscopy derives information concerning the djdyz level. The first application of the combined EPR/NTR-MCD approach was in the assignment of the axial ligands present in the alkaline form of horse heart ferricytochrome c (Gadsby & Thomson, 1990). Ferricytochrome c exhibits a pH-induced exchange of axial ligands in which the native M80 ligand to the heme iron is exchanged for a lysyl residue. This change in axial ligation can be monitored by both EPR and (NIR)-MCD spectroscopy. However, the NIR-MCD absorption maximum for alkaline ferricytochrome c occurs in a region that is not distinguishable from that observed for a low-spin ferriheme centre with bishistidine axial ligation (Cheesman et al., 1991). EPR 39 spectroscopy, on the other hand, can distinguish between these two alternatives because they differ in the position and shape of g±. This complementarity between these two techniques in resolving these two coordination environments was subsequently reinforced by the successful identification of His/amino axial ligation at the active site of cytochrome/(Rigby et al., 1988) prior to the structural characterization of this protein by X-ray diffraction analysis (Martinez et al., 1994). H. Thesis objectives Understanding the manner in which the heme binding environment provided by the apoprotein dictates the ligand binding and catalytic properties of heme proteins remains one of the fundamental objectives of research concerning this family of proteins. As one of the most significant structural characteristics of such proteins is the coordination environment of the heme iron, the application of site-directed mutagenesis to the investigation of the relatively simple heme protein myoglobin has led to the production and characterization of several variants in which the functional contributions of the axial ligands can be assessed. Several recent studies have evaluated the perturbations of ligand binding induced by the substitution of the distal histidine residue (H64) with a variety of amino acids (e.g., Springer et al., 1994). On the other hand, the proximal H93 residue has been studied less extensively. Until recently, the only variants of this type were the H93Y variant of sperm whale (Egeberg et al., 1990; Morikis, 1990) and human (Adachi et al., 1991, 1993) myoglobins and the H93C variant of human myoglobin (Adachi et al., 1991, 1993). The emphasis of these previous studies has been investigation of selected spectroscopic and ligand binding consequences of these substitutions, so several critical structural and functional aspects of these proteins remain to be elucidated. The present study defines the structural, functional, and spectroscopic consequences of replacing several key residues, namely H93, H64, and V68, in the active site of horse heart 40 myoglobin. In this component of the present work, combinations of single and double variants that are expected to alter the polarity and ligation properties of the active site have been assessed. The axial ligand variants (H93Y, H93C, H64V/H93C and H64I/H93C) are also of interest because the coordination environments of other heme proteins (e.g., catalase, cytochrome P-450, chloroperoxidase) are known from X-ray crystallographic studies to involve other amino acid residues than histidine. Variants of myoglobin designed to assess the effects of changes in polarity of the distal heme pocket include: H64V, H64I, V68H and V67A/V68S. Active site substitutions such as these provide insight into mechanisms by which protein structures control the chemical reactivity of heme centres. Such investigations provide a means of determining the extent to which these changes in axial ligation determine spectroscopic, functional and structural properties of the protein relative to the contributions made by the other, extensive differences in structures between myoglobin and other heme proteins. Some of the work described here has been published prior to submission of this dissertation (Bogumil et al., 1995; Hildebrand et al., 1995(a), 1995(b); Lloyd et al., 1995). 41 EXPERIMENTAL PROCEDURES A. Site-directed mutagenesis and myoglobin expression The synthetic gene coding for wild-type horse heart myoglobin was constructed by Dr. Guy Guillemette in the laboratory of Professor Michael Smith (Dept. of Biochemistry and Molecular Biology, U.B.C.) and has been described elsewhere (Guillemette et al., 1991). Oligonucleotide-directed mutagenesis techniques were used to construct all of the variants in this work (Zoller & Smith, 1983, 1984). The oligonucleotides used to construct the variants are listed in Table 2. The codons of interest are underlined. The Kunkel method was used as a means of mutant selection (Kunkel, 1985). The uracil-containing DNA required for this procedure was produced by a strain of E.coli (RZ1032) that was deficient in dUTPase and uracil-N-glycosylase. Transformations were performed with the CaCl2 method (Maniatis, 1989). Single-stranded DNA sequencing of the entire myoglobin gene ensured that no other mutations were introduced by the mutagenesis method (Maniatis, 1989). All enzymes and reagents for mutagenesis were obtained from Pharmacia Corp. All enzymes and reagents used for sequencing were obtained from United States Biochemical Corp. (Cleveland, Ohio). Table 2: Mutagenic oligonucleotides H93Y 5' -TTT-AGT-AGC-GT A-CGA-TTG-CGC-3' H93C 5 -TTT-AGT-AGC-GC A-CGA-TTG-CGC-3' H64V 5 -AAC-GGT-ACC-AAC-ATG-TTT-TTT-CAG-3' H64I 5 -AAC-GGT-ACC-GAT-ATG-TTT-TTT-CAG-3' V68H 5 -CC-TAG-GGC-AGT-TAA-GTG-AAC-GGT-ACC-ATG-3' V67A/V68S 5 -GGC-AGT-TAA-CGA-AGC-GGT-ACC-ATG-3' Wild-type and variant myoglobins were expressed in E.coli (LE392) grown in superbroth 42 [tryptone 10 g/L (BDH), yeast extract 8 g/L (BDH), NaCI 5 g/L (BDH) and ampicillin 100 ug/L (Sigma, A-9518)]. Preparative scale cultures (20 x 600 mL) were grown (20 h) in 2 L Erlenmeyer flasks placed in a shaker-incubator (37 °C, 300 rpm). B. Purification of myoglobin Cells from 12 L of bacterial culture were collected by centrifugation (Sorvall RC-5B with a GS-3 rotor, 5000 rpm) and suspended in buffer [20 mM Tris-HCl buffer (Fischer, BP152-1), pH 8.0 (100 mL)]. To lyse the cells, lysozyme [250 mg; Sigma (L-6876)] was added and the suspension was kept on ice for 2 hours. The cells were frozen in liquid nitrogen and left overnight (4 °C) to thaw. Deoxyribonuclease I [10 mg; Sigma, (D5025)], ribonuclease A [1 mg; Sigma, (R4875)] and 20 mL of 2M MgCl2 solution were added to the thawed cells and the suspension was placed on ice for 2 hours. The resulting cellular debris was removed by centrifugation (Sorvall GS-3 rotor, 8000 rpm, 30 min) and washed once with buffer (20 mM Tris-HCl buffer, pH 8.0). The supernatant fluid was brought to 55% saturation by the slow addition of solid ammonium sulfate and was then stirred on ice for 20 min. The resulting precipitate was removed by centrifugation (Sorvall GS-3 rotor, 8000 rpm, 30 min), and the supernatant fluid brought to 100% saturated ammonium sulfate as before. The precipitated protein was dissolved in a minimum volume of cold (4 °C), distilled water and dialyzed against distilled water (2 x 20 L, 4 °C). The dialyzed supernatant fluid was adjusted to pH 8.0 with 1.0 M NaOH and centrifuged to remove any precipitate that may have formed during dialysis. Measurements of pH were obtained with a Radiometer Model PHM84 pH meter. Before loading the ion-exchange column, the conductivity of the supernatant fluid was checked to ensure that it was below that of the equilibration buffer (below). Conductivity was measured with a Markson Model 10 conductivity meter. The supernatant fluid was applied to a DEAE-Sepharose CL-6B (Pharmacia) column (2.5 x 10 cm) that 43 was equilibrated with 50 mM Tris-HCl buffer (pH 8.0). Myoglobin eluted after repeated washing with the equilibration buffer and was applied directly to a zinc chelate affinity column. The chelating sepharose column (2.5 x 6 cm) (Pharmacia) was charged with 700 mL of 35 mM zinc sulfate/25 mM acetic acid and equilibrated with 1 L of 5 mM Tris-HCl/0.5 M NaCI (pH 8.0). Both apo and holoprotein bound to the column and were eluted with 50 mM Tris-HCl buffer containing 50 mM imidazole and 0.5 MNaCl (pH 8.0). The eluted protein was exchanged into a buffer containing 0.1 M potassium phosphate, 0.1 MKC1 and 10% glycerol (pH 8.0). Protein samples were concentrated with Amicon Centripreps and Centricon 10 microconcentrators. The low level of holoprotein produced in E.coli eliminates any contribution of sulfmyoglobin production during the expression of these variants (Lloyd & Mauk, 1994). To reconstitute the variant apoproteins, a slight excess of hemin (Sigma, H2250) dissolved in a minimum of NaOH (0.1 M) solution was added. Hemin uptake by the apoprotein was monitored by the increase in the intensity of the Soret band. Excess hemin was removed by passage of the reconstitution mixture over a Sephadex G-75 superfine (Pharmacia) column (2.5 x 90 cm) equilibrated with 50 mM Tris-HCl buffer containing 1 mM EDTA (pH 8.0). Extinction coefficients were determined by the hemochromogen method (De Duve, 1948). C. Spectroscopic characterization of variant myoglobins J. Electronic absorption spectroscopy Electronic absorption spectra were recorded at 25 °C with a Cary 219 spectrophotometer interfaced to a microcomputer (On-Line Instruments-Systems, Bogart, GA) and fitted with a water-jacketed cell holder and a circulating water bath. Spectral analysis was performed using Grams (Galactic Industries). Spectra were recorded in quartz cuvettes with a 1 cm pathlength (1 mL or 3 mL volumes) between 700 and 280 nm. The reduced derivatives were produced by the addition of 44 a few grains of solid sodium dithionite (J. T. Baker). The CO bound forms were produced by diluting a stock solution of metMb into buffer which was saturated with CO gas followed by addition of a few grains of sodium dithionite. Oxy-Mb was formed by reducing a metMb sample with an excess of dithionite and removing the excess with a small Sephadex G-25 (Pharmacia) desalting column (0.5 x 8 cm). A stream of 0 2 was then passed over the solution (if necessary) to convert deoxyMb to the oxygen-bound form. Electronic spectra were recorded at 77 K for V68H Mb by Professor Philip Bragg (Dept. of Biochemistry and Molecular Biology, U.B.C.) with an SLM-Aminco Model DW-2C dual wavelength spectrophotometer that was fitted with a sample holder having a 2 mm pathlength. Spectra were collected with buffer in the reference beam at the same temperature as the sample. 2. Electron paramagnetic resonance (EPR) spectroscopy EPR spectra were obtained at 4-10 K with a Bruker Model ESP 300E spectrometer (modulation frequency 100 kHz, modulation amplitude 8 G, microwave frequency 9.46 GHz, power 0.5 mW) equipped with an HP5352B microwave frequency counter and an Oxford Instruments ESR900 continuous-flow cryostat. Protein samples (200 uL, ~1 mM) were prepared in either 20 mM Tris-HCl buffer (pH 8.0), 50 mM sodium phosphate buffer (pH 7.0) or 50 mM glycine buffer (pH 10.5). Spectra were compared in the presence and absence of 50% (v/v) glycerol. Samples placed in quartz EPR tubes were frozen in liquid nitrogen prior to insertion into the instrument. 3. Nuclear magnetic resonance (NMR) spectroscopy NMR spectra were obtained at 20 °C with a Bruker MSL-200 spectrometer operated by either Dr. Juan Ferrer or Dr. Emma Lloyd in the laboratory of Professor Pieter Cullis (Dept. of Biochemistry and Molecular Biology, U.B.C). Protein samples (~2 mM) were prepared by repeated exchanges into 50 mM deuterated sodium phosphate buffer (pH 7.0, uncorrected pH-meter reading). 45 Typical spectra consisted of 30-60 K transients. 4. Magnetic circular dichroism (MCD) spectroscopy Circular dichroism measurements were made with Jasco Model J-720 (280-800 nm) and Model J-730 (800-2000) spectropolarimeters. Magnetic circular dichroism spectra were recorded by placing samples in a magnetic field generated by an Oxford Instruments superconducting magnet (SM-4) capable of 5 T at full field. Sample temperature was controlled by balancing the flow of liquid helium with that of heater output (Oxford Instruments ITC-4 temperature controller) and allowed for a temperature range of 300 K to 4.2 K. Alternatively, a 1.5 T electromagnet (Alpha Magnetics) with a water-jacketed cell holder was used in measurments of samples at ambient temperatures. Protein samples were prepared in 50 mM sodium phosphate (pH 7.0) unless otherwise stated. To acquire spectra in the NIR region, the samples were exchanged into 50 mM deuterated sodium phosphate buffer, pD 7.0. For cryogenic temperatures, the samples were diluted to 50 % with glycerol (v/v). Spectra were measured in quartz cuvettes (Hellma, 1 cm x 2 cm x 1 mm). For the best signal-noise ratio, sample concentrations were adjusted such that the absorbance of the peak maxima was 0.8-1.0 (this was not possible in the NIR region due to the weak transitions; therefore, repetitive scans were averaged and the protein concentration was increased to ~2 mM). Accurate measurements between 300 and 700 nm required that the Soret (300-450 nm) and the visible (450-700 nm) regions be measured separately. Spectra were normalized by dividing the absorbance by the protein concentration, pathlength and magnetic field which resulted in units of M" 1 cm"1 T"1. 5. Fourier transfer infrared (FTIR) spectroscopy FTIR spectra were recorded at 2 cm"1 resolution with a Perkin-Elmer System 2000 spectrometer equipped with a liquid nitrogen cooled mercury cadmium telluride detector. Myoglobin samples were exchanged into 50 mM sodium phosphate buffer (pH 7.0) and concentrated to 46 approximately 3 mM. To a solution of 50 uL protein, a few grains of sodium dithionite were added to reduce the sample and a stream of CO gas was passed over the solution for approximately 1 minute to convert the sample to the carbonyl form. The sample (25 uL) was loaded into a cell with CaF2 windows and a 0.05-mm teflon spacer and placed into a water-jacketed cell holder (Specac, Inc.) connected to a fhermostated, circulating water bath (Lauda Model RS3). Carbonyl myoglobin spectra were collected between 1900 and 2000 cm"1 (25 °C) and corrected for background absorbance by subtracting a metMb spectrum of similar concentration. An average of 500 scans was used for each spectrum. D. Electrochemical measurements 1. Spectroelectrochemical experiments The midpoint reduction potentials of wild-type, H64V, H64I, V68H and V67A/V68S horse heart myoglobin were determined by spectroelectrochemical methods. The apparatus used was an optically transparent thin layer electrode (OTTLE) cell with an optical pathlength of-0.02 mm. The design and construction of the cell has been described in detail elsewhere (Reid, 1982; Lim, 1990). To facilitate the transfer of electrons between the gold-mesh electrode and the protein, small chemical mediators were required. Mediators used were [Ru(NH3)6]Cl3 (Strem Chemicals Inc.) (E° = 51 mV vs SHE; Lim et al. 1972) for wild-type, [Ru(NH3)5Im]Cl3 (E° = 100 mV vs SHE) for H64V and H64I and 2-hydroxy-l,4-naphthaquinone (Sigma, H-0508) (E° = -139 mV vs SHE) for V68H and V67A/V68S myoglobin. Recrystallization of the commercial [Ru(NH3)6]Cl3 mediator is described elsewhere (Pladziewicz etal. 1973). The synthesis of [Ru(NH3)5Im]Cl3 was based on the procedure of Sundberg et al. (1974) and has been described in detail elsewhere (Lim, 1990). The protein samples were exchanged into 50 mM sodium phosphate buffer (pH 6.0 or 7.0 as stated in figure legends). The cell was loaded with -400 uL of a solution containing 500 uM protein, 50 uM 47 mediator and trace amounts of Rhus vernicifera laccase (a gift of Dr. David Thackeray) and catalase (Sigma). Laccase was used to minimize residual dissolved oxygens while catalase removed any hydrogen peroxide generated during the experiment. The OTTLE cell was fitted with a saturated calomel electrode (SCE) (Radiometer Model 4112) as the reference. The temperature of the cell was monitored with the use of a thermocouple that was in direct contact with the protein solution, and was maintained at 25 °C by a circulating water bath (Lauda Model RC3). A typical experiment consisted of taking approximately 8 spectra (280-700 nm, Cary 219 spectrophotometer). A range of potentials between -500 and +200 mV versus SCE were chosen with