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Role of heme propionates in myoglobin electron transfer Lim, Anthony Richard 1990

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ROLE OF HEME PROPIONATES IN MYOGLOBIN ELECTRON TRANSFER by ANTHONY RICHARD LIM B.Sc, The University of British Columbia, 1983 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 March 1990 © Anthony Richard Lim, 1990 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Myoglobin (Mb) is a well characterized hemeprotein found in skeletal muscle. The dimethylester heme-substituted derivative of equine Mb (DME-Mb) was prepared to evaluate the involvement of the heme propionate groups in the electron transfer reactions of Mb. To achieve this goal, an efficient procedure to reconstitute and purify DME-Mb in high yield was developed. The near UV-visible absorption spectra of DME-Mb in various states of ligation and oxidation did not change significantly relative to those of native Mb. The *H NMR spectra obtained for native metMb (heme Fe(III) oxidation state) and metDME-Mb showed differences in the electromagnetic environment of their respective heme groups. The reactivity of DME-Mb was different from that of native Mb. For example the water ligand of metDME-Mb (Fe-H20) has a lower pKa than that of native metMb as determined by spectroscopic pH titrations. The autoxidation rate of oxyDME-Mb (Fe(II)-0 2) is faster than that of native oxyMb. MetDME-Mb apparently has a binding affinity for ferricyanide not evident in native metMb. Compared to native Mb, DME-Mb has decreased susceptibility to the oxidant hydrogen peroxide. The oxidation-reduction equilibrium of DME-Mb has been studied under a variety of solution conditions. At standard conditions (pH 7, 7=0.1 M and 25°C) the midpoint reduction potential (Em) of DME-Mb is 100.0(2) mV vs. SHE, which is 39 mV higher than the Em of native Mb. Analysis of the pH dependence of Em showed differences in the identity or ptfa between titratable groups found in native and DME-Mb. The ionic strength dependence of Em showed a higher net positive charge estimate for DME-Mb than native Mb consistent with the nature of the chemical modification involved. The temperature dependence of Em showed that DME-Mb has a greater difference in stability between oxidation states than native Mb. ii The kinetics of metDME-Mb reduction by Fe(EDTA)2" were also studied under a variety of conditions. At standard conditions, metDME-Mb reacted with the reductant Fe(EDTA)2" at a second order rate constant (Jfc12) two orders of magnitude greater than that of native metMb. After correcting for the differences in reduction potential between reactants, metDME-Mb still reacted at a significantly faster rate than native metMb, indicating differences in their reaction mechanisms. The pH, temperature and ionic strength dependences of Jfc12 for DME-Mb and Fe(EDTA)2" showed that DME-Mb had electrostatic and thermodynamic properties significantly different from that of native Mb. The functional differences between DME-Mb and native Mb can be attributed to the structural and electrostatics properties of the heme propionate groups. The interactions of these groups within the surrounding protein and the external environment are discussed with reference to the structure of Mb available from x-ray crystallographic studies. As a result, it is concluded that the heme propionate groups are involved in the structural stability, electron transfer specificity and reactivity of Mb. iii TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS ACKNOWLEDGEMENTS INTRODUCTION A. Historical Perspective 1 B. Biology of Myoglobin 1 1. Physiology 3 2. Genetics 4 C. Molecular Structure of Myoglobin 5 1. Amino Acid Composition and Sequence of Myoglobin 6 2. X-Ray Crystallography of Myoglobin 6 3. Characteristics of Myoglobin in Solution 10 D. Functional Studies of Myoglobin 11 1. Heme Replacement 11 2. Ligand Binding 13 3. Oxidation State Changes 15 E. Biological Electron Transfer 16 1. Oxidation-Reduction Equilibrium 16 2. Spectroelectrochemical Determination of Reduction Potentials 20 3. Electron Transfer Theory 22 4. Marcus Theory 26 5. Approaches to Studying Electron Transfer Reactions 27 ii iv viii ix x xi iv F. Outline and Purpose of Thesis 29 EXPERIMENTAL PROCEDURES A. General Procedures 31 B. Purification of Myoglobin 32 C. Preparation of Apo-Mb 32 D. Reconstitution of Apo-Mb 33 E. Spectroscopic Characterization of DME-Myoglobin 35 1. Visible Region 35 2. 'H NMR 36 3. EPR 36 F. Determination of the Heme pKa 37 G. Measurement of Autoxidation Rates 37 H. Synthesis of Pentaammineimidazoleruthenium (III) trichloride 38 I. Spectroelectrochemical Experiments 39 J. Reduction Kinetics with Fe(EDTA)2" 41 RESULTS A. Preparation and Isolation of DME-Myoglobin 44 B. Spectroscopic Characterization of DME-Myoglobin 45 1. Visible Region 45 2. *H NMR 47 3. EPR 50 C. Spectrophotometric pH Titration 50 D. Autoxidation Rates 55 E. Synthesis of [Ru(NH3)5Im]Cl3 59 v F. Reduction Potential Measurements 59 1. pH Dependence of Reduction Potential 63 2. Temperature Dependence of Reduction Potential 67 3. Ionic Strength Dependence of Reduction Potential 70 G. Reduction Kinetics Measurement 73 1. pH Dependence of Reduction Rate 76 2. Temperature Dependence of Reduction Rate 80 3. Ionic Strength Dependence of Reduction Rate 80 H. Marcus Analysis of Kinetics 85 DISCUSSION A. Preparation of DME-Myoglobin 86 B. Spectroscopic Properties 87 1. Near UV-Visible 87 2. 'H NMR 89 3. EPR 90 C. Autoxidation Rate 90 D. Electrochemical Studies 93 1. pH Dependence 96 2. Thermodynamics 100 3. Electrostatics 102 E. Reduction Kinetics 104 1. pH Dependence 107 2. Thermodynamics 108 3. Electrostatics 109 F. Marcus Theory Analysis 113 vi CONCLUSIONS A. Emerging Role of Heme Propionate Groups 115 B. Myoglobin 115 C. Cytochrome b5 116 D. Cytochrome c 118 E. Cytochrome c Peroxidase 119 F. Further Studies 119 BIBLIOGRAPHY 121 APPENDICES A. Midpoint Reduction Potentials (vs. NHE) 132 B. Second Order Rate Constants 135 C. Second Order Rate Constants Adjusted for Driving Force 137 D. Derivation of Autoxidation Equation 17 138 E. Marcus Theory Calculation of kff 140 vii LIST OF TABLES I Proposed Assignments for 'H NMR Signals 49 II Parameters from Ionic Strength Dependence of Reduction Potential 73 III Parameters from the Ionic Strength Dependence of Reduction Rate 83 viii L I S T O F F I G U R E S 1. Heme Structure 2 2. Amino Acid Sequence of Myoglobin 7 3. Tertiary Structure of Myoglobin 9 4. Potential Energy Diagram 24 5. Visible Spectra of DME-Mb 46 6. *H NMR Spectra of DME-Mb and Mb 48 7. EPR spectrum of DME-Myoglobin 51 8. pH Titration of DME-Mb Visible Spectrum 52 9. Linear Plot of pH Titration Data 54 10. Autoxidation Spectrum of DME-Mb 56 11. Linear Plot of Autoxidation Data 58 12. OTTLE cell Spectra 61 13. NernstPlot 62 14. pH Dependence of Em with one titratable group 65 15. pH Dependence of Em with three titratable groups 66 16. Temperature Dependence of Em 69 17. Ionic Strength Dependence of Em 72 18. AA vs. Time ± Ferricyanide 74 19. 2 n d Order Plot of AA vs. Time 75 20. pH Dependence of Jt 1 2 77 21. pH Dependence of jfc12(adj) 78 22. Temperature Dependence of k12 81 23. Ionic Strength Dependence of it j2 84 24. Heme Region of Mb 99 25. Dipole Moment of Mb 112 ix A B B R E V I A T I O N S CyDTA trans-1,2, -diaminocy clohexanetetraacetate dipic pyridine-1,2-dicarboxylate DME dimethylester DMSO dimethyl sulfoxide Em midpoint reduction potential EDTA emylenediamine tetraacetate EPR electron paramagnetic resonance Hb hemoglobin His histidine I ionic strength kn second order rate constant o^bs observed pseudo-first order rate constant Lys lysine Mb myoglobin Mb02, oxyMb oxymyoglobin metMb metmyoglobin NaCl sodium chloride NaPi sodium phosphate NMR nuclear magnetic resonance OLIS On-Line Instrument Systems OTTLE optically transparent thin-layered electrode SCE standard calomel electrode SHE standard hydrogen electrode Tris tris(hydroxylmethyl)aminoethane U V ultraviolet ACKNOWLEDGEMENTS I would like to acknowledge the support of my family and the invaluable help I've received during my Graduate Studies from past and present members of the Mauk lab. In particular I would like to thank Dr. Bhavini Sishta for her help in developing the DME-Mb preparation. In addition, I would like to thank Dr. Colin Tilcock for his help with the NMR spectroscopy. Thanks also go to members of the Brayer lab for their assistance and information on the three-dimensional structure of myoglobin, especially Dr. Steve Evans for his work on the dipole moment calculations and Gordon Louie for his help with the Mb radius calculation and molecular graphics program. xi INTRODUCTION A. Historical Perspective Myoglobin (Mb) is a heme containing protein found in the skeletal and cardiac muscle cells of a wide variety of animal species. Until the beginning of this century, the reddish colour of muscle was believed by many to be due to the circulation of blood containing hemoglobin (Hb) through the muscle tissue. Morner (1897; Kagen, 1973) extracted a pigment from dog muscle and compared its visible absorption spectrum with that of hemoglobin. Slight differences in the wavelengths of absorption were found, but the results were thought to be artifacts. Theorell eventually established that myoglobin is a distinct protein (1932; 1934; Kagen, 1973). He subsequently purified Mb from horse heart by crystallization and determined its molecular weight by sedimentation centri-fugation to be ~ 17,500, which was much less than that of Hb. He also measured its oxygen (0 2) affinity and showed it to be greater than that of Hb. Hill (1933), who used the term "muscle hemoglobin," demonstrated that Mb exhibits a hyperbolic oxygen binding curve that is clearly different from the characteristic sigmoidal curve of Hb. B. Biology of Myoglobin Mb consists of two separable components, a globin or apoMb protein component, and a heme prosthetic group. The heme in Mb is more accurately identified as iron protoporphyrin LX (Fuhrop and Smith, 1975), which is depicted in Figure 1. The central iron (Fe) atom of heme is coordinated by the nitrogens of the pyrrole rings. The Fe is the site of 0 2 and other ligand binding. Only the Fe(II) (ferro-) oxidation state binds 0 2. It is this oxygen-bound Fe(II) form (oxyMb or Mb02) that is found in vivo or in 1 Figure 1. Structure of heme. Peripheral pyrrole carbons are labelled 1 to 8. Interpyrrole methines are labelled a to 5. In myoglobin, the heme (protoheme IX) has two propionic acid groups at positions 6 and 7 (R=H). In the dimethylester heme (DME) derivative, R=CH 3. 2 newly prepared samples. The predominant oxidation state in vitro is the Fe(IH) (met- or ferric-) form as ferroMb readily autoxidizes in solution. 1. Physiology Mb content is highest in the cytosol of skeletal (including cardiac) muscle cells with aerobic, mitochondrial driven metabolism (Peter et al., 1972). There is a correlation between cytochrome oxidase activity and Mb content in muscle cells (Lawrie, 1953). By virtue of its 0 2 binding ability, Mb was originally thought to function as a store of 0 2 in the skeletal muscle cells. While storage may be an important function of Mb in diving mammals, which have high muscle concentrations of Mb, the storage capacity of most species is limited (Wyman, 1965). From theoretical calculations and experimental models for oxygen diffusion in cells, Wittenberg (1965; 1970) and Wyman (1965) proposed that Mb has a role in facilitating the diffusion of 0 2 from the cell membrane to the mitochondria. Mb has no direct effect on mitochondrial activity as measured by steady state oxygen consumption and adenosine triphosphate (ATP) production in isolated mitochondria (Cole et al., 1982). However, in isolated cardiac muscle cells (myocytes), oxygen consumption decreases by about a third when Mb mediated oxygen transport is completely inhibited by low levels of carbon monoxide (CO) (Wittenberg and Wittenberg, 1987). Another function has been proposed by Galaris et al. (1989) who have suggested that Mb with ascorbate can act as an electron sink to protect muscle cells from oxidants such as peroxides. Preparations of Mb0 2 tend to autoxidize slowly to the metMb Fe(III) state in vitro (Gotoh and Shikama, 1974). However, in vivo there is no significant amount of metMb (Ray and Paff, 1930), which indicates the existence of a metMb reduction system in muscle cells. Erythrocytes have a metHb reductase system that uses nicotinamide adenine 3 dinucleotide (NADH), a reductase, and cytochrome bs, which is the direct reductant of metHb (reviewed by Hultquist et al, 1984). Hagler et al. (1979) have isolated a metMb reductase with similar characteristics to metHb reductase. Cytochrome b5 can mediate the reduction of metMb by this reductase in vitro (Livingston et al, 1985). However, Hagler et al. (1979) could not detect any cytochrome b5 in muscle cells. 2. Genetics The gene encoding the globin amino acid sequence of Mb has been isolated and cloned from seal (Wood et al, 1982; Blanchetot et al., 1983), man (Weller et al, 1984), and mouse (Blanchetot et al., 1986). In each species, the Mb globin gene is a single copy, which is large (ca. 10 kilobases) relative to the required coding sequence for proteins of similar size (153 amino acids). Each Mb gene is composed of three exons separated by two introns. This pattern is similar to that of the Hb alpha and beta subunit globin genes (Maniatis et al, 1980). Varadarajan et al. (1985) have expressed the human Mb gene in Escherichia coli using a cDNA clone. Springer and Sligar (1987) have expressed a synthetic sperm-whale Mb gene in E. coli with high efficiency. Gilbert (1978) proposed that exons correspond to the functional domains of proteins. By specific enzymatic proteolyis of Hb /?-globin subunits, Craik et al. (1980) isolated a peptide the amino acid sequence of which corresponds to that encoded by the central exon of the Hb p subunit gene and that binds heme tightly and specifically. However, the heme reconstituted Hb fragment was unable to bind oxygen (Craik et al, 1981). From enzymatic digestion of the globin portion of Mb, De Sanctis et al. (1986, 1988) have isolated a "mini-Mb" peptide corresponding to the encoded sequence of the central exon and some (30 amino acids) of the third exon, which encodes the carboxyl terminal sequence. Mini-Mb not only binds heme, but the heme reconstituted fragment also binds 4 dioxygen and carbon monoxide (1988). Expression of the Mb globin gene in vivo begins soon after muscle cell differentiation (Weller et al., 1986). However, in all species studied, including human (Longo et al., 1973), a significant level of Mb does not begin to appear until late in fetal development, mainly in cardiac muscle (review by Kagen, 1973). Unlike Hb, there is no fetal form of Mb. In infancy, the synthesis of Mb in skeletal muscle increases to a level that is sustained through adulthood (Tipler et al., 1978). It appears that in adulthood a steady state level of Mb is maintained with slow (80-90 days) turnover (Akeson et al., 1960; Daly et al., 1967), though the activity of some muscles can influence their Mb content (Hagler et al., 1980). Underwood and Williams (1987) demonstrated that the regulation of Mb globin gene expression in muscle cells appears to be through pre-translational control by correlating Mb mRNA with muscle activity. C. Molecular Structure of Myoglobin The relationship between the structure of Mb and its functional properties is as well characterized as that for any other protein. While the globin (protein) portion of Mb is unique, its heme group is not. The Fe protoporhyrin LX group is a common prosthetic group found in a diverse number of hemeproteins with different functions and from a variety of animal and bacterial species. Examples include: the 6-type cytochromes (such as cytochrome b5), which are involved in electron transfer reactions, catalases and peroxidases, which reduce peroxides. The diversity in function among hemeproteins possessing identical functional groups indicates that the protein environment (sequence and structure) dictates the chemical properties of the active site. 5 1. Amino Acid Composition and Sequence of Myoglobin The amino acid compositions and sequences of myoglobins from a variety of species have been reviewed by Kagen (1973) and Romero-Herrera et al. (1978). Mammalian myoglobins are composed of 153 amino acids, of which a large number (19 or 20) are the basic amino acid lysine. The two most studied mammalian myoglobins are those of sperm whale and horse. The amino acid composition and sequence of sperm whale Mb were determined by Edmundson (1961; 1965). The amino acid composition and sequence of horse heart Mb were originally determined by Dautrevaux et al. (1969) and corrected by Romero-Herrera and Lehmann (1974). The sequences of these two myoglobins differ by 19/153 amino acid residues (see Figure 2). Though horse heart Mb has a high degree of sequence homology with sperm whale Mb, differences in ligand binding have been reported (Goss et al., 1982). These differences presumably arise from differences in the amino acid composition and conformation of the heme pocket of the proteins. 2. X-Ray Crystallography of Myoglobin Sperm whale metMb was the first protein for which a three-dimensional structure was determined by X-ray diffraction methods. This landmark crystallographic work was performed by Kendrew et al. (1958; 1960) who solved the crystal structure of this protein to a resolution of 2 A. Nobbs et al. (1966) solved the three-dimensional structure of the reduced, non-ligated (deoxy-) form of Mb by difference Fourier analysis between the deoxy- and metMb x-ray diffraction data. Both the sperm whale metMb and deoxyMb structures were later refined by Takano (1977a&b) to 2 A. The three-dimensional structure of sperm whale oxyMb was determined and refined by Phillips (1978; 1980) to 1.6 A. Neutron diffraction studies have been done to determine the position of hydrogen 6 horse whale 1 Gly-Lxu-Ser-Asp-Gly<jlu-Trp^3ta<^ Val Glu Leu His Ala 21 horse Ile-Ala-Gly-His-Gly-Gm^31u-Val-Leu-Ile-Arg-Leu^ whale Val Asp-Ile Lys-Ser 41 horse Glu-Lys-Phe-Asp-Lys-Phe-Lys-His-Leu-Lys-Thr-Glu-Ala-Glu-Met-Lys-Ala-Ser-Glu-Asp-whale Arg 61 horse Ixu-Lys-Lys-His<}ly-Thr-Val-Val-lxu-Thr-Ala-Leu-Gly-Gly-Ile-Leu-Lys-Lys-Lys-Gly-whale Val-Thr Ala 81 horse His-His-Glu-Ala-Glu-Leu-Lys-Pro-Leu-Ala-Gln-Ser-His-Ala-Thr-Lys-His-Lys-Ile-Pro-whale 101 horse Ile-Lys-Tyr-Leu-Glu-Phe-Ile-Ser-Asp-Ala-Ile-Ile-His-Val-Leu-His-Ser-Lys-His-Pro-whale Glu Arg 121 horse Gly-Asp-Phe-Gly-Ala-Asp-Ala<Jln<}ly-Ala-Met-Thr-Lys-Ala-I^u-Glu-Leu-Phe-Arg-Asn-whale Asn Lys 141 153 horse Asp-Ile-Ala-Ala-Lys-Tyr-Lys-Glu-Leu-Gly-Phe-Gln-Gly whale Tyr Figure 2. The amino acid sequence of equine (horse) Mb (Dautrevaux et al, 1969) is depicted. For comparision, amino acid differences from the corresponding sperm whale Mb sequence (Edmundson, 1965) are also shown. 7 atoms in sperm whale metMb (Schoenborn, 1971). From the crystals of horse heart Mb prepared by Sherwood et al. (1987), Evans and Brayer have determined the three-dimensional structure of this protein by x-ray diffraction to a resolution of 2.8 A (1988). They have also recently refined it further to 1.9 A (Evans and Brayer, 1990). In both sperm whale and horse heart metMb, the secondary structure consists of a series of eight alpha-helices, labelled A to H, which contain most of the amino acid residues. The tertiary structure has the polypeptide chain folded upon itself into an ellipsoid shape (Figure 3). In general, the folding pattern is such that polar amino acids side-chains are oriented outward, towards the solvent and non-polar side-chains are oriented inward. The heme is situated within a cleft or pocket formed by helices B to C and E to G, which have many hydrophobic amino acids in their sequence. This heme binding region has long been considered hydrophobic, with a presumably low dielectric constant compared to the solvent. However, MacGregor and Weber (1986) have probed the heme binding region of apoMb using a polarity-sensitive fluorescent dye. Their results indicate that the heme binding region may be less hydrophobic than previously believed owing to contributions from the dipoles of the peptide (amide) bonds lining it. The heme is held within the heme pocket by van der Waals contacts with the surrounding amino acid residues. On one side of the heme, the Fe(III) is coordinated to a water molecule, which also forms a hydrogen bond to a "distal" histidine residue (helix position 7E/sequence position 64). On the other side of the heme plane, the Fe is bound to a coordinated "proximal" histidine (8F/93). In the sperm whale metMb model of Takano (1977a) the Fe(III) atom is displaced by about 0.4 A out of the mean heme plane towards the proximal histidine residue. Evans and Brayer (1990) found no such heme Fe atom displacement in horse heart metMb. In Mb one of the heme propionates (P7, see Figure 1) is oriented inward and is hence called the "inner heme propionate" with the 8 F i g u r e 3 . S t e r e o d r a w i n g o f the a - c a r b o n p o l y p e p t i d e b a c k b o n e a n d h e m e o f the e q u i n e ( a q u o ) m e t M b s truc ture d e t e r m i n e d to 2 . 8 A r e s o l u t i o n f r o m x - r a y c r y s t a l l o g r a p h y ( E v a n s a n d B r a y e r , 1988) . F o r re f erence the s i d e c h a i n s o f h i s t i d i n e r e s i d u e s a r o u n d the h e m e , at p o s i t i o n s 93 ( p r o x i m a l H i s ) , 6 4 (d is ta l H i s ) , a n d 9 7 , are d e p i c t e d a n d l a b e l l e d . 9 other (P6) oriented outward (the "outer heme propionate"). The three-dimensional structure of sperm whale deoxyMb is very similar to that of metMb. A significant change is the loss of the water molecule coordinated to the Fe atom. As a result, the distal histidine residue is shifted away from the heme and the Fe(II) is extended further from the plane of the pyrrole rings. In oxyMb, the 0 2 binds the Fe(II) in a bent geometry with a Fe—O—O bond angle of about 115° (Phillips, 1980). The heme prosthetic group in oxyMb is rotated further into the heme pocket with the Fe atom closer to the heme plane. 3. Characteristics of Myoglobin in Solution Functional comparisons of myoglobin crystals and solutions, which are usually at non-physiological conditions, have been made. Observed differences may arise from either differences in structure or environments of these two states. For example differences in ligand binding rates between crystals of Mb/Hb and Mb/Hb in solution (Antonini and Brunori, 1971) have been observed. Pain (1983) has stressed that proteins in solution are less densely packed and have more dynamic structures than indicated by the average crystal structures. Areas of weak contact within a protein can result in "mobile defects" through which small molecules such as ligands can penetrate (Pain, 1987). Such variations in conformation have been observed by Frauenfelder et al. (1979), who have collected and analyzed x-ray diffraction data for Mb at a variety of temperatures. One specific difference between solution and crystal structures of sperm whale Mb is evident from the NMR studies of La Mar et al. (1978; 1983; 1984). 'H NMR spectra of newly reconstituted metMb exhibited two distinct sets of signals from heme methyl and vinyl groups arising from different orientations of the heme upon its entry into the apo-Mb pocket. The two orientations are related by a 180° rotation about the a—T carbon 10 axis of the heme (see Figure 1). Sperm-whale Mb equilibrates with time (~72 hours at neutral pH) to a preferred or major form of heme orientation (~ 12:1 ratio), which is that found in the crystal structure. Newly reconstituted metMb, which has about a 1:1 mixture of isomers, was initially reported to have (after reduction) a higher oxygen affinity than native Mb (Livingston et al., 1984). However, later 0 2 and CO binding studies by two independent groups showed no significant difference between newly reconstituted (and reduced) Mb and native Mb (Light et al., 1987; Aojula et al., 1987). D. Functional Studies of Myoglobin Several approaches have been used to study the manner by which the interactions between the apoprotein and prosthetic group determine the functional properties of Mb. The kinetics and equilibria with gaseous and anionic ligands have been studied to probe the steric and electronic environment of the heme pocket. Electrochemical and kinetic methods have been used to study the reduction and oxidation (i.e. electron transfer) processes of Mb. Heme prosthetic groups with altered porphyrin structures and/or metal substitutions have been used to determine the role of the heme within Mb. Invariably these studies monitor the spectroscopic properties of the substituted forms of Mb. 1. Heme Replacement Following the Hb reconstitution work of Hill and Holden (1926), Drabkin (1945) was able to dissociate (met)Mb into its apoMb (globin) and heme components by extraction into acidified acetone. He then reconstituted Mb by mixing the apoMb and heme and found no change in the UV-visible spectroscopic characteristics of the product relative to the native protein. Antonini and Rossi-Fanelli subsequently showed (1957) that 11 reconstituted Mb has the same oxygen binding properties as native Mb. Heme removal from Mb produces a slightly lower density in the resulting apoMb as determined from ultra-centrifugation measurements (Breslow, 1963) and a loss of a-helical content as determined by optical rotational dispersion observations (Breslow et al, 1965). Metal-free protoporphyrin IX has been inserted into apo-Mb by Breslow et al. (1967) to form porphMb. From circular dichroism studies, these authors found no significant difference in conformation between porphMb and Mb, and they concluded that porphyrin interactions with the globin were a major determinant of Mb tertiary structure. Successful reconstitution of Mb with protoheme has led to the use of "unnatural" or modified derivatives of protoheme to study the influence of heme substituent groups on the structure and function of the protein. Heme modifications employed in this type of work involve chemical modification or substitution of the protoheme methyl, vinyl or propionate groups of the porphyrin ring (see Figure 1). In an extreme example Chang et al. (1984) have reconstituted apoMb with synthetic hemes lacking the methyl and/or vinyl groups. The most common parameters measured to determine the effects of the heme modification are spectrophotometric characteristics and ligand binding properties. Yonetani and co-workers have reconstituted apoMb with several modified hemes (Tamura et al, 1974a&b). The optical absorption spectra of these heme substituted Mbs showed no major differences from that of native Mb. However, substitution of the heme vinyl groups (e.g. with methyl groups) resulted in increased 0 2 affinity, while methyl esterification of the propionate groups had no effect. Miki et al. (1986a&b) have used a variety of hemes modified at the vinyl groups to reconstitute sperm whale apoMb and then determine the three-dimensional structures of these metMb derivatives by x-ray crystallography. In comparing the structures of the heme-substituted Myoglobins these workers observed differences in the heme pocket conformation such as a-helix shifts, which were related to the observed changes in dioxygen binding affinity of the heme 12 substituted oxyMb derivatives. The coordinated Fe atom of the heme can be substituted with other metals to form various metal-substituted forms of Mb (reviewed by Hoffman, 1979). The unique properties of individual metals can be exploited to study the influence of the apoprotein or porphyrin ring on the metal. Cobalt (II) substituted Mb is perhaps the most well characterized form of metal-substituted Mb. This derivative has been useful because it binds dioxygen reversibly and because the paramagnetic properties of Co (II) allows analysis by EPR spectroscopy (Hoffman and Petering, 1970). Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) are sensitive techniques used to probe the electronic and magnetic environment of the heme within the protein. EPR spectroscopy has been used to characterize the affect of heme modifications on the paramagnetic Fe(III) atom (Tamura et al, 1973a). In NMR spectroscopy of metMb the majority of *H resonance signals from the porphyrin ring are shifted downfield (and hence distinct) from the bulk of the *H protein signals. Using a variety of deuterium (2H) substituted hemes, La Mar et al. (1980) have specifically identified several porphyrin resonance signals in the *H NMR spectra for sperm whale Mb. 2. Ligand Binding Both ferri- and ferro-Mb are able to bind a variety of exogenous ligands. The ligand binding properties of Mb reflect the steric and electronic environment of the heme pocket. The influence of the protein on ligand binding can be determined by comparison to model compounds such as the 0 2 binding "picket fence" iron porphyrin synthesized by Collman et al. (1974). As described above, the influence of the porphyrin on ligand binding can be determined at least in part from use of modified hemes. 13 I n m e t M b , a w a t e r m o l e c u l e n o r m a l l y b i n d s as the s i x t h c o o r d i n a t i n g l i g a n d to the i r o n , t h o u g h o t h e r a n i o n i c l i g a n d s s u c h as c y a n i d e a n d a z i d e c a n d i s p l a c e the w a t e r . T h e v i s i b l e s p e c t r u m o f m e t M b has a c h a r a c t e r i s t i c p H d e p e n d e n c e ( B r u n o r i a n d A n t o n i n i , 1 9 7 1 ) . T h i s d e p e n d e n c e i n v o l v e s a n a c i d - a l k a l i n e t r a n s i t i o n o f the w a t e r m o l e c u l e to a h y d r o x y l i o n ( O H - ) that is a c c o m p a n i e d b y a h i g h to l o w s p i n t r a n s i t i o n i n the F e a t o m . D e o x y M b has b e e n u s e d to s t u d y the d y n a m i c p r o c e s s e s i n v o l v e d i n b i n d i n g g a s e o u s l i g a n d s . T h e e n e r g y b a r r i e r s o r steps i n l i g a n d b i n d i n g h a v e b e e n d e f i n e d b y F r a u e n f e l d e r a n d W o l y n e s ( 1 9 8 5 ) as: 1) p e n e t r a t i o n o f the l i g a n d i n t o the p r o t e i n m a t r i x , 2 ) d i f f u s i o n t h r o u g h the p r o t e i n m a t r i x , 3) b i n d i n g to the m e t a l . T h e t h r e e - d i m e n s i o n a l s t r u c t u r e o f d e o x y M b ( a n d m e t M b ) suggest a n e n t r a n c e to the h e m e p o c k e t that is t o o s m a l l to a l l o w e n t r y o f l i g a n d s s u c h as 0 2 . A s a r e s u l t , c h a n g e s i n p r o t e i n c o n f o r m a t i o n m u s t o c c u r to s u c h e n t r y . F r o m e x a m i n a t i o n o f M b - l i g a n d c r y s t a l s t ruc tures ( N o b b s , 1 9 6 6 ) , N M R spec tra ( L e c o m t e a n d L a M a r , 1985) a n d M b m o l e c u l a r m o d e l l i n g s tud ies ( C a s e a n d K a r p l u s , 1 9 7 9 ) , a v a r i e t y o f a m i n o a c i d r e s i d u e s i d e c h a i n s h a v e b e e n p r o p o s e d to shi f t to f o r m a c h a n n e l , w h i c h w o u l d a l l o w l i g a n d e n t r y . R i n g e et al. ( 1 9 8 4 ) h a v e c o o r d i n a t e d a p h e n y l g r o u p to the h e m e i r o n o f M b a n d d e t e r m i n e d the three -d i m e n s i o n a l s t ruc ture o f the p r o d u c t b y x - r a y c r y s t a l l o g r a p h y . T h e b u l k o f th is g r o u p m a i n t a i n e d a c h a n n e l l a r g e e n o u g h f o r p a s s a g e o f a d i o x y g e n m o l e c u l e . A w i d e l y s t u d i e d l i g a n d p r o c e s s i n v o l v e s the b i n d i n g o f c a r b o n m o n o x i d e to M b . T h e F e - C O b o n d i n M b C O c a n b e b r o k e n w i t h f l a s h p h o t o l y s i s . A t l o w t e m p e r a t u r e s the free C O r e m a i n s w i t h i n the h e m e p o c k e t f o r a b r i e f p e r i o d o f t i m e b e f o r e it r e - b i n d s the h e m e i r o n i n a p r o c e s s r e f e r r e d to as g e m i n a t e r e c o m b i n a t i o n . B r e a k a g e a n d s u b s e q u e n t r e f o r m a t i o n o f the F e - C O b o n d has b e e n f o l l o w e d b y a v a r i e t y o f t e c h n i q u e s s u c h as F o u r i e r t r a n s f o r m i n f r a - r e d s p e c t r o s c o p y ( F i a m i n g o a n d A l b e n , 1985) a n d e x t e n d e d X - r a y a b s o r p t i o n f i n e s t r u c t u r e ( E X A F S ) e x p e r i m e n t s ( C h a n c e et al, 1 9 8 3 ) . 14 3. Oxidation State Changes Just as the kinetics and equilibria of ligand binding to Mb have been studied intensively, the oxidation-reduction properties of the hemeprotein have also been studied extensively. The redox equilibrium of Mb was first quantitatively characterized by Taylor and Morgan (1942) who measured the reduction potential of horse heart Mb. Brunori et al. (1971) subsequently reported detailed studies of the reduction potential of sperm whale Mb. Kinetic studies of Mb usually involve metMb because it is the predominant form in vitro and it is easier to manipulate. The kinetics of metMb reduction has been examined by use of a variety of reducing reagents such as chromium (II) (Huth et al., 1976), ascorbate (Tsukahara and Yamamoto, 1983), dithionite (Olivas et al, 1977) and Fe(EDTA)2- and Fe(CyDTA)2" (Cassat et al, 1975). When Mb0 2 is separated from its in vivo reduction system, autoxidation to metMb occurs readily. The rate of autoxidation reflects the stability of the Fe-02 bond within Mb. George and Stratmann (1952) demonstrated that autoxidation followed first order kinetics. Wide variations in the rate of Mb0 2 autoxidation have been measured as a function of pH (Sugawara and Shikama, 1979), temperature (Sugawara and Shikama, 1980) and anion concentration (Satoh and Sugawara, 1981). The mechanism of autoxidation involves generation of a superoxide anion (0 2~) from the bound oxygen (Gotoh and Shikama, 1976; Tijima and Shikama, 1987). Another possible oxidation state for the heme Fe in Mb is the Fe(IV) or ferryl state. FerrylMb can be formed by use of hydrogen peroxide (H 20 2) as an oxidant of metMb (George and Irvine, 1952), deoxyMb (Yusa and Shikama, 1987) or oxyMb (Whitburn, 1987). The ferryl state is proposed to be in the form of Fe0 2 + (George and Irvine, 1952). The mechanisms of ferrylMb formation are complex because the reactions do not have constant stoichiometrics. Mb cross-linking (Tew and Ortiz de Montellano, 1988) and pH 15 t i t r a t i o n e x p e r i m e n t s ( U y e d a a n d P e i s a c h , 1981) h a v e s h o w n that t y r o s i n e r e s i d u e s a r e o x i d i z e d i n the r e a c t i o n b e t w e e n m e t M b a n d H 2 0 2 . F e r r y 1 M b serves as a m o d e l f o r s t u d y i n g h e m e c o n t a i n i n g e n z y m e s , w h i c h are i n v o l v e d i n o x y g e n m e t a b o l i s m s u c h as cata lase . It a l s o serves as a s tart ing m a t e r i a l f o r s u l f M b p r e p a r a t i o n s ( B e r z o f s k y et al, 1 9 7 1 ; L i m a n d M a u k , 1986) . E. Biological Electron Transfer T h e p r i n c i p a l f u n c t i o n o f M b is to b i n d a n d t ranspor t o x y g e n . H o w e v e r , M b p a r t i c i p a t e s i n e l e c t r o n transfer reac t ions as r e q u i r e d to m a i n t a i n it i n the f u n c t i o n a l Fe ( I I ) state. M e t a l l o p r o t e i n e l e c t r o n transfer r e a c t i o n s h a v e b e e n the subjec t o f i n c r e a s i n g e x p e r i m e n t a l i n v e s t i g a t i o n d u r i n g the past twenty y e a r s (Scot t et al, 1985) . I n g e n e r a l , s tud ie s o f p r o t e i n s c h a r a c t e r i z e d b y x - r a y c r y s t a l l o g r a p h y h a v e b e e n the m o s t i n f o r m a t i v e as they grea t ly r e d u c e the e l e m e n t o f s t r u c t u r a l a m b i g u i t y . S i m i l a r i t i e s ex is t b e t w e e n the m e c h a n i s m s o f l i g a n d b i n d i n g a n d e l e c t r o n t rans fer b o t h o f w h i c h c a n b e s t u d i e d b y r e a c t i o n k i n e t i c s o r at e q u i l i b r i u m . 1. Oxidation-Reduction Equilibria T w o o x i d a t i o n states o f a c h e m i c a l spec ie s c a n f o r m a r e d u c t i o n - o x i d a t i o n c o u p l e , w h i c h i n s i m p l e s t t e r m s is d e s c r i b e d b y the f o l l o w i n g e q u i l i b r i u m : ox + nc~ red (1) T h e s y m b o l ox represents the o x i d i z e d f o r m o f the r e a c t i n g s p e c i e s , . the s y m b o l red refers to the r e d u c e d f o r m a n d n represents the n u m b e r o f e l e c t r o n s ( e ~ ) t r a n s f e r r e d i n 16 the reaction. Hemeproteins such as Mb can be considered metal centers with complex ligation, which consists of protein and porphyrin. Then, for example, ox can represent metMbFe(III) and red can represent deoxyMbFe(II). The free energy of the reaction, AG, can be defined by: AG = AG° - * r i n ( 2 ) [ox] The term AG 0 is the free energy change under standard conditions (one Molar activities), 'R is the gas constant (8.13143 J K"1 mol"1) and T is the temperature (in Kelvin). The free energy terms can be replaced by electrochemical potential terms by the conversion AG = —nFE to produce the Nernst equation: B - B o + ™ m M (3) nF [ox] where F is the Faraday constant for the charge/mole electrons (9.64870 x 104 C mol"1), E is the potential of the system (relative to the standard hydrogen electrode, SHE) and E° is the standard electrochemical reduction potential for the system. Under non-standard biological conditions, E° is replaced by Em, the midpoint reduction potential at the stated conditions. The direction and magnitude of an electron transfer reaction between different reduction-oxidation couples, which is called a cross-reaction, is determined by the difference between their reduction potentials. Important examples of such cross-reactions in biological systems are the electron transport chains of respiration and photosynthesis. 17 The structural factors that determine the reduction potential of metalloproteins are the focus of considerable experimental and theoretical consideration. The Em of hemeproteins vary to some degree depending on the conditions of measurement e.g. pH, ionic strength and temperature. As many hemeproteins have identical heme groups yet differ widely in reduction potentials, the protein structure and its interactions with the heme must be major factors in deteniiining the reduction potential of a given protein. The influences on the reduction potential of electron transfer proteins and their corresponding contributions to the free energy change have been summarized by Moore et al. (1986). The changes that accompany electron transfer involve: 1) bonding interactions of the reduction-oxidation center (AG c e n) 2) electrostatic interactions of the center with the protein and solvent (AGel) 3) oxidation state dependent conformation changes (AG c o n f) The latter type of change, represented by AG c o n f, is considered negligible for simple proteins (VM a vis the similarity of metMb and deoxyMb crystal structures) and may be intrinsically linked to the interaction changes represented by AG c e n and AGe(. The electrostatics-linked changes can be further delineated into four components: a) interactions with ions in solution (AG i o n) b) interactions with the solvent, i.e. water (AG H£>) c) interactions with charges exposed to the solvent/surface (AGsurf) d) interactions with charges within the protein (AGint) In terms of free energy, AG = AG c e n + AG e l + AG c o n f (4) where AG e l = AG i o n + AG H,p + AG s u r f + AG i n t (5). 18 A d i s t i n c t i o n is m a d e b e t w e e n e lectrostat ic in t erac t ions i n the p r e s e n c e a n d a b s e n c e o f water ( w i t h i n the p r o t e i n ) a r i s i n g f r o m the l a r g e d i f f e r e n c e i n p o l a r i z a b i l i t y ( m e a s u r e d b y d i e l e c t r i c cons tant ) b e t w e e n water a n d the m o r e h y d r o p h o b i c p r o t e i n . S t e l l w a g e n (1978) has a t t e m p t e d to s h o w that the r e d u c t i o n potent ia l s (at p H 7) o f a v a r i e t y o f h e m e p r o t e i n s , i n c l u d i n g M b , v a r y i n v e r s e l y w i t h the d e g r e e o f h e m e e x p o s u r e to the s o l v e n t . H o w e v e r , he c o m p a r e d p r o t e i n s that v a r i e d f r o m each o d i e r s t r u c t u r a l l y i n o ther w a y s than j u s t h e m e e x p o s u r e . T h e s o l u t i o n c o n d i t i o n s u n d e r w h i c h a m e t a l l o p r o t e i n is s t u d i e d (e.g. p H , i o n i c s trength a n d temperature ) c a n i n f l u e n c e the r e d u c t i o n p o t e n t i a l t h r o u g h p e r t u r b a t i o n o f d i e s o l v e n t , the p r o t e i n o r the o x i d a t i o n -r e d u c t i o n c e n t r e i tself . U n d e r n o n - s t a n d a r d c o n d i t i o n s o f p H > 0 , d i e m i d p o i n t r e d u c t i o n p o t e n t i a l s o f h e m e p r o t e i n s v a r y to s o m e extent as a f u n c t i o n o f p H ( C l a r k , 1960 a n d D u t t o n , 1 9 7 8 ) . T i t r a t a b l e o r i o n i z a b l e g r o u p s ( each w i t h a u n i q u e pAT a ) i n h e m e p r o t e i n s i n c l u d e s o m e types o f a m i n o a c i d s , (e.g. h i s t i d i n e ) , the h e m e p y r r o l e n i t r o g e n s a n d s o m e l i g a n d s s u c h as the h e m e i r o n b o u n d w a t e r . B y the a p p r o a c h o f M o o r e et al. ( 1 9 8 6 ) , the effect o f p H o n Em i s m a n i f e s t e d b y c h a n g e s i n the e lec tros tat ic in terac t ions o f the h e m e p r o t e i n . T h e p H d e p e n d e n c e o f the Em o f a h e m e p r o t e i n c a n b e t h e n b e c h a r a c t e r i z e d b y the pK& v a l u e s o f t i t ratable g r o u p s , w h i c h h a v e a n o b s e r v a b l e i n f l u e n c e . T h e pAT a v a l u e s o f t i tratable g r o u p s o f h e m e p r o t e i n s m a y i n m m b e d e t e r m i n e d b y the o x i d a t i o n state o f the h e m e p r o t e i n . T h i s effect o f p H i n v o l v e s the s p e c i f i c re lease a n d / o r u p t a k e o f p r o t o n ( s ) ( H + ) c o n c o m i t a n t w i t h e l e c t r o n transfer; the f o r m e r c a n be r e p r e s e n t e d as: oxK + e - " t i ; red + H + (6) T h e d e g r e e o f p r o t o n a t i o n i n a p r o t e i n i n part d e t e r m i n e s its net c h a r g e . H e n c e , the effect o f p H is part o f the m o r e g e n e r a l effect o f p r o t e i n c h a r g e o n Em. M o o r e et al. 19 (1986) consider electrostatic interactions, represented by AGel, to have a substantial contribution to the reduction potential of electron transfer proteins. Ionizable amino acid residues within hemeproteins give rise to various ionic interactions with the heme, solvent and other residues within the protein (Matthew, 1985). The standard free energy change is composed of a standard enthalpy change, AH0, and entropy change, AS0, which are defined by: AG° = AH0 — TAS° (7) where T is the temperature (in Kelvin).' The enthalpic term represents the net energy change from the chemical changes of the system e.g. electrostatic bonds. The entropic term represents the change in energy distribution (degree of order) of the system e.g. degree of protein solvation by H20. Study of the temperature dependence of the reduction potential allows the components of the free energy change to be quantified. This breakdown allows better understanding of the mechanisms of oxidation state changes. As reviewed by Taniguichi et al. (1980), both AH° and AS" are negative for the reduction of many metalloproteins, including Mb. The increased stability of the reduced state has been attributed to the lowering of the metal charge within the protein interior. 2. Spectroelectrochemical Determination of Reduction Potentials To determine the midpoint potential of a hemeprotein using the Nernst equation, the ratio of reduced:oxidized forms must be measured. An indirect method of determining reduction potentials is to measure the changes in current that accompanies changes in potential. The current represents the magnitude of electron transfer, which is proportional to the change in oxidation states. An example of such a "voltammetric" 20 technique is cyclic voltammetry (Evans et al, 1983). There are two direct approaches to measuring the reduction potential. One is to control the ratio of oxidation states and measure the change in potential. The other method is to control the potential and measure the change in ratio of oxidation states. With both methods, the oxidation state changes can be quantified by monitoring the accompanying spectrophotometric changes. To obtain various ratios of oxidation states, potentiometric titration (Wilson, 1978) or the methods of mixtures (Antonini et al., 1964) is commonly used. In the former method, aliquots of an oxidant or reductant, e.g. ferricyanide, in a controlled and defined oxidation state and concentration are used to titrate (oxidize/reduce) a solution of hemeprotein. After equilibration, the electrochemical potential of die solution is measured. In the method of mixtures, separate solutions of hemeprotein are either fully oxidized or reduced. Mixing various volumes of the two solutions allow various ratios of oxidized and reduced forms to be obtained. After equilibration with an electrode, the potential is measured. Equilibration times can be reduced if a mediator is added to the solution. Mediators are small inorganic metal complexes or organic dyes, which react rapidly with the titrant, electrode and hemeprotein, thereby facilitating coupling of the protein equilibrium to the electrode surface (reviewed by Armstrong et al, 1988). The application of a controlled potential across a hemeprotein solution results in a defined change in the position of the oxidation-reduction equilibrium, which is dependent on the applied potential. Such changes in oxidation state can be readily monitored by changes in the electronic spectra. However, hemeproteins in general do not react with electrodes at appreciable rates owing to the insulation of the protein surrounding the metal center. As a result, electrochemical equilibration times are long. Hill and co-workers have circumvented this problem by chemical modification of electrode surfaces to allow more rapid equilibration (Armstrong et al, 1988). 21 H e i n e m a n et al. ( 1 9 8 2 , 1983) h a v e r e p o r t e d a n a l t erna t ive m e t h o d o f p e r f o r m i n g p o t e n t i o m e t r i c t i trat ions t h r o u g h d e v e l o p m e n t o f O p t i c a l l y T r a n s p a r e n t T h i n - L a y e r E l e c t r o d e ( O T T L E ) c e l l s . T h e s e are m o d i f i e d cuvet tes w i t h a s e m i - t r a n s p a r e n t w i r e m e s h w o r k i n g e l e c t r o d e , w h i c h spans the s o l u t i o n ( a n d there fore the pa th l eng th ) but d o e s no t s i g n i f i c a n t l y in ter fere w i t h l i g h t t r a n s m i s s i o n . T h e p a t h l e n g t h o f the cuve t t e (~0.2 m m ) is short to r e d u c e d i f f u s i o n d i s tances a n d h e n c e e q u i l i b r a t i o n t i m e . R e d o x a c t i v e s m a l l m o l e c u l a r w e i g h t m e d i a t o r s are u s e d to fac i l i ta te e l e c t r o n transfer b e t w e e n the w o r k i n g e l e c t r o d e a n d m e t a l l o p r o t e i n s . 