at least 4 points above and 4 below the midpoint reduction potential. The experiment was initiated by applying a reducing potential across the cell to convert metMb to deoxyMb and reduce any residual oxygen in the cell. The potential was then increased in small increments and spectra were recorded until the protein was fully oxidized. A representative experiment consisted of completely oxidized and reduced spectra with a minimum of 6 intermediate spectra obtained for varying ratios of reduced and oxidized myoglobin. The resulting data were fitted with the Nernst equation to determine the midpoint potential. The calculated potentials were adjusted to the standard hydrogen electrode (SHE) scale by adding 244.4 mV at 25 °C. 2. Photochemical experiments The midpoint reduction potentials of the H93Y, H64V/H93C and H64I/H93C myoglobin variants were measured with a photochemical technique (Mauro et al, 1988). To ensure oxygen-free conditions, the experiment was conducted in a 3 mL, anaerobic cuvette (pathlength 1 cm) with a sealable glass top. The cuvette had an entrance port which could be fit with a serum cap, allowing insertion of an argon purging line and a sample injection line. Solutions prepared for these measurements contained 50 mM sodium phosphate buffer, 10 mM EDTA, 21 uM riboflavin and 15 48 uM H93Y Mb (30 uM protein was used for H64V/H93C and H64I/H93C Mb) (pH 8.0). Prior to initiating the experiment, 3 mL of buffer containing riboflavin and EDTA was placed in the cuvette and purged thoroughly by bubbling with argon. A concentrated protein solution (~ 20 uL) was injected into the cuvette while passing argon over the top of the solution for several minutes. The purging and injection lines were then removed from the cuvette. All manipulations were performed under reduced lighting to prevent premature initiation of the reaction. Spectra were taken in an analogous fashion to the OTTLE experiments, except that the potential was controlled by the ratio of riboflavin to semiquinone. The first spectrum recorded was that of the oxidized protein. Reduction proceeded by direct illumination with a 12 V lamp (Sunnex) of the sample for increasing periods of time. At the beginning of the experiment the illumination times were approximately 5 seconds per data point and increased to approximately 30 seconds by the end of the experiment. The illumination time was determined by the observed change in the Soret band intensity. Isosbestic points between fully oxidized and fully reduced proteins were chosen to monitor riboflavin reduction (452 nm for H93Y, and 457 nm for H64V/H93C and H64I/H93C Mb). The midpoint reduction potential of riboflavin was assumed to be -233 mV versus SHE at pH 8.0 (Draper & Ingraham, 1968). Reduction of the protein was monitored at 598 nm for H93 Y, and at 634 nm for H64V/H93C and H64I/H93C Mb. E. Cyanogen bromide titrations Cyanogen bromide (CNBr) titrations were performed on wild-type, H93C, H64V/H93C and H64I/H93C myoglobin (Shiro & Morishima, 1984). The wild-type protein was exchanged (Amicon microconcentrator) into 50 mM sodium phosphate buffer (pH 6.0), whereas the cysteine containing variants were exchanged into 50 mM Tris-HCl buffer (pH 8.9). A fresh solution of CNBr (28 mM) was prepared by dissolving CNBr (Kodak) in distilled water. A solution of wild-type Mb (8 uM) was 49 titrated with -10 molar equivalents of CNBr and electronic spectra were recorded (300-500 nm, 25 °C) until no further changes were seen. The titration was repeated on samples of H93C, H64V/H93C and H64I/H93C Mb (20 uM). F. Reconstitution experiments Apo-H64I/H93C Mb was prepared by the method of Teale (1959) as follows. A solution of myoglobin was placed into a small separatory funnel on ice and acidified to pH 2.0 with hydrochloric acid (1 M). An equivalent volume of cold 2-butanone (Fisher) was added, the solution was mixed, and the phases were allowed to separate. The upper, organic phase was removed and the procedure was repeated until no further color was extracted from the aqueous phase. The apoMb was then dialyzed at 4 °C against 0.6 mM N a H C 0 3 containing 1 mM EDTA, and 0.5 mM DTT. A second dialysis step was performed against 50 mM Tris-HCl buffer containing 0.5 mM DTT (pH 8.9). The concentration of apoMb was determined assuming a molar absorptivity at 278 nm of 16 mM' 1 cm'1 (Tamura etal, 1973). The reconstitution of H64I/H93C Mb was monitored spectrophotometrically between 300 and 500 nm. A stock solution of hemin (Porphyrin Products) was prepared by dissolving hemin in a minimum of 0.1 M N a O H . A 0.75 molar equivalent of hemin was added to a solution containing 21 uM protein in 50 mM Tris-HCl buffer and 0.5 mM DTT. The spectrum was monitored until no further changes were seen. Alternatively, the same experiment was conducted while monitoring the change in absorbance at 402 nm every 15 seconds for 4 hours. G. Coupled oxidation of myoglobin The coupled oxidation of myoglobin was monitored with a Cary model 3E spectrophotometer in 50 m M sodium phosphate buffer (pH 7.0) using a 1 mL cuvette (pathlength 1 cm) (O'Carra & Colleran, 1969). The buffer was saturated by bubbling a stream of oxygen through the solution for 50 approximately 1 hour. Prior to starting the experiment, 1 mL of 02-saturated buffer was placed in the instrument to equilibrate it at 37 °C. The experiment was initiated by the addition of protein (final concentration 3 uM and 5 uM for wild-type and V67A/V68S Mb, respectively) and ascorbate (Sigma, A-7506, final concentration 1 mM) to the cuvette. Spectra were recorded (280 to 800 nm) over a 7 hour period. Alternatively, the assay was monitored at a single wavelength (408 nm for wild-type and V67A/V68S Mb) for 10 hours. To ensure enough product for FfPLC analysis (below), the reaction was prepared on a large scale and carried out under similar conditions to those just described (O'Carra & Colleran, 1969). A beaker containing 10 mL of 50 mM sodium phosphate buffer (pH 7.0), 10 mg of protein (wild-type or variant) and 2 mg of ascorbate was placed in a water bath at 37 °C and stirred vigorously for 2 hours. The reaction was performed in the dark. H. Biliverdin extraction and HPLC analysis Biliverdin from the large scale coupled oxidation reaction was extracted by a method previously described (O'Carra & Colleran, 1969). The reaction mixture was cooled on ice for 15 minutes prior to extraction. Three mL of acetic acid and 8 mL of 5 M hydrochloric acid were then added to the cooled mixture. The solution was then placed in a 100 mL separatory funnel and extracted with diethyl ether (2x15 mL) (BDH). The ether layer contained any unreacted heme and was discarded. The aqueous layer was then extracted with chloroform (1x5 mL) to remove the biliverdin product. To help separate the phases in the previous step, the solution was placed in 50 mL glass centrifuge tubes and centrifuged for 5 minutes at 1000 rpm. Centrifugation separated the clear, upper aqueous phase from the green, lower organic phase leaving the denatured protein as a precipitate at the interface of the two phases. The chloroform was evaporated to dryness under a stream of argon gas. For FLPLC analysis a fraction of the product as well as the standards (Sigma) 51 (biliverdin, heme and bilirubin) were dissolved in a minimum volume of DMSO (BDH). HPLC was performed with a Beckman System Gold equipped with a diode array detector. Samples were loaded onto a C-18 reverse phase column (Alltech, 4.6 mm x 25 cm, 10 um). All solvents were HPLC grade. Solvent A consisted of 56 parts 0.1 M ammonium phosphate buffer (Fisher) and 44 parts methanol (Fisher). Prior to mixing, the ammonium phosphate buffer was adjusted to pH 3.5 by the addition of phosphoric acid (BDH). Solvent B was 100 % methanol. Solvents were filtered and degassed prior to use. The product and standards in DMSO were diluted into an appropriate amount of solvent A such that 20 uL could be injected onto the column. Approximately 150 pmol of the biliverdin product was applied to the column. The standard consisted of approximately 150 pmol of biliverdin, 50 pmol of hemin and 100 pmol of bilirubin. Detection was by absorbance at 405 nm. 52 RESULTS I. Proximal ligand variants A. TheH93Yvariant 1. Electronic absorption spectroscopy The electronic absorption spectra, wavelength maxima and extinction coefficients o f the H 9 3 Y variant are shown in Figure 6 and Table 3. The spectra recorded for the wi ld- type protein are similar to those reported for sperm whale (Tamura et al . , 1973; Egeberg et al . , 1990) and human (Adach i et al., 1991; Adachi et al . , 1993) myoglobin . Notab ly , the Soret band o f the Fe(III) derivative o f the var iant shifts f rom 408 n m ( p H 6.4) observed for the wi ld- type protein to 403 nm. The extinction coefficient o f the Soret band decreases from 188 m M ^ c m " 1 for the wi ld- type protein to 111 m M ^ c m " 1 for the variant. The electronic spectrum o f the oxid ized ( F e l l l ) form o f H 9 3 Y M b is independent o f p H between p H 7 and 10. Ti t ra t ion o f H 9 3 Y wi th an excess o f cyanogen bromide, wh ich causes a blue shift and decrease in Soret intensity in wi ld- type myoglob in (Shiro & M o r i s h i m a , 1984; Bracete et al . , 1991; A d a c h i & M o r i s h i m a , 1992)(see below) , had no effect on the spectrum o f the variant (data not shown). A l l o f these observations suggest that the ox id ized fo rm o f the variant possesses a five coordinate Fe(III) center. 2. Electrochemistry T h e results o f the photoreduct ion titrations o f the H 9 3 Y variant are shown in F igure 7. Reduc t ion o f the variant shifts the Soret maximum from 403 nm observed for the ox id ized protein to 429 nm. The reduction potential o f the H 9 3 Y variant is -208 m V vs S H E ( p H 8.0, 25 ° C , Nernst slope = 58 m V ) . This value is substantially lower than that obtained for the wi ld- type protein (+45 m V vs S H E , p H 8.0) ( L i m , 1990) and is comparable to the value reported for the corresponding human M b variant (-190 m V vs S H E ( p H 7); A d a c h i et al . , 1991). Th is result is consistent w i th the 53 Figure 6: The electronic absorption spectra of oxidized (solid), reduced (dashed) and CO-bound (dotted) H93Y Mb (7.4 uM). Samples were measured in 20 mM Tris-HCl buffer, pH 8.0 at 25 °C. 54 Table 3: Electronic absorption maxima and molar absorbances for wild-type myoglobin and the H93Y variant as shown in Figure 6. Absorption maxima [nm (mM'1 cm"1)] Protein Soret Visible Wild-type" Fe(III) 408(188) 502(10) 630(4) Fe(II) 435(121) 560(14) Fe(II)CO 424(207) 540(15) 579(14) H93Y b Fe(III) 403(111) 487(15) 524(13) 599(13) Fe(II) 429(113) 556(13) Fe(II)CO 419(188) 539(14) 570(16) ' pH 6.4 (Antonini & Brunori, 1971) bpH7-10. 55 Figure 7: Spectrophotochemical titration and corresponding Nernst plot (inset) of H93Y Mb (pH 8.0, 25 °C). Spectra of the completely oxidized (O) and completely reduced (R) species are labeled. Spectra were also taken at varying ratios of oxidized and reduced Mb by varying the potential as follows: -187.5, -207.8, -222.22, -237.25, -254.4, -275.4 mV versus SHE. The Nernst plot (inset) was derived from the dependence of the absorbance at 599 nm on the solution potential. 56 presence of an anionic phenolate proximal ligand, which is expected to stabilize the Fe(III) form of the protein. 3. Electron paramagnetic resonance (EPR) spectroscopy The EPR spectrum exhibited by the H93 Y variant consists of a mixture of high-spin, rhombic components with ^ -values of 6.94, 6.27, 6.08, 5.89, 5.01 and 1.99 (Figure 8). Addition of glycerol to the sample reduces protein heterogeneity (Bizzarri and Cannistraro, 1993) and resolves components with g-values of 7.13, 6.28, 6.06, 5.93, 4.89, 1.99 and 1.93. The EPR spectrum of the H93Y variant is similar to that of bovine liver catalase, which is also a high-spin, rhombic system (Williams-Smith & Patel, 1975; Blum et al., 1978; Palcic & Dunford, 1978). The EPR spectrum of bovine liver catalase, however, indicates the existence of pH-dependent forms (Blum et al., 1978) of this enzyme, whereas the EPR spectrum of the myoglobin variant is independent of pH between pH 7 and 10. The variant is unstable at pH<7, and no species resembling the hydroxo-bound (low-spin) form of wild-type ferrimyoglobin is observed at alkaline pH. 4. Nuclear magnetic resonance (NMR) spectroscopy The heme substituents of the ferric H93 Y variant in the absence of exogenous ligands exhibit much smaller chemical shifts than those of the wild-type protein in the low field region of the 'H-NMR spectrum and remain essentially unassigned although the largest resonances (labeled "a") are presumably attributable to the heme methyls (Figure 9). The majority of the resolved heme resonances have been assigned unambiguously for the wild-type protein by isotopic labeling experiments (La Mar et al., 1980). The spectrum of the H93Y variant is similar to the spectrum reported for the corresponding variant of human myoglobin (Adachi et al., 1993) and confirms the coordination of the phenolate group of Y93 to the heme iron. In the present case, however, a very broad resonance centered at -40 ppm not observed in the human variant can be identified that has been assigned to the 57 Field (mT) Figure 8: EPR spectra (4 K) of the high-spin ferric forms of recombinant (A) wild-type horse heart myoglobin (50 mM sodium phosphate buffer, pH 6.0), (B) H93Y variant (20 mM Tris-HCl buffer, pH 8.0) and (C) H93Y variant diluted with an equal volume of glycerol (50 % v/v). Protein concentrations were ~1 mM. 58 a a a a i—1—i—•—i—1—r 140 100 A »V«^iiitVw.)>y-' "I 1 1 1 1 1 1 ' 1 1 1 1 1 60 20 -20 -60 i—•—i—1—i—1—r I 60 20 B "i—•—i - 20 "i 1 r 140 100 60 Chemical Shift (ppm) Figure 9: Hyperfine shifted region of the 200 MHz 'H-NMR spectra of (A) recombinant horse heart metMb, and (B) H93Y variant in 50 mM deuterated sodium phosphate buffer at pD 7.0. Protein concentrations were ~2 mM. Labeling of the heme substituents is as follows: (a) methyls, (b) propionate CJL (c) vinyl C K H and propionate C a H, (d) propionate C p H, (e) phenolate meta protons, and (m) meso protons. Current methyl resonance assignments for the variant are based solely on their size. 59 meso protons of the macrocycle in model iron (Til) porphyrin compounds (Arasasingham et al., 1990; Caughey & Johnson, 1969). The large upfield shift of this resonance is characteristic of five coordinate high-spin Fe(III) (Bertini & Luchinat, 1986; Arasasingham et al., 1990; Rajarathnam et al., 1991). The broad signal at 112.5 ppm arises from the two meta protons of the axially coordinated phenolate side chain. Similar chemical shifts have been observed for the meta protons of phenolate derivatives of high-spin ferric porphyrin compounds (Arasasingham et al., 1990; Garcia et al., 1991; Goff et al, 1984). In contrast to the present work, the two meta protons of the axial phenolate ligand in the H93Y variant of human myoglobin do not appear as a single resonance but exhibit different chemical shifts (Adachi et al., 1993). B. The H64V, H93C and H64V/H93C variants 1. Electronic absorption spectroscopy The electronic absorption spectra, absorption maxima and extinction coefficients for the Fe(HI), Fe(II) and Fe(II)-CO derivatives of the variant proteins are shown in Figure 10 and Table 4. The absorption maxima of the H64V variant (Figure 10A) are essentially identical to those of the H64I variant. The Fe(III) forms of both proteins exhibit Soret maxima that are blue shifted (X^ (nm) = 395) and less intense than that of the wild-type protein. Such changes are typical for five coordinate Fe(III) centres and resemble the spectrum of cyanogen bromide modified, wild-type myoglobin (see below). The crystallographically determined three-dimensional structure of the H64V variant of sperm whale myoglobin indicates that the distal water molecule normally bound as the sixth axial ligand of wild-type metmyoglobin is not present in the variant (Quillin et al., 1993). Substitution of the distal histidine with small, nonpolar residues eliminates the hydrogen bond normally found between H64 and the coordinated water molecule. Loss of this interaction destabilizes the binding of water as the sixth ligand in the Fe(III) derivative of the variants. Moreover, the electronic spectra of oxidized 60 Figure 10: The electronic absorption spectra of oxidized (solid), reduced (dashed) and CO-bound (dotted) Mb. [(A) H64V, 7.4 uM, pH 6.5; (B) H93C,8.7 uM, pH 6.5; (C) H64V/H93C, 9.1 uM, pH 7.5] at 25 °C. Samples were measured in 50 mM sodium phosphate buffer 61 Table 4: Electronic absorption maxima and molar absorbances for wild-type myoglobin and the distal and proximal variants as shown in Figure 10. Absorption maxima [nm (mM"1 cm'1)] Protein Soret Visible Wild-type3 Fe(III) 408(188) 502(10) 630(4) Fe(II) 435(121) 560(14) Fe(II)CO 424(207) 540(15) 579(14) H64Vb Fe(III) 395(98) 506(12) 640(3) Fe(II) 434(121) 560(13) Fe(II)CO 425(200) 542(13) 576(12) H93CC Fe(III) 402(78) 498(11) 610(7) Fe(II) 429(115) 558(13) Fe(II)CO 422(171) 541(14) 571(14) H64V/H93CC Fe(III) 389(87) 509(13) 634(8) Fe(II) 427(109) 545(12) 570(13) Fe(II)CO 421(153) 539(13) 569(14) 1 pH 6.4 (Antonini & Brunori, 1971) b 50 mM sodium phosphate buffer (pH 6.5) c 50 mM sodium phosphate buffer (pH 7.5) 62 H64I and H64V exhibit an unusual pH-dependence that is unlike that seen for the wild-type protein (Bogumil etal, 1995). The spectra of the reduced and CO-bound forms of H64V and H64I Mb are essentially identical to those of the wild-type protein. The spectrum of the Fe(III) derivative of the H93C variant (Figure 10B) is quite different from that of the corresponding human myoglobin variant (Adachi et al., 1991). For example, at pH 6.5, the Soret maximum of the horse heart variant occurs at 406 nm while the Soret band of the human variant occurs at 391 nm. Raising the pH of the horse heart protein to 9.4 leads to a reversible shift of the Soret maximum to 399 nm (data not shown). Although raising the pH favors coordination of the proximal cysteine residue to the heme slightly, these data and the lower extinction coefficient suggests that the predominant state of the ferric iron atom lacks the interaction with the proximal cysteine. Interestingly, the electronic spectra of the Fe(II) and Fe(II)-CO derivatives of the horse heart variant are nearly identical to those exhibited by the corresponding human variant, suggesting that these derivatives of both species of variant exhibit similar axial ligation. It has been suggested that reduction of the heme iron in the human variant eliminates coordination by the proximal cysteine ligand. (Adachi et al., 1991, 1993). As the best characterized heme enzyme possessing a proximal cysteine ligand to the heme iron, cytochrome P-450 is unusual in that it lacks a distal histidine residue. In view of the role proposed for the distal histidine residue in other heme proteins in stabilizing ligand binding to the heme iron, the effect of eliminating this distal residue (H64) on coordination of C93 has been evaluated. The spectra of the Fe(III) derivatives of the H64I/H93C, H64V/H93C variants are similar to the spectrum of the Fe(III) derivative of the H93C variant of human myoglobin (Am a x (nm) = 390, pH 6.5-10.0)(Figure 10C). Thus, it appears that substitution of the distal histidine allows quantitative ligation of the proximal cysteine of the horse heart protein when the heme iron is in the Fe(III) state. 63 The reduced forms of the double variants exhibit Soret maxima that are blue-shifted (Am a x (nm) = 427) and decreased in intensity compared to that of reduced H93C. The spectra of the Fe(II)CO forms of the cysteine containing variants lacking the distal histidyl residue are inconsistent with proximal thiolate heme iron coordination. 2. Magnetic circular dichroism (MCD) spectroscopy To address further the nature of the ligation state(s) in these variants, a comparative MCD study was undertaken with the oxidized (Figure 11) and reduced (Figure 12) forms of these proteins. The spectral features of wild-type ferrimyoglobin are typical of six-coordinate, predominately high-spin, heme proteins, and the intense positive ellipticity in the Soret region is typical of oxygen or nitrogen containing ligands (Vickery et al., 1976). The Soret region of the wild-type protein has a broad derivative shaped appearance (X^ (nm) = 402, 417). Notable in the visible region is the broad maximum centered at 470 nm and the intense minimum at 640 nm, the latter of which shifts to lower energy in pentacoordinate systems with a single histidine ligand (see below). The spectrum of met H64V/H93C is similar to that of oxidized cytochrome P-450, chloroperoxidase and thiolate containing protoporphyrin models (Dawson et al., 1976). The strong negative intensity in the Soret region (A^fnm) = 392) is diagnostic of five-coordinate thiolate bound protoporphyrin IX. The visible region (Amax(nm) = 482, 550) is also distinct. The H93C variant shares some spectral similarities with the double variant in the visible region, but the shape and intensity of the Soret band (Am a x (nm) = 404, 420) of the single variant is not consistent with cysteinate axial ligation, and it is more likely that a water molecule acts as the predominant ligand in this variant. The cysteine containing variants were also studied in their reduced forms (Figure 12). Upon reduction of wild-type myoglobin to ferromyoglobin, the ligation state of the heme iron changes to 64 Figure 11: MCD spectra (25 °C, 2.0 T) of oxidized (A) wild-type (pH 7.0), (B) H93C (pH 8.0), and (C) H64V/H93C (pH 8.0) horse heart Mb prepared in 50 mM sodium phosphate buffer. Protein concentrations of 5xlO"5 M and 5x10"* M were used to record spectra in the Soret and visible regions, respectively. 65 Figure 12: MCD spectra (25 °C, 2.0 T) of reduced (A) wild-type (pH 7.0), (B) H93C (pH 8.0), and (C) H64V/H93C (pH 8.0) horse heart Mb prepared in 50 mM sodium phosphate buffer. Protein concentrations of 5xl0"5 M and 5X10"4 M were used to record spectra in the Soret and visible regions, respectively. 66 a five-coordinate species with loss of the distally-coordinated water molecule giving rise to a characteristic MCD spectrum (Vickery et al. 1976). The deoxygenated wild-type protein exhibits an intense Soret band (X^ (nm) = 436 nm) and a distinct visible region (X^ (nm) = -540, 591). A weak derivative feature centered at -567 nm is also observed for this form of the protein. The spectrum of H64V/H93C is clearly distinct from that of the wild-type protein as well as cytochrome P-450 (Dawson et al., 1976) and confirms that cysteinate is not the ligand in the reduced form of the double variant. This variant exhibits a weak Soret band (Xmax (nm) = 436) and broad maxima in its visible MCD spectrum (Am a x (nm) = 521, 560, 585). We propose that the reduced form of this variant possesses water as the axial ligand(s), but the ligation assignment remains tentative. In the case of H93C, however, there are spectroscopic similarities with wild-type myoglobin that suggest that the variant is also a five-coordinate system with an axial histidyl ligand. Most notable is the weak derivative feature centered at -563 nm that is present in the wild-type protein but absent from the corresponding spectrum of the H64V/H93C double variant. These observations are consistent with resonance Raman experiments of the corresponding human variant (Adachi et al., 1991, 1993) that suggest that the distal (H64) residue coordinates to the reduced heme iron. The differences in the Soret and visible MCD spectra between the reduced H93C variant and wild-type protein probably arise from incomplete ligation of the distal histidine in the variant. It is possible that this variant possesses a mixture of histidine and water bound forms in the reduced state. Note that the MCD spectra of oxidized and reduced H64I/H93C are essentially identical to those of the H64V/H93C variant. The MCD spectra of H64V and H64L of human metmyoglobin have been reported previously (Ikeda-Saito et al., 1992) and are similar to H64V and H64I of horse heart myoglobin (Figure 13). The spectra of these variants are characterized by weak Soret bands and are similar to 67 Figure 13: MCD spectra (25 °C, 2.0 T) of oxidized horse heart metMb. Samples of wild-type (solid) and H64V (dashed) were prepared in 50 mM sodium phosphate buffer (pH 7.0) to concentrations of 5xlO"5 and 5X10"4 M for acquisition of spectra in the Soret (350-450 nm) and visible (450-700 nm) region, respectively. 68 spectra recorded for naturally occurring five-coordinate heme proteins (Ikeda-Saito et al., 1992). As mentioned, a notable feature of these pentacoordinate variants is the shift of the 640 nm maximum (wild-type) to 654 nm (H64V and H64I). 3. Nuclear magnetic resonance (NMR) spectroscopy The hyperfine shifted regions of the ^ - N M R spectra of wild-type horse metmyoglobin and its H64V variant (Figure 14A and 14B) are similar to those of sperm whale and human metmyoglobins (Rajarathnam et al., 1991; Ikeda-Saito et al., 1992). A characteristic feature of five-coordinate heme systems is the upfield resonance of the meso protons (Arasasingham et al., 1990). The meso protons that appear downfield at -40 ppm in the spectrum of wild-type, hexacoordinate metMb are shifted to -29 ppm in the spectra of the H64V and H64I variants. In addition, the mean heme methyl resonance shifts approximately 5 ppm down field upon loss of the axially-bound water molecule. The heme methyl resonances of the wild-type protein appear at 92.4, 85.6, 72.7 and 52.5 ppm whereas they occur at 103.0, 88.3, 76.2 and 58.3 ppm in the spectrum of the H64V variant. As mentioned previously, the resolved peaks of the wild-type protein have been fully characterized and each methyl has been assigned (La Mar, 1980). The assignments of the H64V and H64I variants, however, are preliminary (Rajarathnam, 1991) and await isotope labeling experiments to assign all the resolved resonances fully. Current methyl resonance assignments are based solely on their size. The ^ - N M R spectrum of H93C Fe(ffl) (Figure 14C) derivative is not as well resolved as the corresponding spectra of the H64I/H93C and H64V/H93C variants (Figure 14D). The spectrum of the H93C variant exhibits a broad upfield resonance (~ -50 ppm) that is indicative of a five coordinate species. The spectra of the double variants more closely resemble the spectrum of the human myoglobin H93C variant. The downfield regions of these spectra exhibit methyl resonances at 62.9, 54.5, 41.4, and 32.7 ppm. In addition, a broad upfield resonance (~ -48 ppm) is observed that was 69 a a a a 100 50 0 -50 Chemical Shift ( ppm) Figure 14: Hyperfine shifted region of the 200 MHz ! H - N M R spectra of oxidized (A) wild-type, (B) H64V, (C) H93C, and (D) H64V/H93C horse heart metMb in 50 mM sodium phosphate buffer at pD 7.0. Protein concentrations were ~2 mM. Labeling of the heme substituents is as follows: (a) methyls, (b) propionate C a H , (c) vinyl C a H and propionate C a H , (d) propionate C p H (m) and meso protons. 70 not reported for the human variant and that is attributable to the meso protons (Arasasingham et al., 1990; Caughey & Johnson, 1969). 4. Electron paramagnetic resonance (EPR) spectroscopy All of the proteins in this study exhibit high-spin EPR spectra at 4.2 K. Wild-type metMb is a high-spin, axially symmetric system having g-values of 5.99 and 1.99 (Figure 15A). The EPR spectra of the H64V and H64I variants are similar to that of wild-type Mb (Figure 15B; Ikedo-Saito et al., 1992; Bogumil et al., 1995). However, the broadening of the low field region in the spectra of these proteins suggests that the iron centres of the H64 variants are slightly more rhombic than that of the wild-type protein. The EPR spectrum of the H93C Fe(III) variant (Figure 15C) exhibits a mixture of high-spin components. The major species has ^-values of 6.12 and 1.99 and is attributable to a form of the protein in which the C93 ligand is not bound to the heme iron. A minor, highly rhombic species having g-values of 8.44, 3.14 and 1.57 is attributable to a pentacoordinate form of the protein in which the C93 residue is coordinated to the heme iron. There is also evidence for a low-spin species (expanded region) having ^-values of 2.37, 2.24 and 1.94 that presumably arises from a hexacoordinate derivative in which the C93 residue and a distal water molecule are bound to the heme iron. The high and low-spin, cysteine-ligated forms exhibit EPR signals similar to those reported for theH93C variant of human metMb, which is six-coordinate (Adachi et al., 1991, 1993). It has been suggested that the low-spin form of the corresponding human metMb variant results from the effect of freezing the sample (Adachi et al 1991, 1993). The EPR spectra of the H64I/H93C and H64V/H93C double variants are identical (Figure 15D) to each other (g= 8.44, 3.14 and 1.57) and are similar to the high-spin species reported for the H93C variant of human metMb. The horse heart variants also exhibit a minor component having g-71 5.99 i 1 ~ i 1 r 50 150 250 350 450 Magnetic Field ( mT ) Figure 15: EPR spectra (4 K) of the high-spin ferric forms of recombinant (A) wild-type, (B) H64V, (C) H93C (low-spin and g=2 region expanded), and (D) H64V/H93C myoglobin. Samples were prepared in 50 mM sodium phosphate buffer (pH 7.0). Protein concentrations were ~1 mM. 72 values of 6.17 and 1.99 that results from a small amount of a derivative in which the C93 residue is not bound to the iron. This species only appears after heme extraction and reconstitution and apparently results from modification of the cysteine residue in the apoprotein because these signals are larger if no dithiothreitol is used during apo-protein preparation. The EPR spectra of the double variants exhibit no evidence of the low-spin form observed in the spectra of the H93C horse heart and human variants, presumably owing to destabilization of the bound water ligand in variants lacking the distal histidine. 5. Electrochemistry Substitution of the proximal histidine with anionic ligands such as phenolate and thiolate has been shown to cause a large decrease in the reduction potential of the protein (Adachi et al., 1991; Hildebrand et al., 1995). For the H64V/H93C (Figure 16) and H64I/H93C variants of horse heart Mb, introduction of a proximal cysteine residue lowers the reduction potential to -217 and -219 mV vs SHE, respectively (pH 8.0, 25 °C, Nernst slope = 48 and 44 mV, respectively). This is a substantial decrease relative to that of the wild-type protein (45 mV vs SHE at pH 8.0) (Lim, 1990) and is further evidence of thiolate ligation. These values are similar to that reported for H93C in human Mb (-230 mV; Adachi et al., 1991). The potentials of the H64V (Figure 17) and H64I variants are +87 and +95 mV vs SHE, respectively (pH 8.0, 25°C, Nernst slopes = 63). Removing the distal histidine is expected to increase the reduction potential of myoglobin by eliminating the distally-bound water molecule and decreasing the electron density around the iron. Due to active site heterogeneity, the reduction potential of the H93C variant of horse heart myoglobin was not measured. 6. Cyanogen bromide (CNBr) titration As shown in Figure 18A, addition of -10 equivalents of CNBr to wild-type horse heart metMb causes a blue-shift in the Soret band (Am a x (nm) = 395) and decreases its intensity (Shiro & 73 Figure 16: Spectrophotochernical titration and corresponding Nernst plot (inset) of H64V/H93C Mb (pH 8.0, 25 °C). Spectra of the completely oxidized (O) and completely reduced (R) species are labelled. Spectra were also taken at varying ratios of oxidized and reduced Mb by varying the potential as follows: -165.0, -185.3, -198.4, -210.4, -214.7, -223.9, -231.7, -235.7, -250.7 and -262.9 mV (vs SHE). The Nernst plot was derived from the dependence of the absorbance at 634 nm on the solution potential. 74 Figure 17: Spectroelectrochemical titration and corresponding Nernst plot (inset) of H64V Mb (pH 7.0, 25 °C). Spectra of the completely oxidized (O) and completely reduced (R) species are labeled. Spectra were also taken at varying ratios of oxidized and reduced Mb by varying the potential as follows: 169.9, 144.1,. 119.2, 95.1, 69.5 and 44.2 mV (vs SHE). The Nernst plot was derived from the dependence of the absorbance at 395 nm on the solution potential. 