3. Electron Transfer Theory A b i m o l e c u l a r e l e c t r o n transfer c r o s s - r e a c t i o n b e t w e e n a n e l e c t r o n d o n o r , D, a n d a n e l e c t r o n a c c e p t o r , A, c a n b e represen ted as f o l l o w s (Scot t et al, 1985) : *« A + D [AD] —> [A~D+] A~ + D+ (8) T h e i n i t i a l s tep i n v o l v e s f o r m a t i o n o f a n e n c o u n t e r o r p r e c u r s o r c o m p l e x o f the reactants . E l e c t r o n transfer then o c c u r s as a n i n t r a m o l e c u l a r p r o c e s s w i t h a rate cons tant o f ket. A f t e r e l e c t r o n t rans fer , the s u c c e s s o r c o m p l e x t h e n d i s soc ia te s to g i v e the f i n a l p r o d u c t s . E l e c t r o n trans fer is t h o u g h t to resu l t f r o m s o m e d e g r e e o f o v e r l a p b e t w e e n d o n o r a n d a c c e p t o r o r b i t a l s , w h i c h a l l o w s d e l o c a l i z a t i o n o f the e l e c t r o n . I f there is a b r i d g i n g l i g a n d b e t w e e n the reac t ive centers i n a n a c t i v a t e d c o m p l e x , e l e c t r o n transfer is s a i d to o c c u r b y a n i n n e r - s p h e r e m e c h a n i s m . I f there is n o s u c h l i n k b e t w e e n reac t ive centers , e l e c t r o n transfer o c c u r s b y a n o u t e r - s p h e r e m e c h a n i s m . 22 The potential energy states of the nuclei in the complex can be represented as a function of nuclear configuration in two-dimensions as illustrated in Figure 4. Changes in nuclear positions arise from vibrational movements of the nuclei. The energy difference between the ground states of the precursor and product complex is A£°, which is simply the difference in reduction potential between reactants. The energy difference between the ground state of the precursor complex and the intersection of coordinates is the activation energy, AE* (or AG * ). The energy difference designated AE between the unreacted and reacted complexes at their intersection represents the degree of electron orbital overlap. In an adiabatic reaction, there is sufficient electronic interaction that AE is large. As a result, the probability of the reaction (electron transfer) proceeding at the intersection is one, which implies a rapid reaction rate. In a non-adiabatic reaction, the precursor complex can exist at high energy levels without electron transfer. As a result, the probability of reaction is less than one. The aforementioned description of electron transfer arose largely from the work of Taube on the reactions of metal complexes (reviews 1977, 1984). Metalloproteins can be considered to a large extent as elaborate metal complexes, whose reduction-oxidation reactions can be analyzed in a similar manner. Electron transfer reactions of metalloproteins are often non-adiabatic. The reasons for their non-adiabatic behavior involves the protein environment or medium through which electron transfer occurs. The metal center in a metalloprotein is held in a relatively fixed position by the protein (and porphyrin ring if present). Proteins effectively act as insulators by separating charged reactive metal centers at finite distances and fixed orientations. Electron transfer in such cases involves no direct contact or bonding between reactive centers and is therefore considered to occur by an outer-sphere mechanism. 23 A-D n u c l e a r c o n f i g u r a t i o n Figure 4. Potential energy diagram for non-adiabatic electron transfer. D is the electron donor and A is the electron acceptor. 24 According to transition-state theory (review by Moore and Pearson, 1981), formation of an activated complex for a bimolecular reaction, such as that described in Equation 8, involves a free energy of activation, A G This energy term determines the cross-reaction rate, kn, which is defined by the equation: * 1 2 = *_7-e-< A G'^ ( 9 ) Nh The term R is the gas constant, T is temperature, N is Avogadro's number (6.022 x 1023 mol"1) and h is Planck's constant (6.625 x 10 _ 3 5.J s). The term RT/Nh in Equation 9 represents the vibrational frequency of the reactants. The free energy of activation consists of enthalpic and entropic energies of activation symbolized by the terms AH * and AS * respectively. The activation energies involved in a reaction can be determined from the temperature dependence of the reaction rate constant defined by Equation 9. Though the kinetics of electron transfer reactions may follow Equation 9, the complex mechanisms involved are not obviously defined. As reviewed by Newton (1968), an electron transfer reaction involves three general processes: 1) work to combine reactants and separate products 2) reorganization of the reaction complex to allow electron transfer 3) electron transfer. The work and reorganization parameters involve energy changes symbolized by the terms w and A (or AG R*) respectively. The work terms describe the energy involved in moving charged particles together (wl2) or apart (w2i). The reorganization energy represents the geometrical changes in the donor' and acceptor required for electron transfer to occur. The free energy of the cross-reaction can be defined as: A G 1 2 = AGR*+ Aw (10) where AH> = w12 - w21 (11) 25 4. Marcus Theory An alternative approach to characterize reaction kinetics has been developed specifically for outer sphere electron transfer by Marcus (reviewed by Marcus and Sutin, 1985). In "relative" Marcus Theory the rate and activation energy of an electron transfer cross-reaction are viewed as a function of the corresponding parameters for the self-exchange reactions of the reactants. An example of a self-exchange reaction involves iron isotopes (Taube, 1977): *Fe(H20)|+ + Fe(H 20)| + —> *Fe(H 20)l+ + Fe(H 2 0)l + The main feature of a self-exchange reaction is that it has no thermodynamic driving force, i.e. AE°=0. The cross-reaction and self-exchange rates of electron transfer are related by the expression: *12=V/(*11*22^12/) (12) where m / = (In AT 1 2 ) 2 (13) 4\n(knk22/Z2) The parameters and Jfc^  are the self-exchange rates of the reactants; kl2 is the cross reaction rate. They are determined by the work and reorganization energies involved in electron transfer. The parameter Kn is the equilibrium constant of the cross-reaction, which is determined by A G ° (or AE°) of the reaction. In Equation 13, if the electron reaction is bimolecular, Z is the collision frequency between the reactants; if it is intramolecular i.e. after precursor complex formation, Z is the vibrational frequency defined in Equation 9. The term / usually has a value of one if die reactions involved are adiabatic or are uniformly non-adiabatic. 26 A useful feature of relative Marcus theory is that if the characteristics of the self-exchange reactions are defined, the rate of the cross-reaction can then be predicted with reasonable accuracy. Alternatively, a self-exchange reaction rate for a reactant can be calculated from the cross-reaction rate and the self-exchange rate of the other reactant (Wherland & Gray, 1976). The self-exchange reaction rate of a reactant is characteristic of its inherent reactivity and hence mechanism of electron transfer. Relative Marcus theory has been used in this manner in many studies of reactions involving metal complexes and metalloproteins (reviews by Wherland and Gray, 1976, Marcus and Sutin, 1985, and Scott et al, 1985). 5. Approaches to Studying Biological Electron Transfer The study of biological hemeprotein electron transfer can be approached by two general methods, the study of intermolecular reactions and intramolecular reactions (Scott et al, 1985). To study intermolecular reactions, suitable electron donors or acceptors must be employed. A variety of electron donors can be generated using flash photolysis to electronically excite otherwise unreactive molecules e.g. Ru(bpy)3+ (English et al, 1982), a variety of flavins (Tollin et al, 1986). Photoactive zinc protoporphyrin has been substituted into hemoglobin, which was then used to reduce cytochrome b5 (Simolo et al, 1984). Another type of electron donor is the hydrated electron generated by pulse radiolysis of an aqueous solution (McLendon and Miller, 1985). Another source of electron donors/acceptors are stable, substitutionally inert metal complexes that are well characterized in terms of their structural and reduction-oxidation properties. The observed kinetic behavior of the protein-complex reaction can be corrected for the contributions of the complex, allowing the intrinsic features of the metalloprotein to be analyzed by for example Marcus theory. Examples of such metal 27 complexes include the iron(II/III) emylenediarninetetraacetate complex, Fe(EDTA)1 , tris(l,10-phenanthroline)cobalt(II/III), Co(phen)3 + / 3 + and hexacyanoferrate (II/III), Fe(CN)|"/4~, which are well characterized and commonly used reactants in metalloprotein kinetics. With the use of such reagents, the effects of electrostatic charges and complex formation on electron transfer rates can be studied. In addition, specific amino acid residues within proteins can be modified. The effects of such substitution on electrochemical and kinetic properties of the resulting derivatives can then be evaluated. For example Butler et al. (1983) prepared cytochrome c derivatives with covalently attached dinitrophenyl derivatives on specific lysine residues. The effects of the location of the lysine derivative were determined "by oxidation kinetics with various cobalt complexes. Another protein modification technique is site-directed mutagenesis, which allows substitution of amino acid residues within proteins e.g. Mb Val-68 mutants (Varadarajan et al., 1989), cytochrome c Arg-38 mutants (Cutler et al., 1989). To carry out bimolecular electron transfer reactions, which often occur at high rates, rapid mixing systems are employed (review by Chance, 1974). Such systems mix the reactants in a rapid and complete manner to produce a homogeneous reaction whose rate can be measured. To measure the rates of electron transfer involving hemeproteins, advantage is taken of their oxidation state dependent spectrophotometric changes. An alternative approach to biological electron transfer is to study intramolecular reactions occurring within a hemeprotein. The effects of distance, orientation, intervening medium (e.g. amino acid residues) on electron transfer rates within hemeproteins can be studied because the sites of electron transfer are fixed and can be localized by x-ray crystallography models. There is a paucity of naturally occurring proteins with two redox active sites that are well characterized. As a result, chemical modification techniques are used to attach a second (detectable) redox-active center at a 28 definable position in a protein. Examples of such modified hemeproteins are the zinc and iron porphyrin Hb hybrids (McGourty et al, 1983), ruthenium modified cytochrome c (Nocera et al, 1984) and ruthenium modified Mb (Mayo et al, 1986). F. Outline and Purpose of Thesis Previous studies of bovine liver cytochrome bs by Reid et al (1984) demonstrated that esterification of the heme propionate groups produces a substantial increase in the reduction potential with a significant reduction in the electrostatic potential surface of the hemeprotein. Based on the structural comparison of hemeprotein three-dimensional structures by Rossman and Argos (1975), the similarity in structure between cytochrome bs and Mb suggests that the heme propionate groups of Mb may be of comparable significance in Mb. In the three-dimensional structure of horse heart and sperm whale Mb, the heme propionate groups are located within the. hemeprotein somewhat similar to the positions they occupy in cytochrome b5. Heme propionate-7 is oriented inward to the heme pocket on the proximal side of the heme and forms a hydrogen bond with His-97 and serine-92. Heme propionate-6 is oriented towards the heme pocket opening and forms a hydrogen bond with arginine-45 in sperm whale and with lysine-45 in horse heart Mb. Comparison of the three dimensional structures of sperm whale metMb (Takano, 1977a) and deoxyMb (Takano, 1977b) crystals does not reveal any significant change in the environment of the heme. However, similar comparison of deoxy- and oxyMb by Phillips (1980) indicates that with dioxygen bonding, the environment of the heme becomes more compact with decreases in hydrogen bonding lengths. This structural change with ligand binding should stabilize the oxygenated form of the protein relative to the oxidized form. As oxygenation is formally equivalent to oxidation, this 29 observation suggests the heme propionate esterification should have an effect on the oxidation-reduction properties of Mb similar to that previously reported for cytochrome by Tamura et al. (1973a&b) reported that heme propionate esterification Mb had no significant effect on the ligand binding properties of sperm whale Mb. These workers however, did not investigate the effects of this modification on the reduction potential or electron transfer kinetics of this protein. Furthermore, the protein that Tamura et al. studied appears to have been contaminated by significant amounts of apoMb. Tsukahara et al. (1986) have reported initial results concerning the reduction of sperm whale DME-Mb (prepared by the method of Tamura et al.) by ascorbate that reveal a significant increase in the reaction rate relative to that of native Mb. The present study provides a comprehensive investigation of the effect of hemeprotein esterification on the electrochemical and kinetic properties of horse heart Mb in an attempt to resolve these apparently contradictory observations. In the course of this work, an efficient method of preparing DME-Mb has been developed. In addition, the effect of heme propionate esterification on the rate of autoxidation, the electrostatic properties of Mb and on the pK& of the axial water ligand of metMb has been assessed. The results of this study establish that the disruption of normal hydrogen bonding patterns of the heme propionates within myoglobin by esterification has important consequences for the functional properties of this protein. 30 EXPERIMENTAL PROCEDURES A. General Procedures Reagent grade chemicals were used in all procedures unless otherwise stated. Glass-distilled water was further purified by passage through a Barnstead Nanopure water purification system until the resistivity was greater than 15.5 Mohm. Measurements of pH were obtained with a Radiometer Model PHM84 pH meter and GK2321c combination electrode. Conductivity was measured with a Markson Model 10 portable conductivity meter (Markson Scientific Inc.). Protein solutions were centrifuged to remove any precipitated material. The centrifuges used were a Sorvall RC-5B superspeed centrifuge (20-30 minutes x 10,000 rpm, SS-34 rotor), which was refrigerated at 4°C, and a Silencer H-25F1 table-top centrifuge (10 minutes x 10,000 rpm), which was used for volumes less than one mL. Protein solutions were dialyzed with Spectrapor type 1 dialysis membranes. Ultrafiltration and concentration of protein solutions were carried out a 4°C with a Millipore Minitan ultrafiltration system (for volumes >500 mL), an Amicon ultrafiltration cell with a YM-5 membrane (volumes >10 mL) and Centricon 10 microconcentrators (volumes <10 mL). Absorbance readings and electronic spectra were recorded on a Shimadzu Model UV-250 spectro-photometer equipped with a jacketed cuvette holder connected to a MGW-Lauda model RM3 water bath. Spectra were also recorded on a Cary-219 spectrophotometer equipped with a jacketed cuvette holder connected to another MGW-Lauda model RM3 water bath. The Cary-219 was interfaced to a Zenith Model Z-248 personal computer. Software for instrument control, data collection and analysis was developed by On-Line-Instrument-Systems (OLIS; Jefferson, Georgia). 31 B. Purification of Myoglobin Horse heart myoglobin (Mb) (Sigma Type III) was purified by the procedure of Tomoda et al. (1981) as follows. Lyophilized Mb was dissolved in 10 mM potassium phosphate (KPi) buffer pH 6.8 (4°C). A 15 x 5 cm CM-50 Sephadex (Pharmacia) column was equilibrated with the same buffer. To the Mb solution were added a few grains of ammonium bisdipicolinatocobaltate, [Co(dipic)]NH4, to ensure complete oxidation to metMb (Mauk et ah, 1979). The solution was then centrifuged to remove insoluble material. The metMb solution was applied to the column and washed with the 10 mM KPi buffer. Reddish coloured bands of material eluted with this buffer while the main metMb band remained bound. The metMb was eluted with a pH gradient of 100 mL 10 mM KPi buffer pH 6.8 and 100 mL 10 mM dipotassium hydrogen phosphate (dibasic). Fractions with A 4 0 9 / A 2 g 0 absorbance ratios (at pH 6) of greater than 5.6 were pooled, concentrated, exchanged into water, frozen in liquid nitrogen, and stored at —70°C. C. Preparation of ApoMb ApoMb was prepared by the method of Teale (1959) at 4°C as follows. In a 30 mL conical glass centrifuge tube fitted with a ground glass stopper, an aqueous solution of metMb was acidified to pH 1.5 with 1 M hydrochloric acid (HCI) and then an equivalent volume of ice-cold 2-butanone (Burdick and Jackson Laboratories Inc.) was added with gentle mixing. After cooling on ice, the less dense 2-butanone and the aqueous solution separated to form two distinct layers. The 2-butanone layer, which contained extracted heme, was removed. The extraction was repeated twice with successively smaller volumes of 2-butanone. The resulting straw coloured apoMb solution was dialyzed against water containing 0.6 mM sodium bicarbonate (NaHC03) and 1 mM ethylenediaminetetraacetic 32 disodium salt and then against water with 0.6 mM bicarbonate. After dialysis, the apoMb solution was centrifuged to remove any precipitate. The concentration of apoMb was determined by measurement of absorbance at 278 nm based on an extinction coefficient of 16 (mM cm)"1 (Tamura et al., 1973a). ApoMb solutions prepared in this manner had concentrations of 300 to 500 pM and were used immediately afterwards in reconstitution experiments. D. Reconstitution of ApoMb Iron (III) protoporphyrin IX dimethyl ester (DME-heme; Porphyrin Products, Logan, Utah) was dissolved in warm methanol (55-60°C) to a concentration of about 1 mg/150 /zl. The solution was centrifuged to remove any undissolved DME-heme, which could be later taken up with a small volume (/*Ls) of methanol. A 1.25 molar excess of DME-heme/methanol solution was slowly added to the apoMb solution (at 4°C) with gentle stirring. After two hours, another equivalent of DME-heme/methanol was added. The total volume of methanol added was <10% of the total protein solution volume. After two to three hours, any newly reconstituted metMb was reduced by adding a few drops of concentrated (49.3 mM) Fe(EDTA)2' solution. Immediately afterwards, the now reddish solution was dialyzed against two changes of 0.6 mM NaHC0 3 to remove the methanol. The dialysate was then centrifuged. Excess DME-heme formed a soft pellet, which was discarded. One molar equivalent of DME-heme was then added to the solution with another one half molar equivalent added two hours later. In total, the apoMb was exposed to four molar equivalents of DME-heme. To maintain reconstituted DME-Mb in the reduced state, a few drops of concentrated (49.3 mM) Fe(EDTA)2" were added to the solution before it was dialyzed against 15 mM tri(hydroxylmemyl)aminoethane (Tris) buffer pH 8.4 (at 4°C). The dialysate was then 33 centrifuged to remove excess DME-heme, which formed a firm pellet. The supernatant solution was gently decanted, and the reconstituted DME-Mb was reduced with the addition of Fe(EDTA)2". Immediately afterwards, the solution was passed through a 10 x 1.5 cm DE-52 cellulose (Whatman) column equilibrated with 15 mM Tris buffer (pH 8.4). A few grains of recrystallized potassium hexacyanoferrrate (III) (ferricyanide), K3[Fe(CN)6], was added to the eluant. The generation of metDME-Mb was followed by the slow change in solution colour from red to brown. The solution was then concentrated and exchanged into 20 mM sodium phosphate (NaPi) pH 7.2 (at 4°C) by repeated ultrafiltration. The solution was centrifuged and applied to a 95 x 2.5 cm G-75 Sephadex (Pharmacia) column equilibrated with 20 mM NaPi pH 7.2 buffer. Fractions of DME-Mb with A 4 0 7/A 2 7 8 ratios greater than 5 (at pH 6) were pooled, concentrated and exchanged into 50 mM sodium chloride (NaCl). To remove any associated ferricyanide from metDME-Mb, a method similar to that developed by Linder et al. (1978) for hemoglobin was used. At room temperature, an 8 x 1 cm column of 20-50 mesh Dowex 1-X8 anion exchange resin was cleaned just before use. The resin was initially washed with one column volume each of the following solvents in the order indicated: water, ethanol and water. This treatment was followed by three cycles of washing in the sequence of 1 M sodium hydroxide (NaOH), water, 1 M HCI and water. The resin was then washed with 1 M NaCl followed by 50 mM NaCl until the conductivity of the eluant matched that of the 50 mM NaCl solution. The concentrated metDME-Mb solution was passed through this column. The resulting eluant was collected, concentrated, frozen, and stored in liquid N2. The DME-heme was checked for homogeneity by using thin layer chromatography. Three types of heme were examined, DME-heme, hemin (Fe protoporphyrin LX) and DME-heme extracted from DME-Mb. The first two heme samples were used as supplied from commercial sources and dissolved in acidic 2-butanone (pH 1.5). The third sample was 34 obtained by extracting an acidified and desalted DME-Mb solution with 2-butanone. Samples were taken up by capillary tubes and spotted onto an ITLC (Instant Thin Layer Chromatography) silicic acid sheet (Gelman Instrument Co.). Sufficient material was spotted to allow visualization. The sheet was placed in a Gelman chromatography chamber pre-saturated with the running solvent, which was 2,6 Lutidine:H20 in a 20:1 ratio (Asakura and Lamson, 1973). After the solvent front had moved a sufficient distance (~8 cm) for significant migration of the samples, the sheet was removed and dried in air. The Rf values for the samples were determined as the ratio of dieir migration distances to that of the solvent. E. Spectroscopic Characterization of DME-Myoglobin 1. Visible Region The visible absorption spectra of metDME-Mb and oxyDME-Mb were recorded on the Cary-219 spectrophotometer. Initially, a sample of metDME-Mb was reduced with dithionite and converted to oxyDME-Mb by the procedure described in section G (vide infra). The visible absorption spectrum of the oxyDME-Mb (80 pM) was recorded using OLIS software. Then the sample was oxidized to metMb with the addition of solid potassium ferricyanide and its visible absorption spectrum recorded. The extinction coefficient of the metDME-Mb (acid form) Soret band was determined using the pyridine hemochrome method described by Fuhrop and Smith (1975) as follows. The Soret absorbance of-a concentrated metDME-Mb solution was measured. A measured portion (1.35 mL) of this solution was transferred to a cuvette and the pyridine hemochrome derivative of DME-heme was formed by mixing in sequence: pyridine (306 /xL), NaOH (144 pL) and solid dithionite. Immediately after mixing, the absorbance of the solution was measured at 562 nm, the visible wavelength of maximal absorbance for the 35 pyridine hemochrome derivative (Tamura et al, 1973a). Based on the extinction coefficient reported (Tamura et al, 1973a) for this pyridine hemochrome derivative (e 562=34.3 mM^cm"1), the concentration of the metDME-Mb solution was determined. The Soret extinction coefficient was then calculated for metDME-Mb. For comparison, this procedure was repeated for native metMb. 2. 1H NMR Spectroscopy Samples of protein were exchanged into D 20 buffer (KPi in D 20 ( 2H 20) at pD=7.0 and 7=0.1 M) through repeated (>8) cycles of centrifugation in the micro-concentrators. 'H NMR spectra of horse heart metMb and metDME-Mb were collected by Dr. Colin Tilcock in the laboratory of Dr. Pieter Cullis of the Biochemistry Dept., U.B.C. using a Bruker Model WP-200 NMR spectrometer. An external sample of 3-2,2-dimethyl-2-2-silapentane-5-sulfonate (DSS) was used to calibrate the instrument. Greater than 10,000 transients were collected per sample before Fourier transformation. 3. EPR Spectroscopy MetMb and metDME-Mb samples were prepared in sodium phosphate (pH 6.0) and sodium borate (pH 9.0) buffers (7=0.1 M), placed in EPR tubes (Ace Glass Inc.) and then frozen in liquid N2. EPR spectra of horse heart metMb and metDME-Mb were collected by Dr. Linda Pearce using a Varian Model E109 spectrometer (equipped with a temperature controller) in the laboratory of Dr. Wu of the Faculty of Food Sciences, U.B.C. The standard used was 2,2-Di(4-fm-octylphenyl)-l-picrylhydrazyl (DPPH) free radical (Aldrich Chemical Co., Dziobak and Mendenhall, 1982). 36 F. Determination of the Heme pKa To determine the pATa of the water molecule bound to the heme Fe in metDME-Mb, a spectroscopic pH titration was performed as follows. A sample of metDME-Mb was dialyzed overnight against 0.1 M NaCl and then placed into a custom-modified quartz cuvette, 4 cm x 1 cm x 1 cm (pathlength). This cuvette had a 12 cm vertical extension of glass that ended in a round 4.5 cm diameter opening. The opening was threaded to allow placement of a plastic cover (Ace Glass), which had two channels drilled through it to accommodate a MI-412 micro-combination pH electrode (Microelectrodes Inc.) and die glass tip of a Digipet 1.0 mL ultramicro-pipet (Manostat). The electrode was connected to a Radiometer Model PHM-84 pH meter. The ultramicro-pipet was used to deliver 0.1 M NaOH (Dilut-it, Baker) in small volumes with an accuracy of ± 0.2 ttL. Mixing was achieved after each addition of base with a magnetic micro-stirbar placed in the cuvette and driven by the stirrer assembly of the Cary-219 spectrophotometer. The pH of the initially acidic (pH 5.8 - 6.0) metDME-Mb solution was gradually raised by addition of ~0.5 /*L volumes of 0.1 M NaOH. The solution was stirred after each addition of base until the pH reading stabilized, at which time the stirrer was turned off. After the pH had stabilized again, the spectrum was recorded and digitized using the OLIS software. The pH was raised until no further changes in the spectrum were observed. A similar titration was performed with native horse heart metMb as a control. G. Measurement of Autoxidation Rates The in vitro autoxidation rate of oxyDME-Mb was measured as follows. MetDME-Mb was reduced by excess sodium dithionite (Na2S204) in the presence of air to produce 37 oxyDME-Mb. As recommended by Brown and Mebine (1969), an ion-exchange chromatography column was used to remove excess dithionite and dithionite by-products, which accelerate autoxidation of oxyMb. DEAE-Cellulose (DE-52, Whatman) was chosen because it also permitted separation of metMb from oxyMb (Gotoh & Shikama, 1974). To remove trace metals from solution, the buffer used (NaPi, pH 7.0, 7=0.1 M, containing 0.1 mM Na2EDTA) was passed through a 12 x 1.75 cm column of Chelex 100 chelating resin (Bio-Rad), which had previously been cleaned by 1 M HCI. Solid dithionite (Baker, stored under vacuum) was added to a small volume (30-50 nL) of concentrated metDME-Mb solution. Immediately afterwards, the solution was applied to a 6 x 1 cm column of DEAE-cellulose that had been equilibrated with NaPi buffer (pH 7.0, 7=0.1 M) containing 0.1 mM Na2EDTA. The oxyDME-Mb passed through the column as a reddish band behind a leading edge of brown colour. The oxyDME-Mb was collected into a clean cuvette and immediately placed into the thermostatted (25 °C) sample holder of the Shimadzu spectrophotometer. After allowing 30 minutes for the solution to reach thermal equilibrium, the visible spectrum (700-450 nm) of oxyDME-Mb was scanned repetitively over one hour intervals for 24 hours. The autoxidation rate for native Mb was measured by identical procedures. H. Synthesis of Pentaammineimidazoleruthenium (III) trichloride The synthesis of [Ru(NH3)5Im]Cl3 was based on the procedure of Sundberg et al. (1974) as follows. A 30 mL glass reaction vessel with a glass frit bottom was charged with 1.2 g freshly prepared zinc amalgam and 20 mL 0.05 M HCI. In this solution was dissolved 72.5 mg (0.25 mmol) of cUoropentaammineruthenium (III) dichloride, [Ru(NH3)5Cl]Cl2 (Strem Chemicals Inc.) and 90 mg (1.32 mmol) of imidazole (Im), which had been recrystallized from benzene. A stream of deoxygenated (vide infra) argon was 38 bubbled through the frit into the solution. After four hours, the solution was filtered and diluted with 50 mL of water. The reaction was then repeated to obtain more material. After combining the product from the two preparations, air was bubbled through the solution for one hour. The solution (pH 6.8) was then applied to an 8.5 x 2.5 cm BioRex-70 column, which had been washed with water. The column was first washed with 1 M HC1 followed by 4 M HC1 to elute the [Ru(NH3)5Im]Cl3. The eluant was dried by rotary evaporation to an orange coloured paste. The [Ru(NH3)5Im]Cl3 was redissolved in a minimal volume of water, crystallized with ethanol, filtered, and dried under vacuum. /. Spectroelectrochemical Experiments Potentiometric titrations were performed with an optically transparent thin-layer electrode (OTTLE) cell as described by Reid et al. (1984). The electrochemical cell consists of a lucite frame onto which were mounted two quartz plates (Wilmad) separated by two teflon spacers (Dilectrix Corp.) and a gold mesh (500 lines/inch, Buckbee Mears Co., Minneapolis, Minnesota) working electrode. A 5 cm length of copper wire was soldered to the bottom of the gold mesh. All components were sealed with epoxy cement (Epoxi-Patch, The Dexter Corp.). Two receptacles were machined into the top of the OTTLE cell body. A buffer-filled glass adapter with glass frit bottom was inserted into each receptacle. A Radiometer Model 4112 saturated calomel reference electrode (SCE) was placed in a water-jacketed salt bridge fitted with a platinum wire junction. This salt bridge was filled with saturated potassium chloride (KC1), inserted into one of the adapters and clamped to a ring stand for support. The water jacket was connected to a MGW-Lauda Model RC3 circulating water bath to maintain the reference electrode at 25 °C. A 39 platinum counter electrode was inserted into the other adapter. The potential across the working electrode was controlled by a Princeton Applied Research Model 173 potentiostat and measured with a Keithley Model 177 digital microvoltmeter to an accuracy of ± 0.1 mV. Near UV-visible absorption spectra (700-340 nm) were recorded using the Cary-219 spectrophotometer in the reduced height beam mode. The OTTLE cell was mounted onto a custom-made jacketed aluminum cell holder. The cell holder was connected to a MGW-Lauda Model RM3 circulating water bath to allow temperature control of the OTTLE cell. The temperature of the OTTLE cell solution was measured to ± 0.2°C using a Fluke Model 2175A digital thermometer and a subminiature copper-constantan thermocouple, which was inserted into a side aperture of the cell. Solutions of metDME-Mb and native metMb were exchanged into the desired buffer by use of a Centricon microconcentrator (Amicon Corp.). NaPi buffer was used in the pH range 5.5 to 8.0. From pH 8.0 to 9.5, the buffer consisted of 0.025 M boric acid titrated with 0.1 M NaOH to the desired pH; solid NaCl was added to raise the ionic strength to the desired level. The volume of protein solution used in the OTTLE cell was 400 fiL. Typical protein concentrations were 100-150 pM with the higher concentrations needed at basic pH values to compensate for lower extinction coefficients. Mediators used were [Ru(NH3)5Im]Cl3 for DME-Mb titrations and [Ru(NH 3) 6]Cl 3 for native Mb titrations at concentrations of 0.5 /*M and 4 respectively. Higher concentrations of mediator were used at lower temperature or basic pH to compensate for slower equilibration times. Protein solutions were made anaerobic by the action of Rhus vernicifera laccase, which in the reduced form is able to reduce oxygen to water (Reinhammar and Malmstrom, 1981). This enzyme was a gift from the laboratory of Prof. Harry B. Gray of the California Institute of Technology. Trace amounts of laccase (0.1 /*M in the protein 40 solution) were used with slightly higher concentrations needed under conditions of low temperature or acidic pH to compensate for lower activity. The electrochemical measurements of native Mb required the presence of the enzyme catalase to prevent formation of a third species of Mb that interfered with the metMb and deoxyMb equilibrium. A 10:1 dilution of a catalase semi-crystalline solution (Sigma type C-100) was prepared using the appropriate buffer. In general 1 /<L of diluted catalase solution was added to the protein solution prepared for the OTTLE cell (ca. 200 Sigma units of activity). Greater amounts were used under conditions of extreme pH or low temperature to compensate for lower activity. The procedure used to measure the reduction potentials of DME-Mb and native Mb were identical. A typical electrochemical measurement of DME-Mb would begin with the application of a potential of —500 mV vs. SCE across the cell. This potential reduces metDME-Mb to deoxyDME-Mb and activates, by reduction, the laccase. The reduction process was monitored by observing the absorbance changes at the Soret wavelengths of the met-and deoxy- forms of DME-Mb. The reduced DME-Mb was then re-oxidized in stages by changes of 20 mV starting from a potential approximately 50 mV higher than the measured midpoint potential to a potential 50 mV lower. Complete oxidation of DME-Mb was achieved by applying a potential of +200 mV vs. SCE to the OTTLE cell. A complete spectroelectrochemical experiment consisted of the fully reduced and oxidized spectra and six intermediate spectra representing various ratios of reduced:oxidized DME-Mb. 41 /. Fe(EDTA)2' Reduction Kinetics Kinetic experiments involving the reduction of DME-Mb by Fe(EDTA)2" were performed on a Dionex Model D-110 stopped flow apparatus. Fittings, transfer lines and seals in the apparatus were modified to improve anaerobicity (Reid and Mauk, 1982; Reid, 1984). The optical system was designed by OLIS and consisted of a tungsten-halogen light source, its power supply, a monochromator, and a photomultiplier tube. This system was interfaced with a Data General Nova 2/10 minicomputer (with a Tektronix Model 4006 graphics monitor). The instrument was controlled and data were collected and analyzed by software developed by OLIS (Reid and Mauk, 1982; Reid, 1984). Solutions were made anaerobic by bubbling them with nitrogen gas (N2) that had been scrubbed of oxygen and humidified by passage through two gas washing bottles each containing a solution of vanadate(IV)/amalgamated zinc (Meites and Meites, 1948), a third gas washing bottle containing a solution of methyl viologen, proflavin and EDTA (Sweetser, 1967) and a condensation trap containing buffer. All solutions were transferred using Hamilton gas-tight syringes and stainless steel needles. A Fe(EDTA)2" concentrated stock solution was prepared for each experiment by mixing deoxygenated solutions of NaPi buffer-EDTA and ferrous ammonium sulfate, [Fe(NH4)2]S04, as described by Wherland et al. (1975). The desired concentrations of Fe(EDTA)2" were made by dilution of the stock solution with deoxygenated NaPi buffer. Fe(EDTA)2" dilutions and protein solutions were prepared under a continuous stream of scrubbed and humidified N 2 in serum bottles capped with clean rubber stoppers. Protein solutions of 2.5 to 3.0 /*M were initially bubbled gently with deoxygenated N 2 for 30 minutes, after which the stream of N 2 was passed over the solution surface. Deoxygenated solutions of protein and Fe(EDTA)2' were drawn into 42 separate drive syringes, which were immersed in a water bath. The water bath was maintained at constant temperature by a Haake Type 3 circulating water bath. Solutions were equilibrated for 20 minutes at 25 °C before use and for 30 minutes at other temperatures. Reactions were performed under pseudo-first order conditions with Fe(EDTA)2" in at least 50-fold excess over protein. Reduction of metDME-Mb was monitored at the Soret wavelength (407 nm) and an absorbance decrease of 0.12 was routinely observed. The average of at least seven traces was used to calculate an observed first order rate constant, Jfcobs. For each experiment, a 10 fold range of [Fe(EDTA)2"] was used to obtain six values of Jfcobs. Each plot of A:obs versus [Fe(EDTA)2"] was subjected to weighted linear least-squares analysis to determine the slope, which represented the second order rate constant, £ 1 2. v 43 RESULTS A. Preparation and Isolation of DME-Myoglobin The apoMb prepared as described in the experimental procedures section showed no significant residual heme as determined by examining the Soret (near UV) region of the spectrum. Dimethylsulfoxide (DMSO) was initially used as a solvent for reconstitution of the apoMb with the water insoluble DME-heme because it had been successfully used in the reconstitution of apo-cytochrome b5 to form DME-cytochrome b5 (Reid et al., 1985). However, the use of DMSO resulted in poor reconstitution of apoMb, with low yield of metDME-Mb. With DMSO as a solvent, there was an apparent aggregation of the DME-heme in the presence of apoMb. This aggregate resulted in a greenish film that stained glassware, ultrafiltration membranes and Sephadex gels. The use of warm methanol as a solvent for the DME-heme reduced the amount of staining. However, the solubility of the DME-heme was lower in methanol than in DMSO and this necessitated the procedures described to remove excess DME-heme. Reduction of newly reconstituted metDME-Mb to oxyDME-Mb improved die yield considerably from about 10% to 70% of apoMb used. This increased yield probably reflected a more stable interaction between apoMb and the reduced form of DME-heme in the presence of methanol. The oxyDME-Mb did not bind to the DEAE-cellulose column under the conditions used to remove excess DME-heme and Fe(EDTA) 2 _ / 1", unlike metDME-Mb, which bound irreversibly. However, oxyDME-Mb tended to precipitate onto ultrafiltration membranes, which necessitated its oxidation (by ferricyanide) back to metDME-Mb prior to concentration. 44 Ferricyanide was used as an oxidant in spite of its apparent binding to metDME-Mb because the oxidant normally used in this laboratory to oxidize native Mb, [Co(dipic)2]", failed to oxidize oxyDME-Mb to any significant degree. Passage of metDME-Mb solutions through the Dowex X-8 ion-exchange column to remove ferricyanide resulted in a slight but consistent increase in the A407/A2g0 ratio of about 2-4%. Failure to remove ferricyanide in this manner resulted in significantly altered electrochemical and kinetic properties of the reconstituted protein. Dialysis was not effective in removing residual ferricyanide from metDME-Mb. Thin layer chromatography of the heme samples showed single spots for the DME-heme and DME-Mb extracted heme samples, but the native heme sample was streaked. The Rf values determined for DME-heme, native heme, and DME-Mb extracted heme were 0. 93, 0.085. and 0.93 respectively. These values agree closely with those reported by Asakura and Lamson (1973) for the same experimental conditions and indicate that the DME-heme within DME-Mb was not modified (i.e. not de-esterified) during reconstitution. B. Spectroscopic Characterization of DME-Myoglobin 1. Near UV-Visible Region The visible electronic spectrum (700-450 nm) of metDME-Mb and oxyDME-Mb are shown in Figure 5. The metDME-Mb spectrum is shown as a function of pH in Figure 8. The deoxyDME-Mb and metDME-Mb spectra can be seen in the spectroelectrochemical titration in Figure 12. Comparison of these spectra with the corresponding Mb spectra shows only slight shifts in the wavelengths of absorbance maxima, e.g. Soret band is at 407 nm for metDME-Mb and at 409 nm for metMb. The extinction coefficient for the Soret band of metDME-Mb was 168 mM"1 cm"1 and 190 mM"1 cm"1 for native horse heart metMb. 45 Figure 5. The absorption spectra of oxyDME-Mb (a and p peaks labelled) and metDME-Mb in the visible region. Spectra were recorded under conditions of pH 7.0, 7=0.1 M and 25 °C in sodium phosphate buffer. The concentration of DME-Mb was 80 i*M. 46 2. lHNMR The *H NMR spectra of both native metMb and metDME-Mb were partially obscured by the strong signal of residual water (around 7 ppm). However, the downfield region of the metMb and metDME-Mb (pD 7.0, 7=0.1 M) lR NMR spectra, which contain the majority of heme signals, were distinct and are shown in Figure 6 (A & B). The !H NMR spectrum of horse heart metMb is similar to that of sperm whale metMb (La Mar et al, 1980) in terms of chemical shifts (ppm), relative intensities and positions (Appm between peaks). Solely on this basis, proposed identification of some of the resonances in the horse heart metMb spectra are given in Table I. The corresponding *H NMR spectra of metDME-Mb, shows a different pattern of resonances from that of metMb. However, the methyl resonances are still recognizable though specific assignments cannot be made (Table I). The signals for the protons of the two methyl esters could be represented by the resonance peak labelled 4 (Figure 6A) centered at 51 ppm due to its uniqueness (compared to the native metMb lH NMR spectrum) and its relatively high intensity (about double that of the methyl peaks). 47 I 2 3 4 5 A 8 0 6 0 4 0 2 0 p p m F i g u r e 6. T h e d o w n f i e l d p o r t i o n s o f the J H N M R spec tra o f m e t D M E - M b ( s p e c t r u m l a b e l l e d A) a n d n a t i v e m e t M b (B). T h e c h e m i c a l shifts (6) are m e a s u r e d i n par t s p e r m i l l i o n ( p p m ) f r o m a n e x t e r n a l D S S s a m p l e set at 5=0. T h e c o n d i t i o n s w e r e 7 = 0 . 1 M p o t a s s i u m p h o s p h a t e b u f f e r , p D 7 . 0 a n d 2 5 ° C . 48 Table I. Proposed assignments for lU NMR resonances labelled in Figure 6. protein peak chemical proposed heme label shift (ppm) *H assignment met-Mb a 92 methyl H's b 85 methyl H's c 71 methyl H's d 51 methyl H's e 74 propionate H a f 58 propionate H a g 45 (broad) vinyl H a and propionate H a h 30 (broad) vinyl H a and propionate H a 1 18 propionate H/?s metDME-Mb 1 81 methyl H's 2 77 methyl H's 3 69 methyl H's 4 51 2 methyl H's 5 49 methyl H's 6 61 vinyl or propionate H's 7 43 (broad) vinyl and/or propionate H's 8 32 (broad) vinyl and/or propionate H's 49 3. EPR T h e f irst d e r i v a t i v e o f the E P R a b s o r p t i o n s p e c t r u m o f m e t D M E - M b ( p H 6 . 0 , 7 = 0 . 1 M ) is s h o w n i n F i g u r e 7 . T h e m a g n e t i c m o m e n t o f a p a r a m a g n e t i c spec ie s is r e p r e s e n t e d b y the g - f a c t o r , w h i c h is d e f i n e d b y : hv = g M B ^ r (W) w h e r e h i s P l a n c k ' s cons tant a n d v is the f r e q u e n c y o f a b s o r p t i o n . T h e t e r m hv represents the e n e r g y d i f f e r e n c e b e t w e e n the t w o o r i e n t a t i o n s o f the m a g n e t i c m o m e n t , i.e. the r e s o n a n c e c o n d i t i o n . T h e t e r m fiB is the B o h r m a g n e t o n ( 9 . 2 7 4 x 10" 2 1 J T 1 ) ; the t e r m Ht is the m a g n e t i c field s t rength at r e s o n a n c e (the i n f l e x i o n p o i n t o f the f i r s t -d e r i v a t i v e s p e c t r u m ) . T h e E P R s p e c t r u m o f m e t D M E - M b ( p H 6 . 0 , 7 = 0 . 1 M ) y i e l d e d g = 5 . 9 , w h i l e at p H 9 . 0 , g=5.7. C. Spectrophotometric pH Titration T h e effect o f p H o n the v i s i b l e s p e c t r u m o f m e t D M E - M b is s h o w n i n F i g u r e 8 . T h e c h a n g e s i n the s p e c t r u m o f na t ive m e t M b w i t h p H are s i m i l a r . T h e t i trat ions o f the o r i g i n a l 2 . 0 m L o f m e t D M E - M b a n d m e t M b s o l u t i o n s r e q u i r e d less t h a n 2 0 /iL o f 0 . 1 N N a O H e a c h . T h i s v o l u m e o f a d d e d N a O H represents less t h a n a 1% c h a n g e i n total v o l u m e a n d as a resu l t spec tra w e r e no t c o r r e c t e d f o r d i l u t i o n . T h e e q u i l i b r i u m represen ted b y the s p e c t r o p h o t o m e t r i c c h a n g e s is as f o l l o w s : m e t D M E - M b - H 2 0 ± ~ m e t D M E - M b O H - + H + (15) 5 0 1000 2000 3000 Magnetic Field (Gauss) Figure 7. The EPR spectrum of metDME-Mb (0.6 mM). Conditions were pH 6.0, 7=0.1 M sodium phosphate buffer frozen in liquid nitrogen (-180°C). The EPR instrument settings were microwave power of 2 mWatt, magnetic field scanning range of 1000 to 3000 Gauss, and temperature of -180°C (liquid N 2 cooling). 51 0.60 "T ' 1 1 1 1 1 r-O C D _Q O CO _Q < 0.45 0.30 0.15 0 — i — i 440 500 560 620 680 Wavelength (nm) 740 Figure 8. The visible region absorption spectra of metDME-Mb (43 pM) as a function of pH (NaOH titration). At 25°C and in 0.1 M NaCl, spectra were recorded in sequence at pH 5.87 (spectrum labelled A), 7.47, 8.17, 8.55, 8 82 9 16 10 12 and 10.47 (B). ' 52 The concentrations of acid and base forms of metDME-Mb or metMb were derived from absorbance values. These values were measured at 580 nm, which for metMb and metDME-Mb demonstrated the greatest change in absorbance with pH. The absorbance values at the extremes of pH were assumed to represent complete (100%) acid or base forms of metDME-Mb or metMb. The relationship used to determine the heme pKa is: pH = pKa + n log A a ~ ~ A (16) A-Ab The terms Ab and Aa represent the absorbance values of the base and acid forms respectively; A represents the absorbance value at an intermediate pH. The parameter n represents the number of protons transferred in the reaction and is expected to have a value of one. A linear regression plot of pH versus log[(Ab-A) / (A-Aa)] for the metDME-Mb titration is shown in Figure 9. The term n in equation (16) is derived from die slope of this plot and the pKa is derived from the y-intercept. For the metDME-Mb titration the calculated pKa is 8.49 ± 0.05 with n = 0.98(6). From a similar analysis of the metMb titration, the calculated pKa is 8.88 ± 0.02 with n = 1.00(2). 53 Figure 9. The data from the pH titration of metDME-Mb (Figure 8) plotted according to Equation 16 by linear regression analysis. A a and Ah are the absorption values of metDME-Mb'H20 (pH 5.87) and metDME-MbOH (pH 10.47) at 580 nm respectively. A is the corresponding absorbance value at intermediate pH values. The spectroscopic pKa of metDME-Mb is determined from the y-intercept of the linear regression analysis. 