75 Morishima, 1984; Bracete et al., 1991; Adachi & Morishima, 1992). This modification is believed to involve cyanation of the distal histidine to produce a pentacoordinate heme iron as the result of disruption of the hydrogen bond between H64 and the water molecule normally coordinated to the heme iron. This experiment was repeated with the H93C variant (Figure 18B) in an attempt to explain the mechanism of ligation of the double variants. At pH 8.9, the Soret maximum is shifted from 399 nm in the unmodified variant to 405 nm following cyanation to produce a spectrum similar to that observed for the H93C variant at low pH (Figure 10, Table 4). Cyanogen bromide treatment, therefore, disrupts the bond between the iron and the proximal ligand. The same effect is seen for the double variants (Figure 18C), in which case the Soret maximum shifts from 389 nm to 404 nm. It appears that in the H93C variants, CNBr causes cyanation of the sulfur atom, which is known to be more reactive toward this reagent than is histidine (Shiro & Morishima, 1984), and that this reaction, therefore, competes with ligation of C93 to the iron. 7. Reconstitution ofH64I/H93CMb During reconstitution of the double variants with heme, time dependent changes in the electronic spectra of these proteins are observed (Figure 19). Reaction of hemin with apo-H64I/H93C Mb leads to a spectrum with an initial Soret band (X^ (nm) = 402) that blue-shifts very quickly (Amax (nm) = 389). The initial spectrum is similar to that of the H93C variant at pH 6.5 (Figure 10 and Table 4) and CNBr modified forms of the H93C, H64I/H93C and H64V/H93C variants (Figure 18B) and is attributable to heme that resides in the active site of the protein but that is not coordinated to the proximal protein ligand. Within an hour following reconstitution, the Soret maximum of these variants shifts to 390 nm. Thus, it appears that reconstitution of these variants occurs in two discrete steps: heme uptake, which is fast, followed by heme ligation to C93, which is slow. To monitor the 76 1.5 o X V H O X < 0.5 0 0.8 0.4 0 0.8 0.4 0 300 400 Wavelength (nm) 500 Figure 18: Titration (25 °C) of metmyoglobin with 10 equivalents of cyanogen bromide: (A) wild-type (50 mM sodium phosphate, pH 6.0), (B) H93C (50 mM Tris-HCl, pH 8.9), and (C) H64V/H93C (50 mM Tris-HCl, pH 8.9). Arrows indicate the direction in which the Soret maximum shifts during the reaction. 77 300 400 500 Wavelength (nm) Figure 19: Kinetics of heme binding to the H64I/H93C variant of horse heart myoglobin. Sequential spectra recorded after the addition of hemin at time = t0. The time after addition of hemin at which each scan was begun is as follows: a, 1.5; b, 7; c, 12; d, 18; e, 24 min. The direction of the shift in the Soret maximum is indicated by the arrows. Inset: Change in the absorbance at 402 nm over a four hour period. 78 heme uptake and ligation, a similar experiment was performed while monitoring the changes at 402 nm (Figure 19, inset). Although only small changes are seen in the spectrum after one hour, the absorbance at 402 nm continues to decrease over a period of at least four hours. For an accurate estimate of the absorbance of the fully-ligated derivative, the spectrum was determined after 24 hours. A monophasic process adequately describes the reconstitution process between approximately one and four hours following heme addition. The rate constant derived from such an analysis of this slower phase is OxlO" 4 s'1, and it presumably relates to cysteine ligation to the heme iron. 8. UnusualpH-dependence of H64VandH64IMb The pH-dependence of the electronic and EPR spectra of H64V ferrimyoglobin are shown in Figures 20 and 21. The isosbestic points observed in the electronic spectra recorded between pH 7.2 and 10.3 provide strong evidence that just two species are in equilibrium (Harris & Bertolucci, 1978). As the pH is raised the Soret maximum shifts from 395 nm to 404.5 nm. The alkaline forms of H64V and H64I exhibit identical electronic spectra (Am a x (nm) = 404.5, 502, 640). The pKa of this transition obtained by fitting the absorbance change at 395 nm to a one proton titration (Figure 20, inset) was 9.3 and 9.2 for the H64I and H64V variants, respectively. As previously mentioned, the EPR spectra of H64V and H64I Mb at pH 7.0 are axially symmetric with a small amount of rhombic distortion that causes line broadening in the low field region. Raising the pH to 10.5 decreases the axial component (g=5.99) and increases the abundance of a single rhombic component with g-values of 6.58, 5.35 and 1.98 in the case of H64V and 6.50, 5.46 and 1.98 in the case of H64I Mb (Bogumil et al., 1995). For neither variant was there any indication of any low-spin component at alkaline pH, that would signify the formation of the hexacoordinate, hydroxo species. On the basis of the available evidence one can assign this pH dependent behavior of the variants to the deprotonation of the proximal histidine, which converts the neutral ligand to the imidazolate form. 79 Figure 20: The pH-dependence of the electronic absorption spectra of the H64V variant. In the inset, the absorbance at 395 nm is plotted vs pH. The data were fitted to a one proton titration (continuous line). Above pH -10.5 the absorbance decreases as a result of protein instability. 80 Figure 21: EPR spectra (4 K) of high-spin ferric forms of recombinant H64V Mb at (A) pH 7.0, and (B)0.1M glycine, pH 10.5 in 50 mM potassium phosphate buffer. Protein concentrations were ~1 mM. 81 II. Variants designed to increase the polarity of the distal active site of myoglobin A. The V68H variant 1. Electronic absorption spectra The electronic absorption spectra of the oxidized and reduced derivatives of wild-type and V68H myoglobins are shown in Figure 22. The spectrum of the oxidized wild-type protein (Am a x (nm) = 408, 502, 630) is consistent with a high-spin ferric heme with water as the sixth ligand. The oxidized V68H derivative has a spectrum that differs substantially (A.max (nm) = 412, 527, -560) from that of the wild-type protein. Both the red shifted Soret band and the visible maxima are characteristic of low-spin heme iron centers and are essentially the same as those observed for bovine liver microsomal cytochrome bi (X^ (nm) = 412.5, 532, 560 nm; Ozols & Strittmatter, 1964) which has two axial histidine ligands (Mathews et al,- 1972; Argos & Mathews, 1975; Mathews, 1980). However, the variant exhibits an additional feature at -630 nm that is not seen in cytochrome b5 and is indicative of high-spin iron. Similar transitions have been identified in imidazole bound Mb (Am a x (nm) = 535, 568 and 647; Vitello et al, 1992; Beetlestone & George, 1964; Diven et al, 1966) which is known to exist as an equilibrium mixture of high and low-spin forms. However, from these data it is not possible to distinguish between high-spin metaquo Mb and high-spin bishistidine Mb. The Soret maximum (428 nm) in the reduced form of the variant lies between that expected for ferrous wild-type Mb (435 nm) and ferrous cytochrome b5 (423 nm) (Ozols & Strittmatter, 1964), but the broad features in the visible region preclude qualitative interpretation at this temperature. To determine the origin of the high-spin component in the oxidized derivative, the electronic spectra of the oxidized and reduced variant were studied as a function of temperature (77-323 K). Small but systematic changes in absorption maxima were identified. As the temperature was raised, the maxima at 527 nm and -560 nm decreased in intensity while the band at -630 nm simultaneously 82 Figure 22: Electronic absorption spectra of wild-type and V68H myoglobin. Oxidized (dashed lines) and reduced (solid lines) forms are shown. Samples (25 °C) of (A) wild-type Mb and (B) V68H variant were measured in 50 mM sodium phosphate buffer pH 6.0 and 7.0, respectively. (C) The low temperature (77 K) spectra of V68H Mb were measured in 50 mM sodium phosphate buffer (pH 7.0). For clarity, the spectrum of the reduced derivative in the visible (475-700 nm) is offset by +0.1 O.D. units against the oxidized derivative. Absorption maxima (nm) are indicated. 83 increased. A shift (Amax(nm) = 412 (10 °C), 411.5 (50 °C)) and a decrease in intensity of the Soret maximum was also observed. As the temperature is lowered further, the spectra exhibit additional changes. At 77 K, the oxidized derivative exhibits maxima (A^Cjim) = 414.5, 526, 560) that compare with those for cytochrome b5 at 77 K (Xm a x (nm) = 413, 531, 559; Davydov et al., 1980). The disappearance of the broad maximum at -630 nm is particularly noteworthy. These observations are consistent with the existence of a thermal equilibrium between high and low-spin iron, with the S =1/2 form dominant at 77 K and the S =5/2 form increasingly favored as the temperature is raised. In the reduced derivative at 77 K (Figure 22C), splitting of the broad feature at 560.5 nm is apparent (Xm a x (nm) = 429, 516.5, 527, 534.5, 555, 561) and results in a spectrum that is similar to the corresponding spectrum of reduced cytochrome b5 at low temperature (Am a x (nm) = 422, 514, 524, 530, 550, 555; Davydov et al., 1980). Although the maxima differ slightly, the distinct cc and P components that characterize ferrous (low-spin) cytochrome £>5can be identified clearly in the variant. Titration of the V68H variant between pH 5.0 and 10.0 resulted in no significant spectroscopic change. Beyond these limits, a decrease in the Soret maximum was observed that could be attributed to protein instability (data not shown). Exposure of the reduced protein to carbon monoxide resulted in formation of a species with a spectrum characteristic of carbonyl Mb (Am a x (nm) = 422, 541, 536) (data not shown). 2. Magnetic circular dichroism (MCD) spectroscopy Additional evidence for bishistidine coordination in the variant is provided by MCD spectroscopy (Figures 23 and 24). In the near-IR region at 300 K (Figure 23), wild-type metaquo Mb exhibits a positive peak at 1038 nm and a negative trough at 1183 nm that are characteristic of high-spin ferric heme iron (Eglinton et al., 1983). The oxidized V68H variant retains this high-spin transition (1041 nm and 1143 nm) as a minor feature but has an additional, dominant transition at 84 +0.6 < 800 1000 1200 1400 1600 Wavelength (nm) 1800 Figure 23: Near-JJR MCD spectra of wild-type (dashed lines) and V68H (solid lines) metMb (25 mM sodium phosphate buffer, pD 7.0 + 50% glycerol v/v, 300K, 4.8 T). 85 500 600 700 Wavelength (nm) Figure 24: Visible MCD spectra of reduced (solid line) and oxidized (dashed line) wild-type and V68HMb (25 mM sodium phosphate buffer, pH 7.0). (A) Wild-type Mb, 300 K; (B) V68H + 50% glycerol v/v, 300 K; (C) V68H variant + 50% glycerol v/v, 77 K. 86 1607 nm that is similar to those observed for imidazole-Mb (1600 nm) and cytochrome bs (1630 nm) (Gadsby & Thomson, 1990). This observation, therefore, provides strong evidence for bishistidine ligation at ambient temperature. Although from the MCD data alone it is not possible to identify the nature of the high-spin component (i.e., metaquo vs. high-spin bishistidine), there was no indication of any formation of hydroxide-bound Mb in the near-ER MCD spectrum as it was titrated to pD = 8.6. This result suggests that the high-spin transition does not derive from metaquoMb. The MCD spectrum in the visible region (450-700 nm) of oxidized and reduced V68H Mb (Figure 24) exhibits little resemblance to that of wild-type myoglobin (Vickery et al, 1976). The spectrum of the oxidized form at 300 K (Am a x (nm) = 487, 551, 570, 639) resembles other bishistidine systems. At 77 K the spectrum intensity increases and sharpens (Amax(nm) = 484, 541, 563). Note the disappearance of the band at 639 nm. With the exception of the 592 nm band and the overall intensity, the spectrum of the reduced form (A m a x (nm) = 511, 550, 561, 592) is essentially identical to that of reduced cytochrome bs (Davydovetal, 1980; Dawson & Dooley, 1989). At 77 K the spectrum sharpens (Am a x (nm) = 510, 549, 558) and is also very similar to that of cytochrome b5 at 77 K (Davydov et al, 1980). Although the origin of the 592 nm band is uncertain, the fact that it is absent at 77 K may indicate a spin-equilibrium as identified for the oxidized form. Together with the low temperature electronic absorption data (Figure 22C), this result provides strong evidence for low-spin, bishistidine ligation in the reduced V68H variant at 77 K. 3. Electron paramagnetic resonance (EPR) spectroscopy The EPR spectrum (10 K) of oxidized V68H Mb suggests that at this temperature the variant is a mixture of two species (Figure 25). The major component is high-spin with ^ -values (g = 5.99, 1.99) similar to those of wild-type Mb (g= 5.99, 1.99). A minor low-spin component (g = 3.24, 2.07) 87 200 300 400 Field (mT) Figure 25: EPR spectra of (A) wild-type met Mb (50 mM sodium phosphate buffer, pH 7.0, 4.2 K, [Mb]~2mM) and (B) the V68H variant (25 mM sodium phosphate buffer, pD 7.0 + 50% glycerol v/v, 10 K, [Mb]~2mM). The same sample of variant protein was used for NTR-MCD and EPR. The expanded region shows the spectrum of the minor, low-spin component. 88 is also apparent although its intensity is quite small at 10 K, and it almost completely disappears on cooling to 4.2 K (data not shown). Interestingly, the EPR spectrum of this low-spin component resembles that reported by Walker and co-workers (gmax~3A) for model compounds in which the planes of the coordinated imidazole groups are orthogonal to each other (Walker et al, 1984; Palmer 1985), in contrast to the EPR spectrum exhibited by cytochrome bs (gz~3.06; Gadsby & Thomson, 1990; Rivera et al, 1992; Lloyd et al., 1994) in which the planes of the coordinated imidazoles are essentially parallel. As the electronic spectra at 77 K clearly indicate the presence of bishistidine ligation, the variant evidently undergoes a temperature dependent ligation change with the histidine ligand largely displaced at 10 K. 4. Electrochemistry A family of spectra collected during a spectroelectrochemical titration of the V68H variant is shown in Figure 26 along with the corresponding Nernst plot (Figure 26, inset). The reduction potential of the variant was determined from the data to be -110 mV vs SFfE ( pH 7.0, 25 °C, Nernst slope = 61 mV), which is considerably lower than the corresponding value of 61 mV determined for the wild-type protein (Lim, 1990). The reduction potential for cytochrome b5 under identical conditions is 4 mV (Reid et al., 1982). 5. Nuclear magnetic resonance (NMR) spectroscopy The 'H-NMR spectrum of the oxidized variant (Figure 27) is similar to that previously reported for the H64V/V68H double variant of human Mb (which also exhibits a spin equilibrium) (Qin et al. 1994). In the case of the human variant, the mean methyl chemical shift and the temperature dependence of the heme resonances are consistent with the existence of a thermal spin equilibrium between a low-spin and high-spin states. The heme methyl resonances of the horseheart wild-type protein appear at 92.4, 85.6, 72.7 and 52.5 ppm whereas they occur at 48.2, 39.4, 26.3 89 o o 300 400 500 600 Wavelength (nm) 700 Figure 26: Spectroelectrochemical titration and the corresponding Nernst plot of the V68H variant (pH 7.0, 25 °C). Spectra of the completely oxidized (O) and reduced (R) species are labeled. Spectra were also taken at varying ratios of oxidized and reduced Mb by varying the potential as follows: -193, -176, -153, -133, -101, -75 and -55 mV (SHE).The Nernst plot was derived from the dependence of the absorbance at 412 nm on the solution potential. 