54 D. Autoxidation Rates The autoxidation of oxyDME-Mb was monitored by recording the visible spectrum of a solution of oxyDME-Mb at one hour intervals, as illustrated in Figure 10. To calculate the oxyDME-Mb autoxidation rate, the amount of oxyDME-Mb (relative to the total amount [DME-Mb] = [oxyDME-Mb] + [metDME-Mb]) has to be determined at each time interval. The visible spectrum of oxyDME-Mb is characterized by its prominent alpha (a) and beta (/?) absorption bands (Figures 5 and 10). The a/p absorbance ratio has been the traditional standard for determining the proportion of oxyMb in a sample of Mb. The fraction of oxyDME-Mb present in a mixture with metDME-Mb can be correlated to the corresponding o /p absorbance ratio, A[a /p) , by the relationship: [oxyDME-Mb] = Cx-A(al p)C2RTx [DME-Mb] C, - 1 + (l-C2)A(a/p)R-1 The derivation of Equation 17 is given in Appendix D. The parameter Cj represents the ratio of the extinction coefficients of metDME-Mb and oxyDME-Mb at the o band; C 2 is the corresponding ratio at the P band. The parameter R is the ratio of the extinction coefficients of oxyDME-Mb at its o and p bands. Using data reduction routines of the OLIS software, the absorbance spectra of oxy-and metDME-Mb (Figure 5) were used to generate spectra of various oxy- and metDME-Mb mixtures. A total of nine such spectra were generated ranging from 1 part metDME-Mb^ parts oxyDME-Mb to 9:1 in 1/10 intervals. For each simulated spectrum generated, an A ( o / p) value was determined. The nonlinear least squares fitting program MINSQ (Micromath Scientific Software, Salt Lake City, Utah) was used to fit the data to Equation 17. Calculated values for Ci , C2 and R are listed in Appendix D. 55 450 500 550 600 650 700 WAVELENGTH (nm) Figure 10 . The autoxidation of oxyDME-Mb (45 / J M ) to metDME-Mb depicted by sequential recordings of the absorbance spectrum in die visible region. Spectra were recorded at one hour intervals. Conditions were pH 7 . 0 , 7 = 0 . 1 M and 2 5 °C in sodium phosphate buffer. 5 6 The A ( o / p) value of each spectrum in Figure 10 was determined. The fraction of oxyDME-Mb present at each time interval was calculated using Equation 17 with the previously calculated parameter', values. These data were fitted by a linear regression program to the first order kinetics expression: - i n [°*y™E-Mb] t = k t ( l g ) [DME-Mb]c The parameter t refers to time (in hours) and k represents the first order rate constant of autoxidation. The ratio [oxyDME-Mb]t/[DME-Mb]0 represents the amount of oxyDME-Mb at time t in relation to the starting amount at t = 0 and is equivalent to the fraction of oxy DME-Mb calculated from Equation 17. The time t = 0 refers to the time the first spectrum was recorded and not the time of formation of oxyDME-Mb. Figure 11 shows the plot of the oxy DME-Mb autoxidation data. The data were analyzed by linear regression analysis to obtain a value for the slope k. At pH 7, 7=0.1 M and 25 °C, oxy DME-Mb has an autoxidation rate of * = 2.6(1) x 10"2 hr'1, which corresponds to a half-life for oxyDME-Mb of 27 hours. Under the same conditions and method of analysis, oxyMb has an autoxidation rate of k = 9.8(1) x 10"3 hr 1, which gives a half life for oxy Mb of 71 hours. 57 0 5.0 10.0 15.0 20.0 25.0 30.0 t ime (hours) Figure 11. The oxy DME-Mb autoxidation data derived from Figure 10, plotted according to Equation 18 and fitted by linear regression analysis. The autoxidation rate, *, is given by the value of the slope. 58 E. Synthesis of [Ru(NH3)5Im]Cl3 The yield of [Ru(NH3)5Im]Cl3 based on the amount of starting compound used, [Ru(NH 3) 5Cl]Cl 2, was 54%. The near UV-visible spectrum of [Ru(NH3)5Im]Cl3, which consists of two absorption bands at 299 and 430 nm, matches that reported by Sundberg et al. (1974). The calculated extinction coefficients (in water) were e299 = 1905 mM"' cm"1 and £ 4 3 0 =271 mM"1 cm"1. For comparison, Sundberg et al. reported corresponding values of 1880 mM"1 cm"1 and 250 mM"1 cm"1 respectively. F. Reduction Potential Measurements As described in the experimental procedures section, electrochemical measurements of native Mb required the presence of catalase. Without catalase present, the reduced Mb absorption spectra exhibit a shoulder centered around 600 nm and oxidized Mb spectra exhibit a shoulder centered at 630 nm instead of a broad peak. In addition, isosbestic points between the spectra of deoxyMb and metMb were not observed during the potentiometric titration in the absence of catalase. The interfering species of Mb was probably ferrylMb. This identification is based on the catalase activity of peroxide decomposition (Saunders, 1973) and comparison to the published visible spectrum of ferrylMb (Tamura et al, 1973a). The DME-Mb spectra obtained from a representative spectroelectrochemical experiment are shown in Figure 12. Corresponding spectra of native Mb are similar. At the completion of the titration, the Soret absorbance was compared to that at the beginning of the titration. There was only slight (max. 5%) decrease in the Soret absorbance, which indicated that both Mb and DME-Mb were relatively stable under the experimental conditions used. 59 The Nemst equation, which defines the reduction-oxidation equilibrium, can be written as: Ea = Em + (2.303 x lO'3) _?L log A r e d ~ A (19) nF A o x - A The ratio of absorbance values in Equation 19 is equivalent to the ratio of concentrations in Equation 3. The terms A r e d, A o x and A represent the absorbance values of the fully reduced, fully oxidized and mixed oxidation state respectively for metDME-Mb or native Mb at a given wavelength. Calculations are generally based on the change of absorbance at the Soret band of met(DME-)Mb because it exhibits the greatest change in absorbance upon change of oxidation state. The terms Ea and Em represent the applied potential (mV vs. SCE) and midpoint reduction potential respectively. The number 2.303 x 10"3 is a conversion factor used to adjust for the mV scale and base 10 logaridim used in Equation 19. The values of log[(Aox-A) / (A-Ared)] were calculated and plotted versus the corresponding applied potentials to obtain a Nernst plot for each spectroelectrochemical titration. Figure 13 is the Nernst plot of the data from Figure 12. Linear regression analysis was used to fit the data and calculate the y-intercept and slope. The calculated y-intercept represented the midpoint reduction potential (mV vs. SCE). Calculated potentials were adjusted to the standard hydrogen electrode (SHE) scale by the conversion mV vs. SHE = mV vs. SCE + 244.4 mV at 25°C. Under standard conditions (pH 7.0, 7=0.1 M and 25°C) the midpoint reduction potential of DME-Mb is +100.2 (2) mV (vs. SHE) and that of native Mb is +60.9 (2) mV (vs SHE). 60 Figure 12. The absorbance spectrum of DME-Mb (150 nM) at varying ratios of the oxidation states metDME-Mb (spectrum labelled O) and deoxyDME-Mb (R) as generated by an applied potential (Eapp). The DME-Mb sample was in a spectroelectrochemical (OTTLE) cell as described in text. In sequence, the applied potentials (mV vs. SCE) were: -500.0 (R), -200.0, -180.0, -160.0, -140.0, -120.0, -100.0 and +200.0 mV (O). Conditions were pH 7.0, /=0.1 M, 25°C in sodium phosphate buffer with 0.5 fiM Ru(NH3)5Im and 0.1 laccase. 61 -1.20 -0.80 -0.40 0.0 0.40 0.80 1.20 log((A 0 -A)/(A-A r )) Figure 13. The data derived from Figure 12 plotted according to the Nernst Equation (19). AQ and Ar are the absorbance values of metDME-Mb and deoxyDME-Mb respectively at 407 nm (the Soret band of metDME-Mb). A is the absorbance at 407 nm of various ratios of metDME-Mb and deoxyDME-Mb as determined by the applied potential (£ a p p). The applied potential values are referenced to both the standard calomel and hydrogen electrodes (SCE and SHE respectively). The midpoint potential is determined from the y-intercept of the linear regression analysis fit. 62 The close agreement between the calculated slope and the coefficient value of the logarithmic term in Equation 19 was used as a criterion for the reliability of the electrochemical potential measurement, e.g. at 25°C, the expected slope is 59 mV. Another criterion was the correlation coefficient of the plotted data to the linear (first order) fit. Repeated measurements of Em (for a given sample under identical conditions), which met the above criteria, agreed within ± 3 mV. A list of measured reduction potentials for both DME-Mb and native Mb (Lim and Mauk, 1986) is given in Appendix 1. All measured potentials were derived from Nernst plots with correlation coefficients >0.995 and slopes with a <3% difference from the expected slope. J. pH Dependence of Reduction Potential The pH dependencies of the midpoint reduction potential for DME-Mb and native Mb are shown in Figure 14. The pH of the protein solutions after spectroelectrochemical measurements did not change significantly from their initial pH. At pH 8.0, the reduction potential of DME-Mb was measured using both phosphate and borate buffer. There was no significant difference between the midpoint reduction potentials determined in these two buffers. This absence of specific buffer effect allowed the continuous fit of the data gathered throughout the pH range measured for both native and DME-Mb. The pH dependences of the DME-Mb and native Mb reduction potentials were initially fitted with a least-squares routine to the equation: Em=E0 + RI ln + [ H + ] (20) nF Kox + [H +] 63 Equation 20 describes the effect of a single titratable group within a hemeprotein on its measured Em (Dutton, 1978). The proton dissociation constant for this group is in turn influenced by the oxidation state of the protein. The terms Kted and Kox represent the dissociation constant of the group when the hemeprotein is in the reduced and oxidized states respectively. The term EQ is the theoretical reduction potential (and hence free energy change) independent of contributions from the free energy change associated with the change(s) in protonation upon reduction i.e. at pH 0. From a least-squares analysis based on Equation 20, the fits of the pH dependence data for native and DME-Mb are shown in Figures 14. The pH dependence of the native Mb reduction potential is consistent with an EQ of 64.6 mV with a pKred of 8.5 (ATred = 3.2 x 10"9 M) and a pKox of 7.7 (ATox = 2.0 xlO"8 M). The pH dependence of the DME-Mb reduction potential is best fitted with an EQ of 134 mV with a pK[ed of 7.8 (Kred = 1.6 x 10"8 M) and a pKox of 6.4 (ATox = 4 x 10'7 M). Another model for this type of numerical analysis was proposed by Pettigrew et al. (1975) based on the method of Clark (1967). This model assumes two ionizations that affect the reduction potential in the oxidized protein and only one ionization in the reduced form. This model is applicable to Mb because the heme bound water ligand is only present in metMb and not deoxyMb. The dependence of Em on pH for this model is written as: R T Em = E0 + 1' ln nF [ H + ] 2 + Kt [H +] 2 + tfol[H+] + Kol Ko2 (21) 64 LxJ X CO > > Ld 120 6.0 7.0 8.0 9.0 10.0 PH Figure 14. The pH dependence of the midpoint potential (Em) of DME-Mb (—O—O—) and native Mb (—•—•—) fitted to Equation 20. Conditions were 7=0.1 M and 25 °C in sodium phosphate buffer. 65 Figure 15. The same data as in Figure 14 fitted to Equation 21. The pH dependence of the midpoint potential (Em) for DME-Mb is labelled ( - O - O - ) and that of native Mb is labelled ( — • — • — ) . 66 The pH dependence data was fitted to Equation 21 using the least squares fitting program MINSQ (MicroMath Scientific Software, Salt Lake City, Utah). In this analysis, the value of Ko2 was fixed at the Ka value for the heme coordinated water molecule of native or DME-Mb, which was determined from titration of the metMb or metDME-Mb visible absorption spectrum (Section C). The fits of the pH dependence data for native Mb and DME-Mb derived from Equation 21 are shown in Figure 15. For die pH dependence of the native Mb reduction potential, values of 7.4 and 7.7 were calculated for the pKol and pKr respectively with J50=65.4 mV. For DME-Mb, the pKol and pKt values were 6.3 and 7.5 respectively with f?0=135 mV. 2. Temperature Dependence of Reduction Potential The free energy change of the reduction process defined in Equation 2 is composed of enthalpic and entropic terms. In an electrochemical cell, these parameters are related by the equation: A G 0 = AH° - TAS°C (22) In this relationship, AS°C is the reaction entropy change for the entire electrochemical cell including that of the reference electrode. In biological applications, the energy terms with the superscript symbol (°) do not represent standard conditions (i.e. one Molar concentrations) but conventional biological conditions, e.g. pH 7, 7=0.1 M. Converting free energy to electrochemical terms (by AG°=—nFEm) yields the electrochemical version of the van't Hoff equation: _ 7AS° _ AH0 (23) m F F 67 The temperature dependences of DME-Mb and native Mb midpoint reduction potentials are shown in Figure 16. These data were analyzed by linear regression, and the slope and y-intercept were calculated for each set. The value of the slope represents AS°C/ F and that of the y-intercept represents —AH/F. At pH 7.0 and 7=0.1 M, the oxidation-reduction equilibrium of native Mb is characterized by AS° C = —22.6(5) eu (—94(2) J mol^K"1) and AH0 = —8.1(2) kcal mol"1 (—33.9(8) kJ mol"1). From Equation 2, the free energy change is AG 0 =—1.36 kcal mol"1 at 25°C. The midpoint reduction potential of DME-Mb exhibited a greater dependence on the temperature than native Mb. The oxidation-reduction equilibrium of DME-Mb is characterized by AS°C = —35.4(8) cal moHK-1 or eu (-148(3) J mol^K"1) and AH° = —12.9(2) kcal mol"1 (-54.0(8) kJ mol1). The free energy change at 25°C is AG°= —2.34 kcal mol"1. The AS°C values can be corrected for the contribution of die reference standard hydrogen electrode (SHE) by the equation: AS 0 = A S ° - (5 H+-'/ 2S H 2) (24) The entropic change in the SHE half-cell at 25°C is +15.6 eu (Taniguchi et al, 1980; Giauque, 1930) and is regarded as a constant. After correcting for the SHE entropic contribution, AS 0 for DME-Mb is —51.0(8) eu (-213(3) J mo^K"1) at pH 7.0 7=0.1 M. Similarly, AS 0 for native-Mb is —38.2(5) eu (—160(2) J mol^K"1). 68 Figure 16. The temperature dependence of the midpoint potential (Em) of DME-Mb (-O-O-) and native Mb (—•—•—) fitted to Equation 23 by linear regression analysis. Conditions were pH 7.0 and 7=0.1 M in sodium phosphate buffer. 69 3. Ionic Strength Dependence of Reduction Potential The ionic strength dependences of DME-Mb and native Mb reduction potentials are shown in Figure 17. These data were fitted to the equation derived by Goldkorn and Schejter (1976): Em = En - 2.3 RTjqj-tf) A /(I) (25) F In this relationship, the term Ea is the midpoint reduction potential at zero ionic strength and the terms qQ and qT are the net charges on the protein in the oxidized and reduced states respectively. The constant A is the Debye-Hiickel constant of ionization, which has a value of 0.509 C^M'^ at 25°C in water. The term J\I) is an ionic strength (/) function with the general form (Tanford & Kirkwood, 1957; Beetlestone & Irvine, 1963): M = — (26) 1 + BRaVl The parameter B is the Debye-Hiickel coefficient, which has a value of 0.329 M'^A"1 in water at 25°C; Ra is the distance of closest approach between the protein and a small ion in solution. Goldkorn and Schejter have used two forms of J\I) depending on the observed degree of dependence of Em on ionic strength. For cytochrome b5 (Reid et al., 1982) and cytochrome c (Margalit and Schejter, 1973), the midpoint reduction potentials of which exhibit a relatively small ionic strength dependence, Ra is considered to be equivalent to the radius of the protein. For cytochrome c-552, the reduction potential of which exhibits a larger ionic strength dependence (Goldkorn and Schetjer, 1976), the term BRa is assigned a value of one. 70 In fitting the current data, various values for the radius of Mb were used. The hydrodynamic radius of Mb (assuming a globular shape) was calculated from the following equations derived from Tanford (1961): if3 = 3 M ( v 2 + 5jt7f) (27) 4JT/V In this relationship the term N is Avogadro's number (6.023 x 1023 mol"1) and the term M is the molecular weight of native Mb, which is 17,641 Daltons based on its sequence. The terms tr2 171 3 1 6 m e partial specific volumes of proteins and solvent (water) respectively. The term 5j is the effective solvation of protein. The standard values from Rosenberg et al. (1976) are, S l = 0.2, t>2 = 0.73 cm3/g, Vf = 1 cm3/g. As a result, Equation 27 reduces to: From Equation 28 the calculated hydrodynamic radius for Mb is 23 A. The average axial radius of the native horse heart metMb crystal structure (Evans & Brayer, 1988) was calculated by Mr. Gordon Louie. First the centre of the metMb crystal structure was determined from the coordinates of the polypeptide chain atoms. Then solvent accessible amino acid residues were identified (88 in total). The distances from the calculated center to the outermost fixed position atom of each solvent accessible amino acid residue were calculated and averaged. A value of 17.8 A for the average axial radius was obtained by this method. R = 0.717 V M (28) 71 0 0,20 0.40 0.60 Ionic Strength (M) Figure 17. The ionic strength dependence of the midpoint reduction potential (Em) for DME-Mb (-O-O-) and native Mb (-•-•-) fitted to Equation 25. Conditions were sodium phosphate buffer, pH 7.0 and 25°C. 72 The ionic strength dependence of Em was fitted to Equation 25 using a least squares fitting routine with each of these values for the protein radius. Figure 17, which shows the fit for Ra = 17.8 A, is representative of all the fits calculated. Estimated values for the E0 and qQ of DME-Mb and native Mb are listed in Table II. Table II. Estimated values for midpoint potential at zero ionic strength (EQ) and net charge of the oxidized hemeprotein (qQ) as a function of various radius (Ra) values used in Equation 26. hemeprotein Ra (A) E0 (mV vs. SHE) q0 native metMb 3.04* 95.9(4) + 2.8(2) 17.8 150 (2) + 14.0 (3) 23 169 (2) + 19.9 (4) metDME-Mb 3.04* 158(6) + 4.3(3) 17.8 250 (3) + 22.7 (4) 23 281 (3) + 32.6(4) * denotes a radius value such that the term BRa = 1 in Equation 26 G. Reduction Kinetics Measurements The effect of residual ferricyanide on the observed kinetics of metDME-Mb reduction was significant. Samples of metDME-Mb that were not stripped of ferricyanide exhibited biphasic pseudo-first order plots for the change in A 4 0 7 with time. Samples that were stripped of ferricyanide by the Dowex X-8 anion-exchange column gave pseudo-first order plots that were linear for about 3 - 4 half-times. Figure 18 compares pseudo-first order plots obtained with and without the presence of ferricyanide. 73 + Fe(CN)l- -Fe(CN)l-Figure 18. Reproductions of the stopped-flow tracings (AA vs. time) and kobs plots (logAA vs. time) for the reduction of metDME-Mb by 2 mM Fe(EDTA)2" at pH 7.0, 7=0.1 M and 25°C in sodium phosphate buffer. In the set of tracings labelled +Fe(CN)|~, ferricyanide was present in the metDME-Mb solution. In the set labelled -Fe(CN)|~, ferricyanide was removed as described in the procedures section. 74 Figure 19. The second order plot for the reduction of metDME-Mb by Fe(EDTA)2". The observed pseudo-first order reaction rate (Jfcob«) was determined for various concentrations of the reductant ([Fe(EDTA)2"]). The second order rate constant was determined from the slope of the linear regression analysis fit. 75 The reduction of metDME-Mb (stripped of ferricyanide) proceeded to completion under all experimental conditions as demonstrated by the identical changes in A 4 0 7 observed at all Fe(EDTA)2" concentrations used in each experiment. Figure 19 shows die dependence of metDME-Mb reduction rate on Fe(EDTA)2" concentration with the fit from linear regression analysis. Such analysis of similar data obtained under various reaction conditions yielded linear fits with y-intercept values hear zero, which is expected for a second order reaction. The second order rate constant for metDME-Mb reduction by Fe(EDTA)2" under standard conditions (NaPi buffer, pH 7, 7=0.1 M and 25°C) is 1.34(2) x 103 M 'V 1. 7. pH Dependence of Reduction Rate The pH dependence of the second order reaction rate for the reduction of metDME-Mb by Fe(EDTA)2" is shown in Figure 20. The variation in with pH is in part due to variation in the driving force of the reaction with pH. The driving force is equivalent to the difference between the reduction potentials (A£ m) of DME-Mb and Fe(EDTA). The A £ M can be calculated from the pH dependence of the Em for DME-Mb and for Fe(EDTA), which has been measured at 7«0.1 M and 25°C by Kolthoff and Auerbach (1951) and at 7=0.5 M and 25°C by Reid (1984). The observed kn can then be corrected for the change in driving force. An adjusted reaction rate, k^K is defined by an equation derived from Marcus theory (Reid, 1984; Reid et al, 1986): ^ = k n ^ l 9 M A E ^ (29) 76 F i g u r e 2 0 . F r a m e A i l lus trates the p H d e p e n d e n c e o f the s e c o n d o r d e r rate cons tant (kn) f o r the r e d u c t i o n o f m e t D M E - M b b y F e ( E D T A ) 2 " f i t ted to E q u a t i o n 3 0 . T h e c o n d i t i o n s w e r e 7=0.1 M a n d 2 5 ° C i n s o d i u m p h o s p h a t e b u f f e r . F r a m e B i l lus trates the resul t s o b s e r v e d f o r the c o r r e s p o n d i n g r e a c t i o n b e t w e e n n a t i v e m e t M b a n d F e ( E D T A ) 2 " ( L i m a n d M a u k , 1986) at 7=0.5 M a n d 2 5 ° C . 77 Figure 21. Frame A represents the data for the pH dependence of the second order rate constant adjusted (tf^ ) for the driving force of the reaction between metDME-Mb and Fe(EDTA)2" at 7=0.1 M and 25 °C. Frame B represents the results of corresponding experiments and calculations for the reaction between native metMb and Fe(EDTA)2" (Lim and Mauk, 1986) at 7=0.5 M and 25°C. 78 A table of adjusted reaction rates is provided in Appendix C and the results are plotted in Figure 21. After correcting for the driving force, it can be seen that about 25% of the change in rate over the observed pH range was due to the pH dependence of the difference in reduction potential between DME-Mb and Fe(EDTA)2~. The pH dependence of the reaction rate for metDME-Mb reduction by Fe(EDTA)2" is based on the assumption that a single titratable group in DME-Mb or Fe(EDTA)2" influences the reaction rate. Based on such a model, Rosenberg et al. (1976) have derived the following relationship between second order rate constant and pH: _ * a[H +] + khKa (30) "•12 ~ [H +] + Ka The term Ka represents the proton dissociation (acid) equilibrium constant for the titratable group in question. The terms ka and kh represent the second order rate constants for the fully protonated and fully deprotonated forms of DME-Mb respectively. Both the adjusted and unadjusted sets of pH dependence kinetics data for DME-Mb were fitted to Equation 30 using a least-squares fitting procedure. For the unadjusted set of data, the values calculated from the fit (Figure 20) were pKa=lA (Ka=4.0 x 10~8 M), fca=1918 M 'V 1 and Jfcb = l . l M 'V 1. From the fit of the adjusted data set (Figure 21), p/sTa=7.6 (tfa=2.5 x 10"8 M), *a=1562 M"1 s'1 and *b=2.8 M"1 s"1. Measurement of the pH dependence of the reduction potential of native Mb allowed re-examination of the Mb reduction kinetics data of Lim and Mauk (1984). The observed pH dependence of the reaction rate, kl2, was adjusted for the driving force (AE m between native Mb and Fe(EDTA)2") using Equation 29. The results are tabulated in Appendix C and plotted in Figure 21. Analysis of the adjusted Jt 1 2 pH dependence for native Mb using Equation 30 resulted in the following parameter values (unadjusted values in parentheses): ptfa=5.9(5.8), fca=163(60) M 'V 1 and fcb=13(8.7) M 'V 1 . 79 2. Temperature Dependence of Reduction Rate Equation 9, which relates kn to the free energy of activation by transition state theory, can be redefined in terms of enthalpic (A//*) and entropic (AS*) activation energies as: ft,, AS* Atf* . ,_ R (31) In * 1 2 = _ - + In T R RT Nh The term /V represents Avogadro's number (6.022 x 1023 mol"1), h represents Planck's constant (6.625 x IO - 3 4 J s), R is the gas constant (8.314 J K^mol'1), and T is the temperature in degrees Kelvin. Figure 22 shows the Eyring plot of —lnft 1 2 versus temperature fitted to Equation 31 by a linear regression analysis. For the reduction of metDME-Mb by Fe(EDTA)2", AH *= +9.2(3) kcal mol"1 (38(1) kJ/mol) and AS *= —13(1) eu (-54(4) kJ mol"1 K 1). 3. Ionic Strength Dependence of Reduction Rate Analysis of the ionic strength dependence of metalloprotein reaction rates was initially based on relationships derived from the Debye-Hiickel theory for the activity of ions in solution (Moore and Pearson, 1981). However, simple Debye-Hiickel theory has proven inadequate for the analysis of metalloprotein kinetics (Feinberg and Ryan, 1981). An expression for the ionic strength dependence of a cross-reaction involving a metalloprotein and a small molecular reagent has been derived from Marcus theory by Wherland and Gray (1976; 1979) and is given in Equation 32: 80 0.25 1 ' «- 1 1 3.2 3.4 3.6 1/T (1/KELVIN) X 1000 Figure 22. The Eyring plot for the temperature dependence of the second order rate constant (kl2) for the reduction of metDME-Mb by Fe(EDTA)2". The data were fitted to Equation 31. The conditions were pH 7.0 and 7=0.1 M in sodium phosphate buffer. 81 ln kn = ln * i n f 3.567 -BR^I -BR^I 1 +_! (32) 1 + BR2y/I 1 + BR^I The parameters Zj and 7^ represent the apparent net charges of the reagents. The term Rl + R2 is the sum of the radii of the reactants and is assumed to represent the distance between the reactive centers. By convention, the protein is designated reagent 1 and the low molecular weight oxidant/reductant is reagent 2. The constant 3.567 is derived from the term lA((p-le), where e is the electron charge and e represents the dielectric constant of water at 25 °C. The constant B is the same Debye-Hiickel constant defined in Equation 26. The term Jfcinf represents the second order rate constant at infinite ionic strength. In a similar manner to the analysis of the ionic strength dependence of the midpoint potential, Em, several values for the radius of Mb were used. The hydrodynamic and average axial radii of Mb were applied to Equation 32 in fitting the ionic strength dependence kinetics data by non-linear regression analysis. Use of these values implies that the net charge of the protein influences the reaction rate. Derivation of Equation 32 involves the simplifying assumption that proteins are spherical ions with uniformly symmetric charge distributions. However, this equation does not consider the effect(s) of individual and localized charges in proteins. If any of these charges influence the cross-reaction rate significantly, for example by acting as binding sites or through short-range electrostatics interactions, they have a dis-proportional influence on the calculated protein charge, Z j . The radii of proteins used in such calculations of ionic strength dependence then become less valid because of the varying influence specific regions of protein surfaces have on reaction rates. 82 To account for a predominant local rather than net charge effect, Segal and Sykes (1977) used the arbitrary value of 5 A for the radius (Ri) of the active site of the blue copper protein plastocyanin. A radius of 5 A for the active site of Mb may be too small because of the size of the porphyrin ring prosthetic group coordinated to the iron molecule. A value of 10 A was chosen for the current analysis because it can accommodate the heme radius and because it is also equivalent to the distances from the heme Fe to the Lys residues 45 and 96, which flank the opening of the heme crevice. The latter reason would also be consistent with a mechanism that has electron transfer occurring at the heme edge. The resulting charges (Zj) calculated for metDME-Mb using the various radius values are listed in Table III. The fit of the data in Figure 23 with Rx = 17.8 A is representative of all the fits because the parameter Rx is treated as a constant in Equation 32. Table III. Estimated values for net oxidized hemeprotein charge (Zj) and second order rate constant at infinite ionic strength (Jfcinf) using various values of hemeprotein radius (R{) in Equation 32. hemeprotein Rx (A) Zx fcinf (M'V1) native metMb 10 +2.0(3) 11(1) 17.8 +4(1) 12(1) 23 +6(1) 12(1) metDME-Mb 10 + 6.1(4) 2.0 (3) x 102 17.8 + 14 (2) 2.2 (6) x 102 23 + 22 (3) 2.2 (7) x 102 83 12 1 • - — * » 0 0.30 0.60 Ionic Strength (M) Figure 23. Frame A represents the ionic strength dependence of the second order rate constant (k12) for the reduction of metDME-Mb by Fe(EDTA)2" fitted by Equation 32 (pH 7.0, 25°C, sodium phosphate buffer). Frame B represents similar results for the reduction of native metMb by Fe(EDTA)2" (Lim and Mauk, 1986). 84 ff. Marcus Analysis of Kinetics The second order rate constant for the reaction between a metalloprotein and a substitutionally inert inorganic complex can yield useful mechanistic information if it is analyzed in terms of relative Marcus theory. Such analysis permits, in principle, allowances for the contributions to this rate made by the thermodynamic driving force of the reaction, the electrostatic interactions between the reactants and the intrinsic reactivity of the inorganic complex. This analysis, however, requires considerable information about the properties of both reactants. This information is readily available in the present case. The electrochemical properties of Fe(EDTA) have been characterized by several investigators (Schwarzenbach and Heller, 1951; Kolthoff and Auerbach, 1952; Reid, 1982). The self-exchange rate of Fe(EDTA)2" has been estimated by Wilkins and Yelin (1968) on the basis of cross-reaction with Fe(CyDTA)1". From the Fe(EDTA)2" and DME-Mb electrochemical and kinetic data, the various activation energies of the reactants and their reactions can then be calculated (Appendix E). From the calculated reaction parameters, the free energy change for the hemeprotein's self-exchange reaction, AG*1( can be corrected for the electrostatic work term (wn) to determine AG** (or AG*jorr). The electrostatics corrected self-exchange reaction rate (at 25 °C and 7=0.1 M), # 1 ° " , can then be calculated from AG*i o r r. For DME-Mb an average value of 0.41 M"1 s"1 was determined for fc^f (from AG*forr= 18 kcal mol'1). 85 DISCUSSION A. Preparation of DME-Myoglobin The procedures used to prepare and purify DME-Mb are a significant improvement over those reported by Tamura et al. (1973a). These investigators reported a yield of less than 5% while the method developed here provides a 70% yield. Furthermore, the purity of the product obtained with the current method is significantly improved as reflected in the higher Soret/A280 absorbance ratio (>5.0 vs. 3.5). A major problem in the reconstitution of apoMb with DME-heme is the insolubility of this heme in aqueous solutions that results from esterification of the propionate groups. Another problem was aggregation of the DME-heme and apoMb, which necessitated the additional step of using ion-exchange chromatography to remove excess heme. The type of aggregation observed with apoMb was not similar to that observed in the reconstitution of apocytochrome b5 (Reid et al., 1984). Excess DME-heme could be separated from reconstituted DME-cytochrome b5 as a well defined band by gel-filtration on a Sephadex G-75 column with none of the streaking problems seen with apoMb. This difference was probably due to non-specific aggregation of apoMb with DME-heme, which could be another cause of the low yield Tamura et al. have reported. Removal of aggregated heme by centrifugation and repeated exposure of the apoMb to freshly dissolved DME-heme presumably also helped improve the yield. However, the step most responsible for the improvement in yield was the reduction of newly reconstituted metDME-Mb with Fe(EDTA)2" (in presence of air) to oxyDME-Mb. In the presence of the DME-heme/methanol, apoMb appears to have a greater affinity for reduced (ferro)DME-heme than for oxidized (ferri)DME-heme. This observation is consistent with the report of Banerjee and Stetzkowski (1970) who found that the heme-binding fragments of sperm whale Mb derived from cyanogen bromide cleavage have much 86 higher affinities for ferroheme than ferriheme. Tamura et al. (1973a) also reduced reconstituted metDME-Mb (with dithionite) just before loading it onto a cation exchange column of CM-50 resin. However, repetition of their procedure resulted in irreversible binding of a large proportion of the oxyDME-Mb to the resin. Difficulty in reconstituting other apoproteins with DME-heme is a common experience. Asakura and Yonetani (1969) reported difficulty in preparing the DME-heme derivative of cytochrome c peroxidase (CCP). Ozols and Strittmatter (1964) were unable to prepare the DME-heme derivative of cytochrome bs. Yonetani has suggested that the heme propionates in CCP direct the heme to the correct position within the heme pocket. In Mb, the heme propionates may have the same function. Control of heme entry by the propionates may explain how heme rotational isomers may form in Mb. Heme rotation around the a—i axis does not alter the positions of the heme propionates relative to the protein (see Figure 1). B. Spectroscopic Properties 1. Near UV-Visible Comparison of the DME-Mb near UV and visible absorption spectra (Figures 5 and 12) to those published for native Mb by Tamura et al. (1973a) shows that there are differences in the wavelengths of absorption maxima, which only differ by only a few nanometers, e.g. the Soret band for native metMb (aquo form) is at 409 nm compared to 407 nm for metDME-Mb (aquo form). The similarity of their spectra indicates that any changes in the environment of the heme within the protein upon propionate esterification are not observable based on the near UV-visible spectrum alone. The near UV-visible spectra of other heme derivatives of Mb prepared by Tamura et al. (1973a) also showed only slight differences from that of native Mb. 87 The Soret extinction coefficient detennined here for native (aquo)metMb, 192 mM"1 cm"1, is similar to that determined by Tamura et al. (1973a), 188 mM^cm"1. The Soret extinction coefficient detennined for (aquo)metDME-Mb (at pH 6), 162 mM^cm"1, was higher than that reported by Tamura et al. (1973a) of 145 mM^cm"1. This difference may be attributable to the reduced level of apoMb contamination in DME-Mb preparations obtained by the methods described. The pH dependent changes in the visible spectrum of metMb are due to die proton equilibrium of the water molecule coordinated to the heme Fe(III) i.e. Fe—H 20 Fe—OH - + H +. The pKa of this water ligand was found to be 8.9 for native Mb in agreement with the findings of Antonini et al. (1971) and Tamura et al. (1973b) who used similar titration procedures. From titration of the metDME-Mb visible spectrum (Figure 8) the pKa of the water ligand in metDME-Mb was found to be 8.5. In contrast, Tamura et al. (1973b) reported a pKa of 7.5 for metDME-Mb. However, their pK& value was calculated from a set of data (absorbance change, pH) containing only three points, which were measured at pH values less than 8. Tamura et al. reported difficulty with DME-Mb stability at alkaline pH, while in the current experiment there were only minor shifts of the isosbestic points at extreme alkaline pH values, indicating only slight denaturation of metDME-Mb. The increase in pKa of the heme coordinated water ligand in metMb over that of free heme (7.6; Falk, 1964) has been attributed to hydrogen bonding with the distal His-64 residue. The difference in pK& between metDME-Mb and native Mb corresponds to a 2.5 fold increase in the Ka of the water ligand. The greater acidity of the water ligand in metDME-Mb may result from some weakening in the hydrogen bonding interaction between the distal His residue and the water ligand e.g. an increased distance of separation between them. 88 2. lHNMR In the !H NMR spectra of sperm whale metMb published by La Mar et al. (1983), the minor component of heme rotational isomerization gives rise to absorption peaks with similar chemical shifts as those of the major heme rotational isomer. There is no evidence of the minor heme rotational isomer in the (aquo)metMb 'H spectra shown in Figure 6B. The absence of the minor components signals is probably due to the limited resolution of the 200 MHz NMR instrument used, which was unable to resolve the expected propionic acid methylene proton signals (labelled g & h in Figure 6B). The metDME-Mb *H NMR spectrum at 200 MHz (Figure 6A) also exhibits no evidence the minor component, which suggests that the heme rotational equilibrium ratio of (aquo)metDME-Mb is no greater than that of native (aquo)metMb. That their heme rotational equilibria may be similar is to be expected since the heme methyl and vinyl groups have more influence on the equilibrium between heme rotational isomers by apoMb than the propionate groups (La Mar et al., 1986). Also, rotation of the heme is about the a - 7 axis, which does not affect the relative positioning of the heme propionates or propionate esters. The proton signals of the heme propionate methyl esters in DME-Mb may be represented by the broad peak centered at 51 ppm (labelled 4 in Figure 6A), which does not appear in the metMb *H NMR spectrum. The 'H NMR spectrum of the model compound DME-protoporphyrin IX Fe(Imidazole)2 (La Mar and Walker, 1979) exhibits a single narrow peak for the proton signals from both methyl esters. The peak broadening seen with DME-Mb may indicate that the local environments of the heme propionate esters within DME-Mb are similar though not identical. One possibility is that in metDME-Mb the heme propionate esters are both extended out towards the heme pocket opening unlike the inner and outer heme propionate arrangement found in native metMb. 89 The downfield !H NMR spectra of the observable heme signals in metDME-Mb show that there are changes in the chemical shift values of all the observable heme methyl groups from that in native Mb. These changes indicate alterations throughout the entire porphyrin environment after propionate esterification though the specific changes can not be determined from the current results. 3. EPR The EPR spectra of metDME-Mb at pH 6 (Figure 7) and 9 were similar to the corresponding spectra of native metMb in terms of shape and calculated g value. These results agree with those of Tamura et al. (1973a) who calculated an identical g value (6.0) for both native and metDME-Mb (and all other heme propionate and vinyl derivatives of Mb) under liquid helium (10 K) temperature. The similarity between the g values of metDME-Mb and native metMb indicates that heme propionate esterification did not grossly perturb the heme Fe environment in contrast to its effects on that of the porphyrin. C. Autoxidation Rate The measured rate of autoxidation for native horse heart oxyMb at pH 7 (NaPi buffer) and 25°C (0.0098(1) hr 1) is slower than the 0.012 hr"1 reported for both bovine (Gotoh and Shikama, 1974) and sperm whale oxyMb (Suzuki and Shikama, 1983). This difference is probably not due to a species difference (e.g. sequence) because of the similarity of the sperm whale and bovine oxyMb autoxidation rates. The different rates measured may be due to the different methods used to prepare oxyMb. Shikama and co-workers isolated and purified oxyMb from fresh muscle thus avoiding the need to reduce 90 metMb with dithionite, the oxidation products of which can accelerate the observed autoxidation rate. That the present study measured a lower autoxidation rate indicates that dithionite and its by-products were effectively removed from the oxyMb solution. The difference in autoxidation rates may also be due to the use of the Chelex 100 chelating resin in the current study to remove trace metals from the buffer solutions, which can also accelerate autoxidation. Analysis of oxyDME-Mb and native oxyMb autoxidation by first order kinetics gave good results. However, slight deviations from the calculated fit of the data (Figure 11) may indicate that autoxidation has a more complex mechanism than simple first order kinetics. Based on the observed pH dependence of the autoxidation rate, Shikama and Sugawara (1978) have proposed an "acid-base catalyzed three state model" for the mechanism of autoxidation in which there is catalysis by protons at low pH and hydroxyl anions at high pH. Protons were proposed to catalyze this reaction by reaction with the bound oxygen molecule: Fe—0 2 + H + Fe(III) + H0 2* 0 2~ + H + The hydroxyl anion promotes autoxidation via a S N2 nucleophilic displacement of a superoxide anion from the Fe atom: Fe-0 2 + OH" -» [HO—Fe—0 2] -+ Fe-OH + 0 2~ The three state component involves two titratable or "prototropic" groups, which participate in what the authors call the "spontaneous" reactions: Mb02(AH, BH) -• metMb + 0 2~ Mb0 2(A- BH) -» " Mb0 2(A- B~) - " 91 The values of the pKa for the two groups has been estimated as 6.75 and 10.4. Based on the thermodynamic analysis of the pH dependence, Sugawara and Shikama (1980) identified the prototropic groups as a histidine and tyrosine residue respectively. The authors proposed the (distal) His-67 residue, as the specific His residue involved in autoxidation and that it participates in hydrogen bonding or direct protonation of the Fe bound oxygen molecule in a reaction similar to the one described above. The action of the His residue is accompanied by a S N2 displacement of the oxygen molecule (as 0 2) by an incoming water molecule, which then becomes the aqueous ligand for metMb. Olson et al. (1988) have prepared a mutant Mb in which the distal His residue has been replaced by a Gly residue. The resulting mutant exhibits a greater dioxygen dissociation constant than the wild-type protein but the effect of the amino acid substitution on autoxidation was not studied. The autoxidation rate for oxyDME-Mb at pH 7 and 25°C (0.026 hr"1, ^=27 hours) was ~2.5 times greater than the corresponding rate for oxyMb. However, the observed rate was much slower than that reported by Tamura et al. (1973a), who estimated the half-life of oxy DME-Mb as only 20 minutes (pH 7, room temperature). Loss of the inner heme propionate negative charge through esterification would eliminate any possible electrostatic interaction with the distal His residue. As a result, there may be an acidic shift of the His residue ptfa, which would then increase protonation of the heme Fe oxygen ligand leading to an increased autoxidation rate. However, a complete pH profile of the DME-Mb autoxidation rate is required to demonstrate such an effect. Another possible explanation could be a change in the geometry of the Fe—0 2 bond from that of native Mb, which is not favorable for electron transfer reactions such as autoxidation (Shikama, 1985). OxyMb prepared from tuna Mb, which has a ~3:2 ratio of heme rotational isomers (Levy et al., 1985), has a greater autoxidation rate than sperm whale or bovine Mb 92 species (Brown and Mebine, 1969). OxyMb prepared from newly reconstituted sperm whale metMb, which has a high degree of heme rotational isomerization, was observed to autoxidize faster than equilibrated oxyMb (Aojula et dl., 1987). These results indicate that the minor heme rotational isomer, while having no difference in 0 2 affinity from the major isomer, may have a less stable Fe-02 bond with a resulting higher autoxidation rate. With metDME-Mb however, the *H NMR results presented previously, even with their limited resolution, do not show any degree of isomerization as high as that of tuna Mb. Unless the predominant rotation of the heme group in DME-Mb is the opposite of that in native Mb, the heme rotational equilibrium is not expected to affect the autoxidation rate of oxyDME-Mb. D. Electrochemical Studies The initiation of metMb or metDME-Mb reduction and laccase activity occurred simultaneously upon application of the potential across the OTTLE cell. As a result, oxygen was still present in solution when metMb or metDME-Mb reduction was begun. Reduction of metDME-Mb initially resulted in formation of oxyDME-Mb. OxyDME-Mb gradually formed deoxyDME-Mb presumably through dissociation of the oxygen ligand and subsequent laccase activity. However, metMb reduction did not result in significant formation of oxyMb as determined by periodic examination of Mb spectra during reduction. The conditions used for electrochemical measurements of native Mb differed from those of DME-Mb in that catalase was needed to prevent the formation of ferrylMb. FerrylMb resulted from oxidation of either the ferrous or ferric states of Mb presumably by reaction with hydrogen peroxide (H 20 2) because of the inhibitory activity of catalase. The H 20 2 could have been formed from incomplete reduction of oxygen by either 93 electrode and/or laccase reactions. The described conditions for the formation of ferrylMb must have been present during the DME-Mb electrochemical experiments. However, there was no sign of ferrylDME-Mb in the spectra collected. Tamura et al. (1973a) observed that in the presence of excess H 20 2, the ferryl form of DME-Mb does not form as readily as native Mb or other heme derivatives of Mb including monomethyl ester heme substituted Mb. The latter observation indicates that the resistance of DME-Mb to oxidation to the ferryl state could result from the esterification of just one of the heme propionates. The observed formation of oxyDME-Mb but not oxyMb within the OTTLE cell is another indication of the difference in reactivity of these hemeproteins to external ligands such as H 20 2 and 0 2. The heme propionate esters may cause these differences by altering the pathway(s) of 0 2 and H 20 2 entry into the heme pocket either by steric bulk alone or more likely by disruption of hydrogen bonding and electrostatic interactions within the polypeptide chain. The measured midpoint reduction potential (£ m) for native horse heart Mb was +60.9 mV (vs. SHE) at pH 7.0, /=0.1 M, NaPi buffer and 25°C. Under identical conditions, a value of +62 mV (vs. SHE) for the Em of sperm whale Mb was reported by Ellis (1986) who used an anaerobic OTTLE cell flushed with nitrogen gas rather than an enzymatic oxygen reduction system. In spite of the difference in anaerobic techniques used, similar values were obtained, which indicate that the small amounts of laccase and catalase added to the OTTLE cell solution did not affect the measurement of the Em for Mb and DME-Mb. The similarity in Em values also indicates that there is no significant species (amino acid sequence) difference in electrochemical behavior of sperm whale and equine Mb. Comparison of the present results with previously published results for Mb is difficult because of the various solution conditions, techniques of protein purification and 94 electrochemistry that have been employed. Using an OTTLE cell under an anaerobic atmosphere, Heineman et al. (1979) measured an Em for horse heart Mb of +46.4 mV (vs. SHE) at pH 7.0, 7=0.2 M (temperature unreported) using phenazine methosulfate as a mediator. Taylor and Morgan (1942) measured the Em of horse heart Mb using anthraquinone-/?