90 a a a a 100 ' 60 ' 20 ' ^O Chemical Shift (ppm) Figure 27: Hyperfine shifted region of the 200 MHz !H-NMR spectra of (A) recombinant horse heart metMb and (B) the V68H variant in 50 mM deuterated sodium phosphate buffer, pD 7.0 . Protein concentrations were ~2 mM. Labeling of the heme substituents is as follows: (a) methyls, (b) propionate CJH, (c) vinyl C a H, (d) propionate C p H, and (m) meso protons. The methyl resonance assignments for the variant are based solely on their size. 91 and 23.6 ppm in the spectrum of the V68H variant. Although most of the resonances have been assigned for the human variant, this is not the case for the horse heart variant. For systems with a thermal spin equilibrium, the fractional high-spin to low-spin population can be estimated by using the change in chemical shift of any group R, o(R)obs, if the chemical shift of the pure high-spin and pure low-spin species are known (Qin et al., 1994): fraction high-spin = [(8(R)obs - 8(R)k )/(8(R)hs - 6(Rf)] (3) In this example, R was assigned to the mean methyl resonance, 8(CH3), which was approximately 34 ppm. For high-spin, six coordinate metMb, 5(CH3)~80 ppm, whereas for low-spin metMb, 5(CH3)~15 ppm. From the mean methyl shift of the horse heart variant, we estimate that approximately 29 % of the protein exists in the high-spin form at 20 °C. B. The V67A/V68S variant 1. Electronic absorption spectroscopy All of the data collected thus far suggest that placement of a polar serine residue on the distal side of the heme results in only subtle structural changes in myoglobin. Electronic absorption maxima and extinction coefficients of the oxidized, reduced and CO-bound forms of the variant are shown in Table 5. The native ligation states in the oxidized and reduced forms of the iron are maintained, as suggested by the respective positions of the Soret bands (A.max (nm) = 408 and 432) and the shape of the MCD spectra (Figure 28). The shape and intensity of the MCD spectrum of the oxidized (Xmax (nm) = 400, 415, -470, 637) form of the variant is very similar to that of the wild-type protein. Although the variant is capable of forming a stable carbon monoxide adduct (Amax (nm) = 421, 539, 576), the oxygenated form appears far less stable (below). Similar to the wild-type protein, the electronic absorption spectrum of V67A/V68S Mb is 92 Table 5: Electronic absorption maxima and molar absorbances for wild-type myoglobin and the V67A/V68S variant. Protein Absorption maxima [nm (mM'1 cm"1)] Soret Visible Wild-typea Fe(III) 408(188) 502(10) 630(4) Fe(II) 435(121) 560(14) Fe(II)CO 424(207) 540(15) 579(14) V67A/V68Sb Fe(III) 408(168) 501(10) 631(5) Fe(II) 432(119) 558(13) Fe(II)CO 421(200) 539(15) 576(13) a pH 6.4 (Antonini & Brunori, 1971) b pH 7.0 93 400 500 600 700 Wavelength (nm) Figure 28: Visible MCD spectrum of the oxidized V67A/V68S variant (50 mM sodium phosphate buffer, pH7.0, 25 °C, 0.81 T). 94 highly pH dependent (Figure 29). Increasing the pH converts the variant from the metaquo form to the hydroxide-bound form (X^ (nm) = 410, 540, 579) with a pKa for this transition of 9.3. The formation of the hydroxide-bound form of the wild-type protein (Am a x (nm) = 411, 539, 585) occurs with a pKa of 8.9 (Antonini & Brunori, 1971). A typical titration can be performed to approximately pH 10.5, after which protein stability appears to be compromised. 2. Electrochemistry A family of spectra collected during a spectroelectrochemical titration of the double variant is shown in Figure 30 along with a Nernst plot of the data (inset). The reduction potential of the variant is -23 mV vs SHE (pH 7.0, 25 °C, Nernst slope = 62 mV), which is considerably lower than the corresponding value of 61 mV determined for the wild-type protein under identical conditions (Lim, 1990). 3. Coupled oxidation of myoglobin The modification of the heme prosthetic group in heme proteins exposed to ascorbate in the presence of dioxygen has been known for 65 years and is referred to as coupled oxidation (Warburg &Negelein, 1930; Lemberg, 1956). The oxidation of heme to biliverdin following aerobic addition of ascorbate was monitored spectrophotometrically for wild-type metMb and the variant (Figure 31). Addition of excess ascorbate to wild-type myoglobin results in complex time dependent changes in the electronic absorption spectrum that involves a slow conversion of metMb to oxygenated Mb (Am a x (nm) = 418, 542, 580) with subsequent slow decay to the Mb-biliverdin complex. The kinetics of this reaction are described adequately as the sum of two single exponential processes (Figure 3 2 A) with k,= 1.6 x io-2 and k2- 1.7 x 10'3 min'1. On the other hand, addition of excess ascorbate to the V67A/V68S variant results in a rapid decrease in the absorbance at the Soret maximum (408 nm) (Figure 31). A similar kinetic analysis assuming two exponential processes results in k, = 1.1 x 10"1 95 Wavelength (nm) Figure 29: The electronic absorption spectra of met V67A/V68S Mb as a function of pH (0.1 M NaCI, 25 °C). Spectra were recorded in the following order: pH 6.96, 7.27, 7.59, 8.00, 8.32, 8.68, 9.06, 9.28, 9.53, 9.80, 10.16, 10.51. The titration was monitored at 580 nm and fit to a single deprotonation process (inset). Above pH -10.5, the variant was unstable. 96 +34 Wavelength (nm) Figure 30: Spectroelectrochemical titration and the corresponding Nernst plot of the V67A/V68S variant (50 mM sodium phosphate buffer, pH 7.0, 25 °C). Spectra of the completely oxidized and (O) and reduced (R) species are labeled. Spectra were also taken at varying ratios of oxidized and reduced Mb by varying the potential as follows: -97.4, -74.4, -50.4, -25.5,-1.2, 22.7 mV (SHE). The Nernst plot was derived from the dependence of the absorbance at 408 nm on the solution potential. 97 350 450 550 650 Wavelength (nm) Figure 31: Changes in the electronic absorption spectra of wild-type and variant Mbs during coupled oxidation of horse heart Mb (50 mM sodium phosphate buffer, pH 7.0, 37 °C) containing 1.0 mM ascorbate. (A) Wild-type Mb (3 uM); (B) V67A/V68S Mb (5 uM). The time after initiation of the reaction at which spectra were recorded were as follows: (a) 0 h, (b) 1 h, (c) 2 h, (d) 3 h, (e) 4 h, (f) 5 h, (g) 6 h, (h) 7 h. 98 0.25 1 1 1 1 1 1 1 0 100 200 300 400 500 600 Time (min) +0.3 £ 0.0 «, -0.3 < 0.10 1 ' ' ' ' ' 1 0 100 200 300 400 500 600 Time (min) Figure 32: Change in absorbance monitored at the Soret maximum during aerobic coupled oxidation of horse heart Mb (3 uM) in the presence of ascorbate (1 mM). (A) Wild-type and (B) V67A/V68S Mb were monitored at 408 nm. The lines through the data points represent the non-linear least squares fits to the data. The residual analysis are shown above the kinetic data. 99 and k2 = 5.3 x 10"3 min"1 (Figure 32B). The assay was repeated with a concentrated sample of the variant to better assess the spectral changes in the visible region (Figure 33). In this experiment the increase in absorbance at 705 nm that is consistent with Fe(III)-biliverdin formation is notable (Sano et al., 1986). Although the Soret region showed no evidence for oxyMb formation in the assay of the variant (Figure 32B), two maxima are present in the visible region at 539 and 575 nm that may indicate the presence of a small amount of oxygenated species (Figure 33). It is possible to produce the oxygenated form of this variant, but it autoxidizes too rapidly for characterization. Alternatively, these bands could be due to a small amount of carbonyl Mb formed during the reaction as the result of heme degradation. There is spectroscopic evidence for the presence of an additional species, as indicated by the broad band at -650 nm, that may be due to one of the intermediates (verdoheme) that is known to form during the coupled oxidation reaction of myoglobin (Sano et al., 1986; Modi et al., 1989). To probe the identities of the final heme degradation products formed during coupled oxidation of the wild-type and variant proteins, the reaction mixtures were allowed to incubate for two hours, and the heme prosthetic groups were then extracted and subjected to analysis by HPLC. Both the wild-type protein and the variant exhibit a product with a retention time of 17 minutes under the conditions employed that is identical to that exhibited by an authentic biliverdin standard (Figure 34). In theory, four isomeric products may be formed in this reaction depending on which meso carbon is oxidized (a,P,y,o). The dimethyl ester (DME) derivatives of the four isomers are separable by HPLC analysis (Noguchi et al., 1982). Esterification of the biliverdin products followed by HPLC analysis results in a single peak on the chromatogram (data not shown) for both the wild-type and variant myoglobins and confirms that the specificity for the a-meso position is unchanged by the active site substitutions. 100 0 1 • • • 1 1 — 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 Wavelength (nm) Figure 33: Changes in the electronic absorption spectra of V67A/V68S Mb (50 uM) during coupled oxidation (50 mM sodium phosphate buffer, pH 7.0, 37 °C) with 5.0 mM ascorbate. The times after initiation of the reaction at which spectra were recorded were as follows: (a) 0 h, (b) 0.5 h, (c) 1.0 h, (d) 1.5 h, (e)2.0h. 101 (i) (ii) (iii) Retention Time (min) Figure 34: HPLC (C-18 reverse phase, 4.6 mm x 25 cm, 10 um) analysis of product isolated from the coupled oxidation reaction of horse heart myoglobin with ascorbate. (A) Standard mixture of authentic (i) biliverdin, (ii) heme, and (iii) bilirubin; (B) product extracted from wild-type Mb, and (C) the V67A/V68S variant. 102 To elucidate the possible influences of the V67A/V68S substitutions on the manner in which exogenous ligands bind to the heme iron, the FTIR spectrum of the carbonyl derivative was examined and the CO stretching frequency, v c o , of the variant was compared with that of the wild-type protein. As previously observed (Caughey et al., 1969), the predominant v c o band of the wild-type protein occurs at 1945 cm'1 (Figure 35). In contrast, the corresponding spectrum of the variant exhibits two equally intense v c o bands at 1948 and 1962 cm"1. The v c o band at 1962 cm"1 differs from that observed for other Mb variants with a serine residue at position 68 (v c o = 1949 cm"1) and is reminiscent of the spectra of variants possessing a threonyl residue at this position ( v c o = 1958 cm"1) (Li et al., 1994). 103 A Figure 35: Infrared spectra (FTIR) of ferrous carbonyl derivatives (average of 500 scans) of (A) wild-type Mb and (B) the V67A/V68S variant (50 mM sodium phosphate buffer, pH 7.0, 25 °C, ~3 mM). 104 D I S C U S S I O N Prior to the advent of site-directed mutagenesis, the only available means of probing the structural contributions of specific amino acid residues in the active site of myoglobin was through investigation of various species of myoglobin or through chemical modification. Comparative studies of various species of protein are of limited value because there are few instances in which sequences differ by just a single residue. Use of chemical modification is generally compromised by the limited specificity with which individual amino acid residues or types of residues can be modified and the difficulty with which the sites of modification in such proteins can be rigorously characterized. This latter difficulty, however, has been greatly remedied through the recent development of electrospray mass spectrometry. The work included in the present study represents some of the first work in which site-directed mutagenesis has been used to study horse heart myoglobin. As such, this work follows on previous work in other laboratories concerning related studies of sperm whale, pig, and human myoglobins. Some of the variants studied in this work involve amino acid substitutions that are identical to those reported previously for these other species of myoglobin and some have not been studied previously in any species of myoglobin. Interestingly, even in those cases where the same mutations have been introduced previously in other species of myoglobin, subtle species specific differences in behavior were observed as a result of the mutation. In the discussion of the current results provided below, an effort has been made to consider the relationship of the new findings to those of related studies with variants of other species and with relevant chemically modified derivatives of myoglobin. 105 I. Proximal ligand variants. A. TheH93Yvariant Replacement of the proximal histidine l igand of myoglob in w i t h a tyrosine residue produces a variety o f interrelated functional and structural changes to the protein that provide insight into the fundamental role that the proximal ligand plays in dictating the properties o f heme proteins in general. Interest ingly, al though the tyrosine side chain is larger than that o f a histidine residue, the heme binding site can adjust to accommodate the new residue through displacement and increased mobil i ty for the F helix, without a concomitant decrease in thermal stability. M o r e o v e r , N M R , electronic, and E P R spectroscopy, as w e l l as the crystallographically determined three-dimensional structure (Hildebrand et al., 1995), all indicate that in contrast to the wi ld- type protein, the ferric form o f the variant is five coordinate under all conditions considered in this study. 1. Spectroscopic considerations. T h e electronic absorption and E P R spectra o f the H 9 3 Y variant a l low comparison to catalases, the natural tyrosinate bound heme enzymes that catalyze the decomposi t ion o f hydrogen pe rox ide in a w i d e range o f organisms. The posi t ion and intensity o f the Soret band in various derivatives o f heme proteins reflect the l igat ion state o f the i ron. The blue-shift and decrease in the extinction coefficient o f the variant relative to the wi ld- type protein is similar to bovine l iver catalase ( A m a x (nm) = 405; e ( m M ' 1 cm' 1 )= 100; B r o w e t t & Sti l lman, 1980). M a g n e t i c techniques, such as E P R spectroscopy, permit direct analysis o f heme protein electronic environments and offer information regarding spin and ligation-state changes induced by proximal ligand substitutions. The spectrum o f the variant confirms that at 4 K the i ron is high-spin (S=5/2) between p H 7 and 10, and exists as a mixture o f rhombical ly distorted components. A rhombic E P R signal is also seen in all known (heme containing) catalases and appears to be a general 106 phenomenon of tyrosyl ligation. Bovine liver catalase possesses two major rhombic high-spin ferric heme signals (g=6.90, 6.55, 5.42, 5.05; Blum et al., 1978). The origin of the mixture in the variant (and in catalase) is unknown, but the fact that it is present in both suggests the tyrosine ligand is the cause and possibly due to small conformational changes in the protein which affect the electronic environment of the heme iron. The NMR spectrum obtained in the current study differs from that reported previously for the corresponding H93Y variant of human myoglobin (Adachi et al., 1993) in that the two meta protons of the proximal phenolate exhibit different chemical shifts (128.1 and 107 ppm) in the human protein. In contrast, the spectrum of the H93 Y variant of the horse heart protein studied here exhibits a single signal at 112 ppm that is attributable to these protons. For the phenolate ligands of Fe(III) protoporphyrin IX model compounds in which there are no steric constraints on the orientation of the axial ligand relative to the porphyrin ring, the two meta protons of the phenolate ligand exhibit the same chemical shift (Caughey & Johnson, 1969; Arasasingham et al., 1990). In related systems where the phenolate ligand is held in a restricted rotation, the two meta protons exhibit different chemical shifts (Goff et al., 1984). With pendant-capped Fe(III) porphyrins in which the axial phenolate is forced into a fixed conformation and the porphyrin ring is perfectly symmetric, these protons exhibit identical chemical shifts because their chemical environments are the same (Garcia et al., 1991). Based on these reports for model compounds, the current result indicates that the environments of these protons are equivalent in the horse heart protein and, therefore, implies that the phenolate ring of the bound tyrosine residue can flip at a rate that is fast on the time scale of the NMR measurement. Note that the lack of an NMR spectrum for catalase (MW~250,000) precludes any comparison to this protein. A more specific mechanistic origin for this observation can be obtained by a comparison of 107 the horse heart and human myoglobin sequences, which differ at 17 positions. Examination of the three- dimensional structure of the horse heart protein reveals that the side chain of 1142 is the only one of these 17 groups that is located in proximity to the proximal ligand to the heme iron. Although the three-dimensional structures of human myoglobin and its H93Y variant are not currently available, we estimate that the closest approach of the 1142 side chain to the Y93 side chain in the horse heart variant is -0.7-1.0 A longer than the shortest distance between the nearest atom of M142 in the corresponding human variant. We, therefore, suggest that the M142 residue in the human protein constrains the rotation of the proximal tyrosine ligand in the H93Y variant of human myoglobin. 