-sulfonate as titrant with toluylene blue and cresyl blue as mediators to obtain a value of +46 mV (vs. SHE) at pH 7, 7=0.2 M and 30°C. Under similar conditions but with Fe(CN)6 as a titrant and thionin as a mediator, Behlke and Scheller (1961) obtained a value of +51 mV (vs. SHE). Brunori et al. (1971) measured an Em for sperm-whale Mb of +47 mV (vs. SHE) at pH 7, 7=0.1 M and 30°C using the method of mixtures with the same mediators as Morgan and Taylor. One problem with the use of dyes as titrants (at concentrations higher than used for mediators) however, is their potential binding to Mb, an interaction that might influence the oxidation-reduction equilibrium. The midpoint reduction potential of DME-Mb was measured as +100 mV (vs. SHE) at pH 7.0, 7=0.1 M and 25°C. The 40 mV difference in reduction potential between native Mb and DME-Mb under standard solution conditions indicates a role for the heme propionate groups in the determination of the Mb reduction potential. When Tamura et al. (1973a) prepared DME-Mb they reported no significant change in its 0 2 affinity from that of native Mb. However, the 0 2 affinity of DME-Mb may bear re-investigation because of the formal relationship between oxidation and oxygenation In comparison to Mb, Reid et al. (1984) reported a 64 mV difference in reduction potential between native cytochrome bs (Em=+5 mV vs. SHE) and DME-cytochrome b5 (+69 mV vs. SHE) under standard solution conditions. The low potential of the native cytochrome b5 and the increase in reduction potential of the DME derivative has been accounted for by a stabilizing coulombic interaction (over a distance of ~5.4 A) in the oxidized state of cytochrome bs between the inner heme propionate (P7) and the heme Fe (vide infra). Based on the crystal structure of horse heart metMb (Evans and Brayer, 95 1988), a carboxylic oxygen of the inner heme propionate is ~6.4 A from the heme Fe. A salt bridge similar to that in cytochrome b5 may also be present in Mb. Such a bond would be of lower potential (stabilization) than that of cytochrome b5 due to the greater distance of separation. An alternative explanation for the difference between the reduction potentials of native and DME-hemeproteins is that the reduction potential is simply a function of the presence and number of heme carboxylate groups, which exert their influence through an inductive electronic effect. Using newly reconstituted cytochrome b5, Walker et al. (1988) have measured the reduction potential of the mixture of rotational isomers and from extrapolation have estimated a 27 mV difference in the Em (7=0.13 M, pH 7.0 and 24°C). An electrochemical titration of a mixture of hemeproteins with different reduction potentials would be expected to exhibit non-Nerstian behavior. Analysis of such a titration using Equation 19 would give values for the slope different from expected values for integer (n) electron transfer. Results of this type have not been reported in previous or current electrochemical measurements of hemeproteins known to have heme rotational isomerization. This absence may be due to either a very small or large difference in reduction potential between isomers or insensitivity in the techniques employed in such titrations to the contribution of the minor isomer. 7. pH Dependence The measured midpoint reduction potentials for both native Mb and DME-Mb decrease in Em with increasing pH from pH 6 to about 9 (Figure 15). Previous measurements of the Em by Brunori et al. (1971) for sperm whale Mb and Behlke and Scheler (1961) for equine Mb have shown a similar pattern with a further decrease in Em at more alkaline pH. This relationship between the pH and reduction potential has been termed the 96 oxidation Bohr effect (Brunori et al., 1971). The decrease in Em at alkaline pH is attributed to the ionization of the water ligand coordinated to the heme iron. The hydroxyl anion presumably negates the net positive charge of the heme Fe(III) to produce an overall neutral ferric form and as a result the reduction potential of Mb decreases. The observed decrease in reduction potential from pH 6 to 7.5, which is greater for DME-Mb than native Mb, is inconsistent with the water ligand being the only pH dependent determinant of the reduction potential. The pAfa values for the reduced and oxidized states of both native and DME-Mb derived from fitting the pH dependence of Em are all less than the pAfa values for the water ligands determined from spectroscopic titrations. This non-correspondence between electrochemical and spectroscopic transitions is not uncommon as discussed by Wilson (1978). The reason for this non-correspondence is that visible spectra only represent the electronic transitions of the group(s) (e.g. heme) that undergo changes in oxidation state. As a result, the visible spectrum is insensitive to perturbations in other parts of the protein such as pH linked conformation changes that indirectly affect the oxidation states. Analysis of the change in reduction potential with pH by Equation 20, which is based on the assumption of a single redox-linked titratable group, appears to have fitted the observed data very well (Figure 14). However, the contribution of the heme bound water to hydroxyl transition in met(DME-)Mb has not been taken into account. As a result, not all of the change in Em within the pH range examined is due to a single titratable group. The values for the pKtei and pK0X for the titratable group are probably overestimated owing to the additional decrease in reduction potential (with increasing pH) from the heme bound hydroxyl anion. The lower pATa of (aquo)metDME-Mb (8.5) compared to that of native (aquo)metMb (8.9) indicates that a larger proportion of the change in Em for DME-Mb in the neutral to alkaline pH range was due to the heme bound hydroxyl than in native Mb. 97 The one titratable group model of Equation 20 has been used to analyze the pH dependencies of the Em for a variety of cytochromes such as b5 (Reid et al., 1982), c-551 (Moore et al., 1980) and some species of c2 (Pettigrew et al., 1978). This model is suitable for die pH dependence of the reduction potential of these cytochromes because the heme group of these proteins do not normally bind external ligands such as water. The diree ionization model represented by Equation 21 is more appropriate for Mb and DME-Mb because it accounts for the presence of the heme boimd water/hydroxyl ligand in the oxidized state of Mb. From Equation 21, the pATol and pATr values were calculated as 7.4 and 7.7 respectively for native Mb. The pK& values for some histidine residues in Mb have been measured by J H NMR titrations for various species" of Mb (Carver and Bradbury, 1984; Bradbury and Carver, 1984). Three out of the eleven His residues of Mb are within the heme pocket as determined from the three-dimensional stmcture of horse heart metMb (Evans and Brayer, 1988). These residues are His-64 (proximal Fe ligand), which does not titrate in the pH range studied, His-94 (distal Fe ligand) and His-97, which is hydrogen bonded to the inner heme propionate. The measured pKa values reported by Bradbury and Carver for His-97 (5.54) is less than the pKol measured here for native Mb. Another candidate for the titratable group in native Mb is one of the heme propionates. In studies examining the pH dependence of the reduction potential of cytochromes c2> Moore et al. (1984) proposed a role for the protonation of heme propionates in regulation of the oxidation-reduction equilibrium of these proteins. From correlation with *H NMR studies these workers conclude that the variation of Em with pH for some species of cytochromes c2 that exhibit a large (~60 mV) change in Em with pH) is attributable to the titration of a heme propionic acid group. The proposed pKR values for these groups were greater than that of heme propionates in solution (pKa&4.5; Falk, 1964). The increase in heme propionate pKa when within hemeproteins was 98 Figure 24. Stereo drawing of selected amino acid residues in the heme pocket of (aquo)metMb determined from the 2.8 A model of Evans and Brayer (1988). The heme is viewed edge on parallel to its a—7 axis. Shown are water molecules, amino acid residue Serine-92, histidine residues within the heme pocket (at positions 64, 94,and 97), and the lysine residues at positions 45 and 96, which flank the heme pocket opening. 99 attributed to the effect of the hydrophobic heme pocket and hydrogen bonding with His residues. Within Mb, the inner heme propionic acid (P7) may have a pKa value within this range because the three-dimensional structure of metMb (Evans and Brayer, 1988) indicates possible hydrogen bonding between this propionate and the side-chains of residues His-97 and Serine-92. For DME-Mb, the calculated pX^ and pKr values generated by the fit to Equation 21 were 6.3 and 7.5 respectively. If the titratable group for native Mb is a heme propionate, a different group or groups must be titrated in DME-Mb. In some species of cytochrome c2, pH dependent changes in Em have been attributed by Moore et al. (1984) to Wstidine residues outside the heme pocket, which can induce conformational changes sufficient to alter the heme environment. Some of the surface His residues of metMb have pKa values (Bradbury and Carver, 1984) within the range of the pKox calculated for DME-Mb. 2. Thermodynamics The standard entropic energy change (AS0) measured for the reduction of native horse heart metMb to deoxyMb (—38.2(5) eu at pH 7.0 and 7=0.1 M) is in close agreement with the corresponding AS 0 value measured for sperm whale Mb (—39(1) eu) by Ellis (1986). With myoglobin, the loss of the heme bound water ligand upon reduction would be expected to result in a positive change in entropy. That the measured AS 0 is negative indicates that there are other processes associated with reduction that have negative entropy changes e.g. conformational changes in the protein. Interestingly, the AS 0 for the reduction of cytochrome b5 (AS°=—37 eu at pH 7 and 7=0.1 M; Reid et al., 1982), which does not undergo a change in heme Fe coordination upon reduction, is similar to that of Mb. 100 A negative net change in entropy is commonly measured for the reduction of many hemeproteins because hemeprotein conformations are believed to become more "rigid" and "compact" upon reduction (Taniguchi et al, 1980). The increase in entropy from the accompanying release of bound water molecules is presumably offset by the decreased entropy from solvent-solvent interactions (i.e. hydrogen bonding). Cytochrome c is one of the few hemeproteins whose structure has been determined by x-ray crystallography in both the oxidized and reduced states. Comparison of the two structures revealed apparently small differences in conformation (Takano and Dickerson, 1981a & b). In solution however, reduced cytochrome c is more resistant to denaturation than the oxidized form (references in Dickerson and Timkovich, 1970), indicating that the former form is more stable. From comparison of the structures of sperm whale met- and deoxyMb, Takano (1977a & b) has also reported differences in the conformations of the polypeptide chains and heme. In neither case has an estimate been made for the entropy changes arising from any observed redox-linked conformational changes. The entropy change (—51.0(8) eu at pH 7) calculated for the reduction of metDME-Mb to deoxyDME-Mb is greater than that for the reduction of native metMb (—38.2(5) eu). This finding may indicate a greater difference in relative stability between the oxidized and reduced states of DME-Mb than those of native Mb, which may be a reflection of a greater flexibility in the conformation of DME-Mb than that of native Mb. This possibility is supported by the observed greater affinity of apoMb for reduced DME-heme over metDME-Mb. Also, in this current study, it was observed that metDME-Mb is less stable than native metMb at high temperature (>35°C, pH 7), as measured by their respective Soret absorbance values over time. The enthalpic change for DME-Mb (—12.9(2) kcal/mol) is lower than that of native Mb (—8.1(2) kcal/mol). This difference may arise in part from the difference in enthalpy between a Fe-H20 and a Fe-OH bond because the pATa of the heme coordinated water 101 ligand of DME-Mb (8.5) is lower than that of native Mb (8.9). Though at pH 7 the percentage of DME-Mb bound with the hydroxyl ligand is low (~3%) and is not much greater than that of native Mb (~1%). 3. Electrostatics Table I shows that the calculated net charges of Mb and DME-Mb derived from the use of full protein radius values are both much higher than the net charge of +5.5 based on the amino acid composition and heme charges. At neutral pH, horse heart metMb has an apparent net charge close to 0 based on electrophoretic mobility (Romero-Herrera, 1974; McLellan, 1984). The equations used in ionic strength dependence analyses are based on the original work by Kirkwood and Tanford (1957) who used the following assumptions: 1) spherical proteins, 2) solvent impenetrable spheres, 3) random (smeared) charge distribution. Though Kirkwood and Tanford in the same paper called these assumptions "invalid" for proteins, Equation 25 has been used with apparent success to analyze the ionic strength dependence of the reduction potential for various hemeproteins such as cytochromes c . (Margalit and Schejter, 1973) and bs (Reid et al., 1982). Clearly Mb has characteristics that do not agree with the above assumptions. Based on its three dimensional crystal structure, the overall shape of Mb is more elliptical than spherical. Also the location of the heme within Mb is more peripheral than central. The heme bound water ligand demonstrates that Mb has some solvent accessibility. The higher molecular weight (size) of Mb relative to cytochromes c would tend to localize the effects of individual charged groups. 102 From Table I, it can be seen that the calculated net charge decreases to seemingly more reasonable values with the use of decreasing protein radius values. This trend indicates that ions can approach the heme iron center more closely than the radius of Mb would indicate. For Mb this is not unreasonable considering that the heme Fe does bind water and other ligands. As a result, the function /(7)=v///(l+v//) may be the most appropriate to describe the ionic strength dependence of the reduction potential of both native and DME-Mb. The choice of the function/(/)=v///(l+v/V) is supported by the work of Shire et al. (1974) who studied the effect of ionic strength on the pKa of the heme bound water ligand of metMb. Shire et al. found that use of the function f(I) =s/ll(l +\fl) fitted their ionic strength dependence better than the function f(I)=\/l/(l + 6\/V), which uses an estimate for the full radius of Mb. The term BRa in Equation 26 is assigned a value of one to derive the function /(/)=v///(l +V7/). One interpretation of this value is that local charges within a small radius (Ra) around the heme determine the Em of Mb. In an alternative view, Goldkorn and Schejter (1976), have interpreted the use of this function as an indication that the medium (solvent and ionic strength) around the heme is the main determinant of the reduction potential. As Moore et al. (1986) have stated, all of these aforementioned factors influence the reduction potential though their relative influence varies from protein to protein. In the case of DME-Mb, esterification of the heme propionates would presumably decrease the negative (or increase the positive) charge around the heme, which would tend to raise the reduction potential of the hemeprotein. In addition, the sensitivity of the Em to the ionic strength of the medium has also been increased as shown by the larger change in reduction potential (AE m=65 mV) for DME-Mb over the ionic strength range examined (0.05-0.5 M) as compared to native Mb (AE m=33 mV). This increase in sensitivity to ionic strength may indicate greater heme exposure to the solvent. 103-E. Reduction Kinetics The presence of ferricyanide resulted in a biphasic reaction between metDME-Mb and Fe(EDTA)2" with AA 4 0 7 vs. time (Figure 18) showing an initial rapid phase of reaction (decrease in A ^ ) followed by a slower phase. The biphasic nature of the reaction is more evident in the corresponding logarithmic plot in Figure (18). After passage of metDME-Mb through the Dowex X-8 column to remove any associated ferricyanide, the observed reaction with Fe(EDTA)2" became monophasic as shown in the AA 4 0 7 time tracing (Figure 18) and the corresponding logarithmic plot. Otherwise, the average rate of the biphasic reaction was about an order of magnitude lower than that observed after ferricyanide removal. Free ferricyanide in solution could have oxidized Fe(EDTA)2" thereby decreasing its effective concentration, which would result in the slower reaction phase. However, buffer exchange of the protein solution should have removed any free ferricyanide. The protein solution itself (<50 uL) was diluted into 20 mL of buffer for use in the kinetic experiments. In addition, the concentrations of Fe(EDTA)2" used were always at least an order magnitude greater than the concentration of metDME-Mb. It is therefore unlikely that any ferricyanide in solution could have caused the observed biphasic kinetics. These results indicate that ferricyanide could have been bound (non-covalently) to metDME-Mb. This is interesting because no such binding has been reported for native Mb. However, ferricyanide has been known to bind ionically to hemoglobin (e.g. Hoffman and Bull, 1976; Linder et al, 1978) and cytochrome c (Stellwagen and Cass, 1975). If metDME-Mb was bound with ferricyanide, the following mechanisms could account for the biphasic kinetics: 1) steric hindrance by Fe(CN)|" of the Fe(EDTA)2" reaction with heme Fe 2) electrostatic repulsion of Fe(EDTA)2" by the negatively charged Fe(CN)^" 104 3) increased reduction potential of metDME-Mb bound with Fe(CN)|" 4) intramolecular transfer between Fe(CN)^"/3" and heme Fe(III / II) In the first two cases, the initial rapid rate would be due to reaction of unbound metDME-Mb. In the third case, ferricyanide binding could have resulted in an activated state-like complex in which the potentials of the two metal centers have changed to a level intermediate between their unbound (free) states. The reduction potential of ferricyanide bound metDME-Mb would increase. As a result, the driving force of the reaction would become more favorable, which would lead to the higher initial rate. In the last mechanism listed, ferricyanide may have sterically blocked the Fe(EDTA)2" reduction of the metDME-Mb heme iron but not its own. The resulting ferrocyanide could then reduce metDME-Mb. Such an intramolecular reaction can be depicted as: Fe(EDTA)2" + Fe(III)(CN)6-DME-Mb Fe(III) -» Fe(II)(CN)6—DME-Mb Fe(III) + Fe(EDTA)1" (33) Fe(II)(CN)6-DME-Mb Fe<III) - Fe(III)(CN)6-DME-Mb Fe(II) (34) The initial rapid rate observed could be due to reaction of unbound metDME-Mb while the slower rate could reflect the rate limiting step in Equations 33 and 34. Alternatively, if metDME-Mb was saturated with bound ferricyanide, the initial phase could be due to the forward reactions depicted in Equation 33 and the slower phase could be due to the contribution of a back reaction electron transfer from deoxyDME-Mb to ferricyanide, which would be favored based on the difference in reduction potential of deoxyMb and ferricyanide. 105 The aforementioned possibilities imply ferricyanide binds specifically to DME-Mb. The crystal structure of native horse heart metMb (Evans and Brayer, 1988) shows that Lys residues 45, which flanks the opening of the heme pocket, forms a salt bridge widi the outer heme propionate (P6). Esterification of the propionate would eliminate its negative charge and may allow the positively charged Lys residue to bind a ferricyanide anion. In cytochrome c, ferricyanide is believed to bind a lysine residue near the heme edge (Stellwagen and Cass, 1975; Eley et al., 1982). The rate of Fe(EDTA)2" reduction metDME-Mb was always greater than that of metMb under all conditions of temperature, pH, and ionic strength examined. In addition, at all concentrations of Fe(EDTA)2" used the reduction of metDME-Mb proceeded to completion in contrast to the incomplete reduction of metMb at low Fe(EDTA)2" concentrations (Lim and Mauk, 1984). There are three general reasons for the increase in reaction rate with heme propionate esterification, all of which are probably significant. First, the driving force for the reaction is more favorable as indicated by the increase in reduction potential of DME-Mb compared to native Mb. Second, electrostatic interactions between Fe(EDTA)2" and metMb are probably enhanced by esterification of the heme propionates and elimination of the negatively charged carboxy late groups. As an example, esterification of the outer heme propionate would release the positive charge of Lys-45 and result in increased electrostatic attraction of an approaching Fe(EDTA)2" anion. Finally, the actual mechanism of electron transfer (i.e. pathway) from Fe(EDTA)2" to the heme iron of metDME-Mb may be altered (from that in native Mb) to allow faster electron transfer. For example, the loss of H-bonding and ionic interactions by heme propionate esterification may allow greater conformational flexibility in DME-Mb than native Mb and result in greater heme access by Fe(EDTA)2". 