2. Structural considerations. The structural analysis of H93 Y myoglobin was conducted by David Burk and Robert Maurus in the laboratory of Professor Gary Brayer and appears, in part, in this discussion to facilitate comparisons to the observed functional and spectroscopic properties. The mechanism by which a tyrosyl residue is accommodated to provide a stable environment for heme binding is apparent from the three-dimensional structure of the variant determined by X-ray diffraction analysis. This structure represents the first refined structure of a proximal ligand variant of myoglobin (Figure 36). The principal conformational change observed is the displacement of the F helix (88-99), on which the proximal histidine ligand resides, away from the heme. This movement of the F helix is accompanied by a shift of the heme iron atom out of the plane of the porphyrin ring toward Y93 and by an increase in the thermal factors of the residues in this helix that is indicative of increased conformational flexibility in the variant. At the same time, the water molecule bound as the sixth ligand in wild-type myoglobin is lost, and the side chain of Y93 rotates relative to the position of the H93 imidazole group so that the angle between the plane of the residue-93 side chain and the porphyrin ring 108 Figure 36: (a) Stereo drawing of the alpha-carbon backbones of the wild-type (thin lines; Evans & Brayer, 1988) and H93 Y variant (thick lines; structural analysis by David Burk and Robert Maurus in the laboratory of Professor Gary D. Brayer) myoglobin structures. Every tenth residue is labeled. Note the displacement of helix F (residues 88-99) caused by the substitution, (b) Detailed stereo diagram showing the immediate environment of the mutation site. 109 increases. The positional changes caused by the mutation are accompanied by several changes in hydrogen hydrogen bonding interactions that involve either substituents of the heme prosthetic group or residues that are in the heme binding pocket. S92 is involved in three of these changes in that hydrogen bonds normally with propionate A and H93 are not present in the variant, while a new hydrogen bond is formed between the S92-OH group and the NE2 atom of H97. In addition the hydrogen bond between the H97 NE2 atom and the propionate A, and the hydrogen bond formed by K45 and the propionate D, are not present in the variant. The loss of three hydrogen bonds formed by heme substituents in the wild-type protein probably contributes to the increased thermal factors observed for atoms of the F helix and may explain the decreased stability of the variant at pH<7. It is also conceivable that the reduced number of interactions between the heme prosthetic group and the apoprotein may contribute to the expression of this variant in E. coli as the apoprotein rather than as the holoprotein as observed for the wild-type protein. Nevertheless, at neutral or alkaline pH, the stability of the reconstituted Fe(III) protein appears to be comparable to that of the wild-type protein. The movement of the F helix away from the heme prosthetic group is accompanied by a displacement of the heme iron atom out of the plane of the heme toward the Y93 ligand. Similar displacement of the heme iron has been observed for five-coordinate model heme complexes (Koenig, 1965; Hoard et al., 1965). The position of the Y93 residue relative to the heme group resembles that of bovine liver catalase. In both proteins, the plane of the tyrosine ring eclipses the Fe-pyrrole nitrogen bonds of pyrroles A and C. However, the tyrosine ligand in bovine catalase forms a hydrogen bond with an adjacent arginine side chain that is analogous to the hydrogen bond formed by the S92- OH group and the proximal histidine residue of wild-type protein. No hydrogen-bonding interactions of this type occur in the H93 Y variant. The movement of the F helix combined with the greater electronegativity of the tyrosyl 110 oxygen atom (relative to histidine) presumably reduce the affinity of the iron for coordination of water as the sixth ligand. Interestingly, the three dimensional structures of the bovine liver (Reid et al., 1981; Murthy et al., 1981; Fita & Rossman, 1985, 1986) and fungal catalases (Vainshtein et al., 1986) indicate that the sixth coordination positions of these proteins are also unoccupied while similar studies indicate that a water molecule is bound as the sixth heme iron ligand in a bacterial catalase (Murshudov et al., 1992). From this limited number of structures it appears that although the presence of a proximal tyrosyl ligand increases the probability of a penta-coordinated heme iron, other structural factors may overcome this propensity. The altered hydrogen bonding interactions of the heme substituents and S92 observed here illustrate the subtle structural changes that can result from modification of critical active site residues in proteins of this type. Similarly, comparison of the current NMR results with those obtained for the corresponding variant of human myoglobin demonstrates the subtle manner in which apparently small species-dependent structural differences can produce significant effects on the dynamics of protein structure. 3. Ferrous H93YMb and catalase activity. The large decrease in the midpoint reduction potential of this variant is consistent with stabilization of the ferric protein by phenolate ligation; however, the value is still far above that of catalase (< -500 mV vs SHE; Adachi et al., 1991). A major determinant of heme protein reduction potentials (and hence function) is the type of proximal ligand to the iron, but clearly this is not the only consideration (Moore & Williams, 1977; Moore et al., 1986). The active site structure of catalase must be responsible for the low potential of the enzyme but it is difficult to speculate on the cause from examination of the structure. A low reduction potential appears to favor formation of the higher oxidation states involved in the mechanism of this class of heme enzyme and distinguishes it 111 from the oxygen binding globins which tend to have higher potentials. Although it is possible to reduce the H93 Y variant, the ligation state of the ferrous derivative remains unclear due in part to the lack of spectroscopic models for comparison. Little spectroscopic evidence is available for the Fe(II) form of catalase, presumably due to the difficulty in completely reducing this enzyme. It is possible that the ligand in H93 Y Mb is displaced upon reduction, but the exact nature of the ligation state may have to await the availability of a high resolution crystal structure of this myoglobin variant. Intriguing functional considerations regarding this variant include its reactions with dioxygen and with hydrogen peroxide. An attempt was made to prepare the oxyMb derivative of this variant by reduction of the metMb derivative with dithionite followed quickly by elution over a column of Sephadex G-25. While this method is sufficient for generation of wild-type oxyMb, the variant rapidly autoxidized (data not shown), presumably owing to its low reduction potential. Thus, characterization of the oxygen binding properties of this variant is not feasible. Addition of a 10-fold excess of hydrogen peroxide to the H93 Y metMb derivative results in a rapid decrease in the intensity of the Soret band along with protein precipitation. There is no evidence for transient formation of the ferryl derivative that such treatment produces for wild-type myoglobin (e.g., Wittenberg, 1978). It seems clear that replacement of the proximal histidine ligand of myoglobin with a tyrosine residue produces a good spectroscopic model of oxidized catalase, but is not sufficient to convert Mb to an effective enzyme. This finding is not surprising in light of the many additional structural differences that distinguish catalases and myoglobin from each other. B. The H64V, H93C and H64V/H93C variants 1. Comparison of horse and human H93C myoglobin. The amino acid sequences of horse heart and human myoglobins exhibit an 89% identity. Nevertheless, subtle differences between the proximal H93 Y variants of horse heart and human 112 myoglobin were discussed previously that were attributed to minor structural differences on the proximal side of the heme binding pocket. The experiments reported here demonstrate the existence of similar subtle differences in the properties of the proximal H93C variants of these two species of metmyoglobin. We have found no experimental conditions that permit quantitative ligation of the proximal cysteine residue to the heme iron as observed for human myoglobin (Adachi et al., 1991, 1993). Evidently, the distance between the iron and the proximal cysteine residue in the horse heart protein is too great to permit its facile coordination. From the spectroscopic results described previously and by analogy to previous results reported for the human variant (Adachi et al, 1991, 1993), we propose that the ferriheme iron center in this variant is predominantly pentacoordinate, with a water molecule bound to the exchangeable, distal coordination position and without coordination of the proximal cysteine residue. Furthermore, we conclude that the distal coordination of this water molecule is stabilized through hydrogen bonding interaction with the distal histidine residue. There also exists some hexacoordinate (cysteine/H20-bound) and pentacoordinate (cysteine-bound) species as minor components, as seen by EPR spectroscopy. From previous crystallographic studies of sperm whale myoglobin variants, it is known that substitution of H64 with apolar residues such as valine or leucine destabilizes distal coordination by an aquo ligand through elimination of the hydrogen bond normally formed with this residue and that in the H64V variant of sperm whale myoglobin, the iron atom is displaced from the pyrrole nitrogen plane by 0.18 A toward the proximal side relative to the wild-type protein (Quillin et al., 1993). In the present study it was found that when H64 is replaced with Val (or Ile) in the H93C variant of horse metmyoglobin, the anticipated displacement of the iron, although small, is sufficient to allow quantitative coordination of Fe(III) by the proximal cysteinyl residue so that the H64V/H93C (and H64I/H93C) double variant possesses a pentacoordinate heme centre in which the distal ligand 113 binding site is vacant. The resultant double variants have spectroscopic properties resembling those of the high-spin form of cytochrome P-450 from Pseudomonas putida. This class of monooxygenase contains a pentacoordinate heme ligated by a cysteinate ligand and lacks a distal histidine or other polar residues capable of interacting with bound ligands (Poulos et al., 1987). The ligation state of the reduced form of the H93C variant involves a novel rearrangement of the active site of the protein. Adachi and co-workers (1993) suggest that the iron atom in the reduced form of the human H93C myoglobin variant is coordinated by the distal H64 residue and that the proximal ligand binding site is unoccupied. Furthermore, binding of CO to this variant was proposed to occur at the proximal side of the heme. The electronic spectra of both the reduced human and horse heart H93C variants indicates that the behavior of both proteins is similar under these circumstances. The MCD spectrum of the reduced H93C variant provides further evidence that it is possible for the distal histidine to coordinate at least partially to the iron under some conditions. Interestingly, the electronic spectra of the reduced H64V/H93C and H64I/H93C variants resembles that of deoxy myoglobin at pH 2.6 in that they possess a blue-shifted Soret and two discernable peaks in the visible region (Han et al., 1990). At low pH, the proximal histidyl residue of wild-type deoxymyoglobin is thought to be replaced by a weak field ligand such as water (Han et al., 1990; Palaniappan & Bocian, 1994). Although the similarity of the electronic spectra of 5 and 6-coordinate model ferroheme complexes (Han et al., 1990; Brault & Rougee, 1974) precludes unambiguous assignment of the coordination state of the ligand-free Fe(II) derivatives of the H64V/H93C and H64I/H93C double variants, the carbonyl derivatives of these double variants can be identified as 6-coordinate based on the observation that the electronic spectra of 5-coordinate ferrous heme carbonyl complexes exhibit Soret maxima at -390 nm (Traylor et al., 1985). 114 2. Trans effects on Proximal Coordination by Cys93. The sine qua non of the cytochrome P-450 family is the red-shifted Soret maximum of the reduced CO derivative (Klingenberg, 1958; Garfinkel, 1958; Omura & Sato, 1962). This spectroscopic feature is generally regarded to be diagnostic of proximal thiolate coordination and has been assigned to a cysteine-heme charge-transfer transition (Hanson et al., 1976). The reduced carbonyl forms of the H93C, H64V/H93C and H64I/H93C horse heart myoglobin variants do not exhibit this characteristic Soret maximum. Instead, the Fe(II)CO derivatives of these variants exhibit electronic spectra similar to those observed for wild-type myoglobin and non-native cytochrome P-450 (cytochrome P-420), respectively, both of which possess proximal histidyl ligands (Wells et al., 1992). Although it is likely that the distal histidyl residue is at least partially coordinated to the heme iron in the CO form of the H93C variant of the horse heart variant, this cannot be the situation in the Fe(II)CO derivative of the double H64V/H93C variant. In this case, it seems likely that the distal coordination position is occupied by a water molecule. Interestingly, a semisynthetic cytochrome c in which one of the natural ligands is replaced with a cysteine residue (M80C) has been proposed to possess a thiolate bound to the heme iron in the oxidized form but not in the reduced form of the protein (Smulevich et al., 1994). These authors suggest that in the Fe(II)CO form of M80C cytochrome c, the cysteine ligand may be either protonated or displaced. 3. Nature of the Cys93-heme iron bond. Modification of C93 in the double variants by reaction with cyanogen bromide suggests that the interaction of this residue with the iron is weak. Weak proximal coordination in this variant may be consistent with either the requirement for a significant conformational change to permit C93 coordination to the heme iron or with a relatively long Fe-S bond. The slow rate of proximal ligand binding to the iron suggests that the proximal C93 residue represents the lower limit on coordinating 115 ligand size that can be introduced successfully at this position. Modeling of the H93C variant of horse heart myoglobin fails to explain the conformational changes required for ligation of the cysteine sulfur in the double variants. Assuming no other conformational changes, the closest approach of a cysteine sulfur at position 93 to the iron is approximately 3.9 A, a distance too great for bond formation. The iron out-of-plane movement observed in pentacoordinate variants is not sufficient to obtain the optimal bonding distance of approximately 2.5 A. Clearly, additional conformational changes are required in the proximal region of the active site to allow cysteine ligation to the heme iron in these variants. The crystal structure of H93 Y myoglobin revealed some flexibility in the F-helix that allows it to move in response to the substitution at position 93. The smaller cysteine ligand may allow movement of the helix to occupy a position slightly closer to the iron, and combined with the iron out-of-plane movement in the double variants, facilitates complete ligation. The range of ligand size that can be accommodated at position 93 suggests a remarkable degree of conformational flexibility in the active site of myoglobin although this flexibility is not sufficient to retain coordination by C93 upon reduction of the heme iron. 4. Unusual pH-dependence ofH64Vand H64IMb. Spectroscopic and structural studies establish that the coordination environment of the heme iron of the H64V and H64I variants are pentacoordinate. As such, the origin of the pH-dependent spectral changes observed for these variants is unrelated to an axially bound water molecule which is known to titrate with a pKa of 8.9 in wild-type metMb (Antonini & Brunori, 1971). MetMb-OH exists as a mixture of high-spin and low-spin iron in thermal equilibrium (Beetlestone & George, 1964; Feis et al., 1994) and no evidence was observed for any low-spin species in the variant proteins studied here. The pH-dependent electronic and EPR spectra of these variants can be attributed to an equilibrium between two ferric, high-spin forms that involves deprotonation of the proximal H93 116 residue to produce imidazolate ligation. The pKa values are in the range expected for deprotonation of the proximal histidine and are consistent with the decrease in imidazole pKa expected to occur upon coordination to a metal ion, and are much lower than that seen for free imidazole (pKa -14) (Sundberg & Martin, 1974). This deprotonation was also observed for the H64T variant of horse heart Mb for which there is a high-resolution crystal structure that confirms the pentacoordinate nature of the heme iron of the oxidized form of this variant (Bogumil et al., 1995). A common structural feature of these pentacoordinate Mb variants is a shorter Fe-His bond length than observed for wild-type, hexacoordinate Mb due to movement of the iron out of the plane of the heme towards the proximal ligand (Bogumil et al., 1995). The H64T structure of horse Mb, for example, has an Fe-His bond length of 1.93 A compared to 2.12 A in the wild-type protein (Bogumil et al., 1995). Similar iron out-of-plane movements were observed in the crystal structures of H64V and H64L variants of sperm whale myoglobin (Quillin et al., 1993). Although similar spectroscopic properties have been reported previously for the H64V and H64L variants of human Mb, no structural explanation has been proposed previously for the unusual behavior at alkaline pH (Ikeda-Saito et al., 1992). Both of the distal variants considered in this analysis exhibited slightly rhombic EPR spectra at neutral pH and significantly increased rhombicity at alkaline pH that is consistent with an increase in anionic imidazolate character as the pH is raised. It is possible that all pentacoordinate, high-spin ferric heme systems exhibit rhombically distorted EPR signals and that the degree of rhombicity is related to the degree of interaction with the axial ligand (Yonetani & Anni, 1987), with stronger interactions increasing the rhombicity. As mentioned previously, pentacoordinate heme proteins with anionic proximal ligands (e.g. catalase and cytochrome P-450) and variants modeling such systems exhibit very rhombic EPR spectra that lend credence to such conclusions. These observations can 117 also be extended to the pentacoordinate, heme enzymes such as cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP), both of which possess a proximal ligand with appreciable imidazolate character and exhibit rhombically distorted EPR spectra (e.g. CcP: g=6A, 5.3 and 1.97; Yonetani & Anni, 1987; HRP: g=6.35, 5.65 and 2.0; Blumberg et al., 1968). Several heme proteins have been reported to exhibit pH-dependent spectroscopic behavior similar to these Mb variants that provides additional, circumstantial, evidence for the deprotonation assignment. The pH-dependence of the electronic spectrum of N-acetyl microperoxidase-8 (Ac-MP-8) exhibits a titratable group with a pATaof ~9 that has been assigned to the proximal histidine (Wang et al., 1992). Ac-MP-8 is an octapeptide, proteolytic fragment of cytochrome c that has been used as a simple, water-soluble peroxidase model. Similar behavior has been reported for ferricytochrome c' (pKa 8-9.1 depending on the species; La Mar et al., 1990) and ferricytochrome bS62 (pKa 9.0; Moore et al., 1985). It has been suggested previously that the deprotonation of the proximal histidine in cytochrome c' is associated with a change in spin states. The neutral form of this protein from some species (e.g. Chromatium vinosnm and Rhodobacter capsiriatus) is known to exist as a quantum mechanically admixed spin state (Maltempo, 1974; Maltempo et al., 1974) with contributions from an intermediate spin state (S=3/2) mixed with that of the high-spin (S=5/2) state to form a new, magnetically distinct spin state. Titration to alkaline pH converts cytochrome c' to a completely high-spin state. Evidence presented for H64V and H64I Mb, however, suggests that the deprotonation of H93 is not associated with a spin-state change at cryogenic or ambient temperatures. First, the EPRg-values are higher than those seen for admixed spin states (Maltempo, 1974; Yoshimura et al., 1990; Monkara et al., 1992) and second, the NMR spectrum at neutral pH shows an upfield resonance for the meso protons (-27 ppm) similar to pentacoordinate, high-spin model heme 118 compounds (Kintner & Dawson, 1991). Although the H64V and H64I variants were not designed as models of the peroxidase family, the increased imidazolate character of these variants at high pH allows comparison to this group of enzymes. The ability to control the protonation state of the proximal histidine of myoglobin is an important structural challenge from a protein engineering standpoint. Clearly, the state of the proximal ligand is an important structural determinant of heme protein function. For example, the high resolution crystal structure of CcP indicates the existence of a strong hydrogen bond between the proximal histidine (HI75) and an aspartic acid (D235) residue (Finzel et al., 1994). Placement of an alanyl residue at position 235 (D235A) removes the H-bonding interaction, abolishes CcP-catalyzed oxidation of horse heart cytochrome c and possibly converts the heme to a hexacoordinate (H20-bound) state (Goodin & McRee, 1993). The stronger ligand field provided by imidazolate in wild-type CcP may be important in maintaining the pentacoordinate nature of the heme. The structural changes caused by the mutation that lead to a decrease in the imidazolate character of the proximal ligand also result in a decrease in the rhombicity and an increase in the midpoint reduction potential of the heme iron (Goodin & McRee, 1993). Thus, it is apparent that the protonation state of the proximal histidine is one of the structural features that distinguishes the peroxidases from the dioxygen binding globins. In the case of the H64 variants of myoglobin, a pentacoordinate heme was produced by decreasing the polarity of the active site whereas a similar coordination state in the peroxidase family has been achieved while maintaining the polarity of the distal active site, which is thought to be another essential structural feature of this class of heme enzymes. Clearly, further peroxidase-inspired modification of the myoglobin active site should address the issue of increasing the imidazolate character of the proximal histidine while maintaining the polarity of the distal region of the active site. 119 IT. Variants designed to increase the polarity of the distal heme binding site. The rationale for the construction of the following variants was to address the question of why myoglobins possess non-polar distal heme binding sites while those of peroxidases are far more polar. Cytochrome c peroxidase, for example, possesses an open active site with several non-coordinated water molecules and polar residues thought to be critical for the reaction with peroxides (Finzel et al., 1984). As such, several variants have been produced in horse heart myoglobin (including V68H and V67A/V68S Mb) to address the effects of polarity on the functional properties of myoglobin such as peroxidase activities. Interestingly, the V67A/V68S variant exhibits a modest increase (~4-fold) in peroxidase activity relative to the wild-type protein, whereas the V68H variant exhibits a 3-fold decrease in activity. An unanticipated feature of the V68H variant was that the proximity of the histidine residue to the heme iron resulted in formation of a reasonable spectroscopic model of cytochrome b5. Similarly, the V67A/V68S variant resulted in a protein with a function (and possibly structure) reminiscent of the heme oxygenase family of enzymes. A. The V68H variant The results presented for the oxidized and reduced V68H variant are fully consistent with the formation of a bishistidine ligated Mb in both oxidation states of the variant in the absence of exogenous ligands. Wild-type metMb is known to exhibit six-coordinate ligation of this type only at high pressure (Zipp et al., 1972) or after reaction with cyanogen bromide and azide (Adachi & Morishima, 1992). While variant myoglobins have been reported in which substitution of the distal histidine residue (H64) results in six-coordinate metMb variants where both proximal and distal ligands are provided by amino acid residues, the new distal ligand is not retained upon reduction of the heme iron in these cases. The double variant H64V/V68H of human metMb also appears to possess bisimidazole axial ligation (Qin et al., 1994), and recent evidence for the human and porcine 120 variants suggests that this variant also maintains a stable interaction with H68 in the reduced form of the protein (Dou et al., 1995). Based on the spectroscopic and crystallographic information that is available for the oxidized form of this double variant, it seems likely that the sixth ligand of the single variant of the horse heart protein studied here is provided by H68 rather than H64. The difference between cytochrome b5, which is entirely low-spin at ambient temperatures, and the variant, which is only partially low-spin, can be rationalized by assuming a weak iron-histidine bond in the myoglobin variant. This assumption is consistent with the observation that strong ligands (e.g., cyanide, CO) can displace the distally bound histidyl residue. A unique characteristic of the variant is the ability of the sixth histidine ligand to coordinate to the iron in both the ferric and ferrous derivatives, as is the case for cytochrome b5 (Argos & Mathews, 1975). Note that, wild-type metaquoMb, imidazole-Mb (Antonini & Brunori, 1978), JV-tetrazole-substituted imidazole-Mb (Adachi & Morishima, 1992), and the H64Y variants of sperm whale Mb (Egeberg et al., 1990) and horse heart metMb (Maurus et al., 1994), in which the distal tyrosine residue is coordinated to the heme iron, all undergo an oxidation-state dependent ligation change upon reduction (from six to five coordinate) and exist as five coordinate high-spin derivatives in the ferrous state. 1. Spectroscopic considerations and ligand binding. Comparison of the visible absorption, NMR and NIR-MCD spectra of the oxidized variant obtained at 293-300 K with the electronic spectrum obtained at 77 K, and the EPR spectrum obtained at 10 K, indicates a variety of thermal effects on the active site of this form of the protein. These spectra are consistent with the presence of bishistidine coordination with a spin-equilibrium at 300 K that becomes predominantly low-spin at 77 K. A schematic of the af-orbital splitting in ferric myoglobin, illustrating the electronic environments present in a thermal equilibrium, is shown below. At 10 K, the EPR spectrum indicates the presence of a large high-spin component and a smaller 121 xz, yz xz, yz xy High-spin Low-spin amount of a low-spin species. Interestingly, the spectrum of the low-spin component, although weak, suggests that the orientation of the axial ligands is such that the planes of the bound imidazole groups are orthogonal to each other (Walker et al., 1985). Consequently, it seems possible that upon cooling the sample to 10 K, the structure of the active site changes such that the distal imidazole ligand moves away from the iron atom to generate a significant amount of high-spin component. As noted above, exposure of the reduced V68H variant to carbon monoxide results in generation of a species with the electronic spectrum of carbonyl Mb, indicating displacement of the distally-bound histidine ligand by CO (data not shown). On the other hand, exposure of the reduced variant to air in the absence of reducing agent leads to the transient formation of a species with the spectrum of M b 0 2 followed by rapid autoxidation (data not shown). The ability of CO to replace the distal histidine ligand in the variant differs from the behavior of reduced cytochrome bs, where there is no response to such treatment. 2. Structural considerations concerning the nature of the V68H bond. Modeling of a histidine residue at position 68 of horse heart myoglobin supports the structural conclusions of this spectroscopic study. Valine-68 is only -3.2 A from the heme iron ligated Watl56 122 and thus the V68H substitution places the imidazole NE2 group very close to the water ligand (~ 2.2 A). A recent X-ray structure of the H64V/V68H variant of porcine metMb provides unambiguous evidence for coordination of the histidyl residue, revealing an Fe-NE2 distance of 2.3 A and a pyrrole nitrogen-to-histidine angle of approximately 55°(Dou et al., 1995). Interestingly, the structure reveals that this substitution causes the heme to move (~0.5 A) and rotate (~8°) away from H68, while at the same time resulting in only minor changes in the polypeptide backbone position relative to the wild-type protein. In terms of heme iron coordination, the position of the helix containing H68 is less than optimal because it precludes binding of the histidyl ligand orthogonally to the heme plane. The unusual orientation of H68 may explain the unique spectroscopic and thermal properties of these variants. The finding that the horse heart variant exhibits a lower reduction potential than the wild-type protein is strongly indicative of bishistidine coordination, although the large magnitude of the decrease (161 mV) is more difficult to understand. In considering the factors most likely to contribute to this relatively low reduction potential, attention is first directed at the proximal and distal histidyl residues. In particular, it is worth noting that there is no evidence for the existence of an imidazolate ligand, which would lower the potential relative to cytochrome b5. Bishistidine coordinated model heme compounds, in which one of the ligands is an imidazolate, exhibit a NIR charge-transfer band in the MCD spectra at -1350 nm (Gadsby & Thomson, 1990) that is not present in the spectrum of the V68H variant. From studies with model heme compounds, the relative orientations of the axially coordinated imidazole groups have been argued to influence the midpoint potential of the heme iron atom, such that the less stable perpendicular orientation is expected to increase the midpoint potential as much as 50 mV over that observed for the parallel orientation (Walker et al., 1986). To the extent that the EPR spectrum obtained at 10 K reflects the coordination geometry of the active site at 300 123 K, the perpendicular orientation of the axial ligands observed in the low-spin component at low temperature does not help explain the relatively low potential of the variant. It is clear that there are structural changes of the active site of this variant at low temperatures and, therefore, the ligand orientation at 300 K and 10 K could conceivably differ. Hydrogen bonding interactions involving either of the coordinated imidazole groups could,.of course, reduce the potential (Valentine et al., 1979; Doeffet al., 1983; O'Brien & Sweigart, 1985), although it seems likely that this property of the proximal histidine residue remains unchanged in the variant. Nevertheless, the hydrogen bonding interactions of the distally coordinated histidine residue cannot be assessed clearly without detailed structural characterization and may be a significant contributory factor. The contribution of the spin equilibrium observed in the oxidized form of the variant to the midpoint reduction potential is difficult to evaluate at present. In the absence of a three-dimensional structure for the horse heart variant, it is also difficult to speculate regarding the contributions of the second, unbound histidine residue in the distal heme pocket and the consequences of its interactions with the adjacent, coordinated histidine residue. This interaction combined with the distorted coordination geometry resulting from the proximity of these two residues also presumably contributes to the spin equilibrium and temperature-dependent behavior of the variant that also relate to the observed midpoint reduction potential. Further analysis of the electronic properties of this variant should include consideration of the temperature dependence of the reduction potential and correlation of these results with corresponding analysis of the temperature dependence of the electronic, MCD, and NMR spectra. Ultimately, it will be necessary to determine the three-dimensional structures of the variant in both the reduced and oxidized states to permit assessment of structural changes introduced at the active site and the consequences of juxtaposition of two histidyl residues in the distal heme pocket. 124 Since the advent of site-directed mutagenesis for the study of structure and function relationships of myoglobin, by far the bulk of the work has focused on structural determinants of ligand binding. Less work has appeared addressing the effects of active site structure on electron transfer rates. Reduction of wild-type myoglobin results in coordination state changes to the iron which contribute, and complicate, structural contributions to electron transfer rates. Studying the electron transfer properties of H64V, H64I and V68H Mb, systems that exhibit no redox dependent ligation changes, could be an informative means of gaining further insight into this important property of heme proteins. B. The V67A/V68S variant J. Structural considerations The previous variants described in this work have resulted in significant changes to the coordination geometry of the heme iron, whereas the effects of the V67A/V68S active site substitutions are more subtle. All of the spectroscopic data available suggest that these mutations produce no change in the nature of the ligands to the heme iron. The oxidized form of the protein remains hexacoordinate (His/aquo) and is converted to the pentacoordinate form upon reduction. The effects of placing a small polar residue in the distal side of the active site appear to involve primarily the ligand binding characteristics of myoglobin. The increase in polarity destabilizes the hydroxide-bound form and increases the pKa for the Fe(III)-H20/Fe(III)-OH" transition. In addition, the midpoint reduction potential of the ferric/ferrous couple is decreased. Although this variant binds carbon monoxide and dioxygen to produce derivatives with electronic spectra essentially identical to the wild-type protein, the Fe(II)-oxy form is very unstable and like V68H Mb autoxidizes very rapidly to the Fe(III) form. Thus it appears that substitution at position 68 in close proximity to the heme has a large effect on oxygen binding to myoglobin. 125 Equivalent variants, V68S and V68T, have been produced in porcine myoglobin to assess the effects of active site polarity on the structure and ligand binding characteristics of this protein (Brantley et al., 1993). Significant increases in the rate of autoxidation were observed for both of these variants and were attributed to destabilization of the bound oxygen ligand. The authors suggested two possibilities for this observation: a direct electrostatic repulsion of the nonbonding electrons on the adjacent oxygen atoms or competition for the NE2 proton of H64, which is known to hydrogen bond with and stabilize the bound oxygen ligand. Although these structural conclusions were inferred indirectly from kinetic data, the crystal structure of the met-aquo V68T variant of porcine Mb provides direct evidence for these arguments (Smerdon et al., 1991). In the ferric form, the nonbonding electrons of the P-OH group are within hydrogen bonding distance to the bound water ligand while the hydrogen atom of the hydroxyl group is hydrogen bonded to the main chain carbonyl of H64 (Smerdon et al., 1991). Such an orientation of the lone pair orbitals would stabilize the water ligand in the oxidized form and destabilize the oxygen ligand in the reduced form. The stabilization of the metaquo ligand presumably accounts for the 60-fold lower azide binding rate of this variant (Smerdon et al., 1991). Although no structures have been determined for the V68S variant in either porcine or horse Mb, one can speculate that a similar interaction in V67A/V68S Mb could explain the altered functional properties of this variant. The proximity of the electronegative hydroxyl group is expected to increase the pKa of the bound water ligand, decrease the reduction potential and destabilize the oxygen ligand in the reduced form. By modeling a serine residue at position 68 it is possible to place the hydroxyl group within approximately 3.1 A of the heme-bound water ligand (Wat 156), a distance that would allow hydrogen bond formation between these groups. The proximity of the serine residue in V67A/V68S Mb may also account for the enhanced rate of coupled oxidation observed for this 126 variant. Elucidation of the exact nature by which the increased polarity introduced by these substitutions produces these functional effects awaits a high resolution crystal structure of this variant. In the meantime it is possible that substitutions of V67 and V68 with smaller residues (e.g. alanine and serine) increases the solvent accessibility of the heme prosthetic group. The structural feasibility of this is difficult to assess from the modeling presented above. 2. Coupled oxidation reaction of myoglobin. The oxidative cleavage of heme to biliverdin is part of the natural process for the degradation and excretion of heme in the form of bile pigments (Schmid & McDonagh, 1979). The reaction of iron protoporphyrin IX, either in heme proteins or complexed as the pyridine hemochromogen, exposure to ascorbate and dioxygen is referred to as coupled oxidation and has been used as a model for the natural heme oxygenase system. The biocatabolic pathway of heme in vivo is: protoheme IX -a-oxyprotoheme IX - verdoheme IX - Fe-biliverdin - biliverdin (Sano et al., 1986)(Figure 37). The reaction of heme in the active site of myoglobin proceeds in a similar fashion but stops at Fe(III)-biliverdin (Sano et al., 1986; Modi, 1993) and, like the heme oxygenase system, is 100% specific for the a-meso position (Yoshinaga et al., 1990; O'Carra & Colleran, 1969). The reaction is initiated by reduction of the ferric heme to the ferrous form followed by dioxygen binding to the iron. Subsequent attack by the iron-bound dioxygen molecule towards the meso carbon produces the highly reactive a-oxyprotoheme intermediate. Formation of verdoheme and Fe(III)-biliverdin proceeds via consumption of two more molecules of oxygen with concomitant formation of carbon monoxide. The reducing equivalents required for the heme oxygenase system are provided by an NADPH-dependent cytochrome P450 reductase. Prior to the discovery of heme oxygenase, the coupled oxidation of hemoglobin and myoglobin was believed to be the principal pathway of heme degradation in vivo 127 H O O C C O O H Heme H O O C C O O H oc-lvydroxyheme H O O C C O O H Verdoheme H O O C C O O H Fe-biliverdin Figure 37: Proposed reaction scheme for the biocatabolic pathway of heme. 128 (O'Carra & Colleran, 1969). The reaction of wild-type myoglobin with ascorbate can be described adequately by a biphasic process, although from the known reaction scheme (Figure 37) it is clear that the reaction is more complex than is assumed by such a kinetic analysis. In the case of wild-type Mb, there is direct spectroscopic evidence for the formation of oxymyoglobin, represented by the first exponential process (k, = 1.6 x lO^rnin"1). In this analysis the remainder of the reaction is represented as a single exponential process (k2= 1.7 x lO^min"1). HPLC analysis confirms that biliverdin is the final reaction product, but in the case of wild-type myoglobin there is no evidence for the presence of any heme intermediates. The reaction of the V67A/V68S variant can also be described as a biphasic process. Unlike the wild-type protein, convincing spectroscopic evidence for oxyMb formation is lacking. Presumably the formation and decay of this derivative is too fast to be observed under these experimental conditions. The initial kinetically-detectable phase of this reaction (k, = 1.1 x 10"1 min"1) may represent the formation of one of the intermediates (e.g. verdoheme). The presence of verdoheme was suggested by electrospray mass spectrometry (data not shown) but the oc-oxyheme derivative was not observed by this technique possibly due to the instability of this precursor in the presence of oxygen. The second process (A:2 = 5.3 x 10"3 min"1) represents the conversion of verdoheme to Fe(III)-biliverdin. It is possible that the proximity of S68 to the oxygen binding site prevents a stable interaction between this ligand and ferrous heme and, instead, directs oxidative attack at the meso carbon to result in efficient coupled oxidation of the variant while maintaining the specificity of the reaction. 3. FTIR analysis of carbonyl myoglobin. FTIR spectroscopy provides a means to investigate the effects of active site substitutions on 129 the ligand binding properties of ferrous myoglobin and provides insight into the mechanism of Fe-biliverdin formation. Although the IR spectrum of carbonyl myoglobin is complex, consisting of four CO conformers (labeled Ao-A3), extensive studies of the carbon monoxide derivatives of numerous myoglobin variants provides the background necessary for comparison to V67A /V68S Mb (e.g. Li et al., 1994). The FTIR spectrum of wild-type myoglobin exhibits a maximum at 1941 cm"1 that consists of a mixture of components with stretching frequencies at 1945 cm"1 (Ax and A 2 , 70%) and 1932 cm"1 (A3, 25%) and 1965 cm"1 (A,,, 5%). The spectrum of V67A/V68S Mb can be compared to that of the corresponding V68S and V68T variants of porcine myoglobin. The V68T variant, for example, has a spectrum with a maximum at 1958 cm"1 and exists as a mixture of components with stretching frequencies at 1961 cm'1 (80%) and 1945 cm"1 (20%), whereas the spectrum of the V68S variant exhibits a band at 1949 cm"1 that is comprised of components at 1961 cm'1 (20%) and 1945 (80%>) (Li et al., 1994). The difference between the two variants, therefore, is in the amount of the A 0 component contribution to the spectrum. The spectrum of the V67A/V68S variant is more complex than that of the wild-type protein, exhibiting two equally intense bands at 1948 cm"1 and 1962 cm"1 (see Figure 35). The band at 1962 cm"1 confirms a substantial contribution from the AQ component to the spectrum of the variant, and in terms of amount may represent a situation intermediate between the two analogous porcine variants. It was not possible to fit the spectrum of the variant using the assigned stretching frequencies of the wild-type spectrum, which suggests that there is some structural heterogeneity in the active site of the variant. If multiple conformations exist for the hydroxyl group of S68 (e.g. pointing towards or away from the CO ligand) a greater number of carbonyl conformations could occur in the active site and consequently a more complex FTIR spectrum would result. The possibility of additional affects from increased solvent exposure must also be considered. Regardless of the origin, however, the spectrum of the variant contains a larger 130 fraction of the A , , conformer than does the wild-type protein. The origin of the various vibrational conformers (AQ-AJ) is related to the variations in the Fe-C and C-0 bond orders and can be affected by steric, electrostatic and hydrogen bonding interactions around the carbonyl and heme groups. The presence of a large AQ component can be interpreted as the result of either a removal of the hydrogen bond (such as in H64V and H64I Mb) or as the result of the addition of an electronegative charge close to the carbonyl that decreases the Fe-C and increases the C-0 bond order to increase the carbonyl stretching frequency through stabilization of Fe 8 ( _ )-C=0 8 < + ). Because the V67A/V68S variant increases the polarity of the active site without removal of the distal histidine, the latter explanation seems more feasible on the basis of structural considerations. Although there is no direct evidence for interaction between the serine residue and a bound dioxygen ligand, studies of the carbonyl derivative may reflect mutation-induced changes at the active site of myoglobin that are related to the enhanced coupled oxidation observed for the variant. 4. Structural similarities between myoglobin and heme oxygenase. At present, the three-dimensional structure of heme oxygenase has not been determined, so current models of the active site structure of this enzyme are based primarily on spectroscopic studies. This class of enzyme has proved difficult to study in the past because it is membrane bound, but this problem has been obviated recently by the cloning of a 30 kDa soluble fraction expressible inE. coli (Wilks & Ortiz de Montellano, 1993). From recent spectroscopic studies, it has become clear that the substrate bound (reconstituted) form of the enzyme exhibits greater active site similarities to myoglobin than to peroxidases or cytochrome P-450. The oxidized form of the iron-protoporphyrin IX cofactor is hexacoordinate at neutral pH, and is coordinated by an essential histidine (H25) and a water ligand that deprotonates with a pKa ~8 (Sun et al., 1993, 1994). Reduced 131 heme oxygenase is pentacoordinate and capable of binding oxygen (Takahashi et al., 1995). The Fe(III)-His stretching frequency of heme oxygenase, observed by resonance Raman spectroscopy, indicates that the proximal ligand has only a weak (or no) H-bond involving ND1 to another group, similar to myoglobin (Sun et al., 1993). A neutral proximal ligand may be an essential determinant of the mechanism of biliverdin formation in heme oxygenase and distinguishes it from the peroxidases and cytochromes P-450 that presumably activate oxygen via heterolytic cleavage of the ligand and ferryl intermediate formation. Interestingly, the carbonyl derivative of ferrous heme oxygenase has a stretching frequency at 1958 cm"1 (Takahashi et al., 1994) that is very similar to that of the V67A/V68S myoglobin variant. As outlined above, there are several structural arrangements which can result in a CO stretching frequency in this region, one of which places electron density close to the ligand. For this reason, it is possible that an electronegative group in the active site of heme oxygenase (e.g. serine or threonine) participates in oxygen transfer from the heme iron to the meso position during heme conversion to biliverdin. DI. Conclusions As noted previously by others, the hydrophobic heme binding environment provided at the active site of myoglobin is optimized for stabilization of dioxygen coordination to a reduced heme iron and to minimize the potential for oxidative chemistry. Therefore, although it is not surprising that modifications of this active site introduced by site-directed mutagenesis generally destabilize the Fe(II)-02 complex, the exact nature of this destabilization and the manner in which it manifests itself are often unpredictable. For example, increasing the hydrophilic character of the distal heme pocket may result in significant alteration in the coordination chemistry of the heme iron (e.g. V68H) or alternatively in greatly increased susceptibility to autoxidation and potential for heme degradation 132 (e.g. V67A/V68S). Although substitutions of the proximal histidine ligand to the heme iron would be expected to result in substantial structural and functional consequences, the range in proximal ligand size that can be accommodated by the active site can be determined only by experiment. Moreover, the species-specific differences observed in functional and spectroscopic properties of the proximal ligand variants is surprising in view of the greater similarities to one another exhibited by the corresponding wild-type proteins. Perhaps the most fundamental conclusion to be drawn from the present work is that despite a detailed understanding of myoglobin and its chemistry from over fifty years of investigation, it is not yet possible to introduce predetermined functional modifications into this protein by mutagenesis from first principles. 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