106 1. pH Dependence The rate constant (t 1 2) of metDME-Mb reduction by Fe(EDTA)2" (Figure 20) decreases with increasing (more alkaline) pH. The causes of this behavior are both kinetic and thermodynamic. Through correction of the second order rate constants for driving force (AEm), the thermodynamic contribution can be eliminated. The residual change in reaction rate constant (Jfcf^ ) with pH can then be attributed to a kinetic origin (Figure 21). To explain this change in rate with pH a single titratable group was assumed to be responsible. This assumption does not imply that other titratable groups cannot influence the reduction rate, only that within the narrow pH range studied (6 to 8) one group has a dominant influence. The change in rate constant observed with changing pH can be attributed to die properties of DME-Mb and/or Fe(EDTA). Within the pH range studied, Fe(EDTA)2" does not have a titratable group, while Fe(EDTA)1" has a bound water molecule with a pATa value of 7.5 (Schwarzenbach and Heller, 1951; Kolthoff and Auerbach, 1952), which influences the Em of the Fe(EDTA)1"/2" couple. However, this effect on the driving force of the reaction was corrected for in calculating ifcf^. Furthermore only Fe(EDTA)2" was used in the reaction with metDME-Mb. As a result, the cause of die observed pH dependence of the reaction rate probably resides in properties of metDME-Mb and not Fe(EDTA)2". This conclusion is reinforced by the substantial difference between the pH dependence of Fe(EDTA)2" reduction of native and DME-Mb (Figure 20). The pH dependence of native horse heart metMb reduction by Fe(EDTA)2" has been measured and analyzed (Lim and Mauk, 1984) by similar methods to those used for metDME-Mb. Esterification of the heme propionates results in two major effects on the pH dependence of the rate constant. MetDME-Mb appears to have a titratable group with a p/ira value 107 of 7.6, while native metMb has a group with a lower pKa value of 5.8. With esterification of the heme propionates, the influence of pH on the reduction rate constant increased. In native Mb, the outer heme propionate (96), which is directed towards the solvent, may be the titratable group. Based on the crystal structure of horse heart Mb, Evans and Brayer have proposed that the outer heme propionate forms a salt bridge with Lys-45. This interaction would negate the positive charge of the Lys residue. The pKa of propionic acid groups in free heme is only 4.5 (Falk, 1964). However, the *H NMR data of cytochrome b5 have estimated a pATa value of 5.9 for the solvent accessible outer heme propionate of this protein (McLachlan et al, 1986). The identity of the titratable group responsible for the pH dependence of reduction by Fe(EDTA)2" of metDME-Mb cannot be determined unambiguously because the pATa values for individual amino acid residues in DME-Mb are unknown. The pATa value of 7.6, produced by fitting the rate data to Equation 30, is suggestive of a histidine residue. With heme propionate esterification not only was the rate of reduction increased, the sensitivity of this rate to pH also increased. The difference between (adjusted) ka and kb increased from ~150 molds'1 for native Mb to ~1560 mol ' V 1 for DME-Mb in going from pH 6 to 8. The heme propionates may be involved in a rate limiting step of electron transfer such as binding of Fe(EDTA)2". After esterification, the influence of a titratable group on the reaction rate appears to have been unmasked, accounting for the increase in rate and sensitivity of the reaction to pH. 2. Thermodynamics The enthalpy of activation (AZ/*=+ 9.2(3) kcal/mol) for the reduction of metDME-Mb by Fe(EDTA)2" is less than that measured for native metMb (+12(1) kcal/mol; Cassat et al, 1975). The entropy of activation (AS* =—13(1) eu) for the reduction of DME-Mb is 108 identical to that reported for native Mb in its reaction with Fe(EDTA)2" (—13(5) eu; Cassat et al., 1975). Interpretation and comparison of the activation parameters derived from Equation 31 are difficult because one cannot distinguish between the individual contributions of reactants. For comparison, the enthalpy and entropy of activation for the reaction between cytochrome bs and Fe(EDTA)2" at pH 7.0 and 7=0.1 M are 5.4-kcal mol"1 and -29.2 eu respectively (Reid and Mauk, 1982). Under the same conditions, the reaction of cytochrome c with Fe(EDTA)2" is characterized by corresponding activation energies of 5 kcal mol"1 and -20 eu respectively (Hodges et al., 1974). These activation enthalpies are lower than those of metMb and metDME-Mb presumably because these cytochromes undergo no change in heme coordination upon reduction. 3. Electrostatics Analysis of the ionic strength dependence of metDME-Mb reduction by Fe(EDTA)2" with Equation 32 using full protein radius values results in an unrealistically large calculated charge for the DME-Mb molecule. Such results are analogous to the large charges calculated from analysis of the electrostatics dependence of the Mb reduction potential when full protein radius values were used. These inconsistencies are similar because the assumptions used by Wherland and Gray (1976) in deriving Equation 32 were also used in deriving Equation 25 for the ionic strength dependence of the midpoint reduction potential e.g. spherical proteins with symmetrical charge distribution. These assumptions also apply to Fe(EDTA)2", which is not a symmetrical complex. Furthermore there is some controversy over whether the net charge of a protein (expressed by use of full protein radii) or the local (active site) charges control its electrostatic behavior in reactions of this type. 109 However, despite these assumptions, the ionic strength dependence relationship shown in Equation 32 has been used successfully for the analysis of many metalloproteins, especially cytochromes. Calculated charges derived from use of the protein radius have generally agreed with those derived from sequence. Based on these results, Feinberg and Ryan (1981) have concluded that the net charge rather than local charges influences the electrostatic behavior of electron transfer proteins. This success may be due to the structural features of the cytochromes analyzed by Feinberg and Ryan, which when compared to Mb are generally smaller (lower molecular weight) and more spherical (better agreement better between hydrodynamic and crystal structure radius values). Consequently the properties of these cytochromes are closer in agreement with the assumptions of Equation 32 than are those of Mb. The apparent success in estimating metalloprotein charges derived from ionic strength dependencies of reduction potentials is based on their comparison to charges derived from the amino acid and heme composition of the protein, which may not reflect the actual charge of the assembled protein. Marcus and Sutin (1985) have pointed out that the agreement between sequence derived charges and calculated net charges may be coincidental. If their signs are identical, the sequence derived charge and the local charge may appear to coincide because they are usually within an order of magnitude of one another. Segal and Sykes (1977) have questioned whether ionic strength dependence equations such as 32 can be applied to reactions involving proteins. Regardless of the view taken (or values used for the radius), Equation 32 does fit the observed ionic strength dependence of the reaction kinetics of many diverse metalloproteins. Equation 32 provides an estimate of the electrostatic contribution to the reaction kinetics and is particularly useful for comparison of the kinetic behavior of modified proteins with those of the native form because of the smaller structural differences involved. The current results for DME-Mb show that esterification of the 110 heme propionates significantly alters the electrostatic behavior of Mb. Given the discrepancy between the calculated and sequence charges of DME-Mb, the local charges around the heme may exert more influence on the kinetics of Mb than does the net charge of the protein. The electrostatic behavior of a metalloprotein, as reflected by the reaction kinetics (and i? m ) , may be influenced more by its net dipole moment rather than net charge. If all the charged groups of a protein contribute to its electrostatic properties, then the net dipole moment may be a more accurate representation than the net charge, especially for a large molecule such as a protein. Koppenol and Margoliash (1982) have calculated the dipole moment of horse cytochrome c as being of surprisingly high magnitude (~300 Debye) with the positive end of the vector directed towards the heme edge, where electron transfer is theorized to occur. Such an orientation is favorable for interactions with reactants that have a negative charge such as Fe(EDTA)2". Van Leeuween et al. (1981) have used the results of Margoliash and Koppenol to analyze the ionic strength dependence of cytochrome c reaction kinetics based on its dipole moment rather tiian net charge. As dipole calculations of this type has not previously been reported for Mb, the dipole moment of native horse heart Mb was calculated using computer programs developed by Northrup et al. (1986). This method calculates partial charges and dipole moments for all the atoms in a protein and then derives an estimate for the net dipole moment. The calculated dipole moment magnitude of cytochrome c was 245 or 286 Debye depending on whether formal charges were assigned to the heme propionyl groups. The Evans and Brayer (1989) model of horse heart metMb (1.9 A resolution) was used as a source of coordinates for the dipole moment calculation. The charge of each heme propionyl group was set at +0.5. This Mb model was also used in an attempt to simulate the effect of heme propionate esterification by setting the charge of the heme propionyl 111 Figure 25. The dipole moment for native Mb estimated using the methods of Northrup et al. (1986). The vector calculated for native Mb is labelled Mb and is depicted in relationship to a stereo drawing of a model of Mb (Evans and Brayer, 1988). The vector of the calculated dipole moment of Mb after elimination of the charge contribution of the heme propionates is labelled Mb'. 112 groups as zero. The dipole moment calculated for native Mb was 2.8 x 102 Debye with the positive end of the vector pointed away from the heme (see Figure 25) and exiting the surface of the protein near residue glutamine-91. This result was surprising because the magnitude and direction of the estimated dipole moment was similar to that of cytochrome c. Elimination of the heme propionates' charge contribution (to simulate esterification) increased the magnitude of the estimated dipole moment to 3.9 x 102 Debye. In addition, the orientation of the dipole moment vector becomes slightly more parallel to the heme plane and exits the protein surface near residue Serine-92. Elimination of the heme propionate charges by esterification may also result in a similar change in dipole moment for DME-Mb assuming no major structural changes from that of native Mb. Such a dipole change could explain the increased reactivity of DME-Mb towards anions such as Fe(EDTA)2" and Fe(CN)f\ There may be a role in electron transfer for both the net dipole moment and local charges as described by Northrup et al. (1988). Reports from this group suggest that the dipole moment attracts or steers reactants together in a nonspecific and unreactive configuration i.e. the precursor complex. This initial bmding allows more efficient attainment of the reactive state via reorganization of the precursor complex through involvement of shorter range electrostatics interactions. F. Marcus Theory Analysis The values of A^j" calculated using different values of the protein radius and corresponding calculated charge were all within an order of magnitude of each other. Similar results were obtained by Wherland and Gray (1976) for the reduction of cytochrome c by a variety of inorganic complexes including Fe(EDTA)2". These results 113 indicate that the Wherland-Gray adaptation of Marcus theory is at least valid for comparison purposes. As developed by Gray and co-workers (Wherland and Gray, 1976; Cummins and Gray, 1977), the electrostatics-corrected self-exchange rate (*iorr) for a metalloprotein calculated on the basis of its cross-reaction with a substitutionally inert transition metal complex is a useful kinetic parameter to detect mechanistic features of a reaction. The basis for the usefulness of k^°n relative to the second order rate constant resides in the elimination of contributions to this rate of the thermodynamic driving force of the reaction, the electrostatic interactions between protein and small molecules and the intrinsic reactivity of the small molecular reagent. The A^orr of 0.41 M ' V 1 at 7=0.1 M calculated for DME-Mb (using full radius value and corresponding calculated charge) is larger by an order of magnitude than the calculated k\OTT of native Mb, 0.020 M 'V 1 . This difference is significant in that it exceeds the uncertainties involved in measuring and calculating the various parameters used in their calculation (Appendix E). This result indicates that esterification of the heme propionates increases the rate of reduction by Fe(EDTA)2" by altering the mechanism of electron transfer between Fe(EDTA)2' and the heme iron to a more favorable pathway. As discussed previously this alteration may involve disruption of the hydrogen bonds and electrostatic interactions of the heme propionate, which may alter the conformation of the protein. At pH 7, 7=0.1 M and 25°C, the electrostatics corrected self-exchange rates for native and DME-Mb are both significantly smaller than those for cytochrome b5, ^orr=4.3 M ' V 1 (Reid and Mauk, 1982) and c, *forr=6.2 M ' V 1 (Wherland and Gray, 1976). These differences presumably arise from the activation barrier to the change in axial ligation of the heme Fe that occurs on reduction of metMb to deoxyMb as cytochromes c and b5 undergo no such redox-linked change in axial ligation. 114 CONCLUSIONS A. Emerging Role of Heme Propionate Groups In the course of hemeprotein evolution protoheme DC has been used as a prosthetic group in proteins with a wide range of functional properties. In principal, the heme propionic acid groups can form specific H-bonds and as propionates can form specific salt bridges either of which can be intra- or intermolecular. As discussed above, heme propionate groups in hemeproteins have been shown to function in structural roles, in regulation of reduction potential and in influencing mechanisms of electron transfer. In addition, heme propionates appear to be essential for formation of the initial heme-apoprotein complex of non-covalent (fc-type) hemeproteins possibly by directing the heme to the correct position within the heme pocket via hydrogen bonds with amino acid residues. Once positioned, the heme is held inside the pocket by hydrophobic and Van der Waals interactions involving the heme vinyl and methyl groups. The propionates may interact with the net positive charge of the ferric iron via coulombic interactions and thereby modify the reduction potential of the hemeprotein. The arrangement of the propionates may allow their participation in complex formation with other proteins through electrostatic/dipole interactions and thereby facilitate electron transfer. Examples of these mechanistic roles for heme propionates are given in the following brief summary. B. Myoglobin In the formation of the apoMb—heme complex only one of the heme propionates appears essential because of the relative ease in preparing the monomethyl ester heme derivative of Mb compared to the dimethyl ester derivative (Tamura et al, 1973a). 115 Esterification of the heme propionates decreased the stability of the resulting metDME-Mb compared to native Mb. The heme propionates can influence the reactions of Mb with small molecules. For example, the pATa of the water ligand bound to the heme Fe is lower in DME-Mb than native Mb and the autoxidation reaction is accelerated in oxy DME-Mb relative to native oxyMb. Finally, esterification of the heme propionates apparently allows high affinity binding of ferricyanide anion to DME-Mb. The reduction potential of Mb is increased by 40 mV (pH 7, 7=0.1 M, NaPi buffer, 25 °C) with heme propionate esterification. This increase may be due to the loss of the propionate negative charge stabilizing influence on the positive charge of the heme Fe. Analyses of the ionic strength dependence of both the reduction potential and second order reaction rate constant with Fe(EDTA)2" showed that the propionates influence the apparent net charge of Mb. The pH dependence of the reduction potential and second order reaction rate constants showed that esterification increases the sensitivity of these parameters to changes in pH and that the calculated pA^s of titratable groups were altered. Temperature dependence studies showed a decreased stability of the oxidized met form relative to the reduced deoxy form of DME-Mb. From application of Marcus theory, it is apparent that the corrected self-exchange rate of DME-Mb is significantly greater than that of native Mb. This result indicates that the heme propionates also influence the pathway or mechanism of electron transfer exhibited by Mb. C. Cytochrome b5 Cytochrome bs is a hemeprotein that functions in fatty acid reduction (Strittmatter et al, 1974), the cytochrome P-450 catalytic cycle in liver (Bonfils et al, 1981) and the metHb reductase system of the erythrocyte. The three-dimensional structure of microsomal cytochrome b5 reported by Mathews and Argos (1975) indicates that the heme 116 propionates in cytochrome b5 are oriented in a manner similar to those of Mb. Based on the electron density difference map between the oxidized and reduced cytochrome b5, Argos and Mathews (1975) proposed that within ferricytochrome b5, the inner heme propionate has a direct stabilizing influence on the heme Fe(III) via a coulombic interaction of 5.4 A separation (from an oxygen atom of the propionate group to the heme Fe). The increase in reduction potential of DME-cytochrome b5 over that of native £ 5 can be attributed to the loss of this stabilization (Reid et al, 1984). The outer heme propionate influences the binding of small molecules and other proteins. The increase in rate of reduction of DME-cytochrome b5 by Fe(EDTA)2* over that of native b5 (Reid et al, 1984) has been attributed to both a loss of the heme propionate electrostatic repulsion of the Fe(EDTA)2" and a change in the electron transfer pathway (as represented by * i o r r ) . Salemme (1975) has proposed a structure for the complex formed between cytochrome bs and cytochrome c, which involves the outer heme propionate of the bs. Analysis of the binding between cytochrome c and DME-65 (Mauk et al, 1986) has indicated that the outer heme propionate is integral to the formation of the Salemme complex but not other possible complexes. The differences in reduction kinetics (with various flavins) between the cytochrome bs—c and DME-b5—cytochrome c complexes has also been attributed to the effect of propionate esterification on the structure of the cytochrome b5-c complex (Eltis et al, 1988). The outer heme propionate of cytochrome b5 has also been implicated by modelling (Poulos and Mauk, 1983) and *H NMR (Livingstone et al, 1985) studies as being involved in complex formation with metHb and metMb respectively. 117 D. Cytochrome c Cytochrome c is a component of the electron transport chain of phosphorylative oxidation. The three-dimensional structures for several cytochromes c from various species have been solved (review by Mathews, 1985). In cytochromes c, the heme group is joined covalently to the polypeptide chain by thioether linkages formed between the heme vinyl groups and (conserved) cysteine residues. Unlike the 6-type hemeproteins, the heme propionates of cytochrome c probably do not have a role in the initial heme—apoprotein complex, which is believed to be formed by a heme attachment enzyme (Pettigrew and Moore, 1987). In the heme pocket of eukaryotic cytochrome c, the heme propionate groups are directed inward, away from the solvent. They contribute to the final conformation of the heme protein, which is tightly folded, by forming hydrogen bonds with amino acid residue side chains from different helices of the polypeptide chain. In tuna cytochrome c, one propionate (P7) forms a salt bridge with an arginine (position 38) residue and hydrogen bonds with the sidechains of a tyrosine (48) and a tryptophan (59) residue. The other propionate (P6) hydrogen bonds to two threonine residues (48 and 78). The role of the propionate P7 may be to moderate the positive charge on the arginine-38 residue on the reduction potential of the heme iron of cytochrome c. Replacement of this arginine residue with other amino acid residues by site directed mutagenesis results in as much as a 50 mV reduction of the reduction potential from that of the wild-type protein (Cutler et al, 1989). 118 E. Cytochrome c Peroxidase Cytochrome c peroxidase (CCP) is a monomelic, monoheme (A-type) yeast mitochondrial protein that catalyzes oxidation of cytochrome c via peroxides (Yonetani, 1976). CCP has a i-type heme, which is non-covalently bound to the apoprotein. Yonetani (1964) prepared the dimethyl heme derivative of CCP, DME-CCP, and found its activity to be less than 1% of native. The three dimensional structure of CCP (Finzel et al., 1984) shows that the heme propionates in this protein are directed towards the heme pocket opening but buried by H-bonds so as to exclude contact with the solvent. Disruption of this network of H-bonds by site-directed mutagenesis has been shown to alter the heme Fe spin and ligand binding properties of CCP (Smulevich et al., 1988a&b). F. Further Studies Interpretation of the effects of heme propionate esterification is hampered by a lack of a three-dimensional structure for the DME-heme protein derivatives. Preliminary results have shown that metDME-Mb can be crystallized. In the absence of structural data, one can use the three-dimensional model of native horse heart Mb as a basis for mechanistic interpretation. However, the assumption that the only difference between their structures is the addition of methyl esters is simplistic. Of particular interest is whether esterification leaves the inner and outer propionate arrangement found in native Mb intact in DME-Mb. Of interest would also be the possible complex between metDME-Mb and ferricyanide. One means of studying this complex would be to soak crystals of metDME-Mb in a ferricyanide solution to prepare complexes suitable for x-ray diffraction. Alternatively, NMR techniques similar to those applied to characterization of ferricyanide binding to 119 cytochrome c (Eley et al., 1982) could be employed to map the ferricyanide binding sites on the surface of DME-Mb. A DME-Mb-ferricyanide complex can be used to analyze electron transfer as a function of distance in a manner similar to the intramolecular electron transfer kinetics studies of hemeproteins with small inorganic complexes covalently attached to the protein surface (review by Mayo et al., 1986). 120 BIBLIOGRAPHY Akeson, A., v. Ehrenstein, G., Hevesy, G., and Theorell, H. (1960), Arch. Biochem. Biophys., 91, 310-318. Antonini, E. and Brunori, M. (1971), Hemoglobin And Myoglobin In Their Reactions With Ligands, Chapter 5, North-Holland Publishing Co., Amsterdam. Aojula, H.S., Wilson, M.T., and Morrison, I.E.G. (1987), Biochem. J., 243, 205-210. Argos, P. and Mathews, F.S. (1975), /. Biol. Chem., 250, 747-751. Armstrong, F.A., Hill, H.A.O., and Walton, N.J. (1988), Acc. Chem. Res., 21, 407-413. Asakura, T. and Lamson, D.W. (1973), Anal. Biochem., 53, 448-451. Asakura, T. and Yonetani, T. (1979), /. Biol. Chem., 244, 4573-4579. Berzofsky, J.A., Peisach, J., and Blumberg, W.E. (1971), /. Biol. Chem., 246, 3,361-2311. Blanchetot, A., Wilson, V., Wood, D., and Jeffreys, A.J. (1983), Nature, 301, 732-734. Blanchetot, A., Price, M., and Jeffreys, A.J. (1986), Eur. J. Biochem., 159, 469-474. Bonfils, C, Balny, C, and Maurel, P. (1981), /. Biol. Chem., 256, 9457-9465. Bradbury, J.H. and Carver, J.A. (1984), Biochemistry, 23, 4905-4913. Breslow, E. (1963), /. Biol. Chem., 239, 486-486. Breslow, E., Beychok, S., Hardman, K.D., and Gurd, F.R.N. (1965), /. Biol. Chem., 240, 304-309. Breslow, E., Koehler, R., and Girotti, A.W. (1967), /. Biol. Chem., 242, 4149-4156. Brown, W.D. and Mebine, L.B. (1969), /. Biol. Chem., 244, 6696-6701. Brunori, M., Saggese, U., Rotilio, G.C, Antonini, E., and Jeffries, W. (1971), Biochemistry, 10, 1604-1609. Butler, J., Chapman, S.K., Davies, D.M., Sykes, A.G., Speck, S.H., Osheroff, N., and Margoliash, E. (1983), /. Biol. Chem., 258, 6400-6404. Carver, J.A. and Bradbury, J.H. (1984), Biochemistry, 23, 4890-4905. Cassat, J.C, Marini, CP., and Bender, J.W. (1975), Biochemistry, 14, 5470-5475. 121 Chance, B. (1974), Techniques of Chemistry, vol. 6, 3rd edition, Part II, edited by Lewis, E.S., John-Wiley & Sons. Chang, C.K., Ward, B., and Ebina, S. (1984), Arch. Biochem. Biophys., 231, 366-371. Clark, W.M. (1960), Oxidation-Reduction Potentials of Organic Systems, Chapter 4, Robert E. Krieger Publisliing Company, New York. Cole, R.P., Sukanek, P.C, Wittenberg, J.B., and Wittenberg, B. (1982), /. Appl. Physiol., 53, 1116-1124. Collman, J.P., Gagne, R.R., Reed, C.A., Robinson, W.T., and Rodley, G.A. (1974), Proc. Natl. Acad. Sci. USA, 71, 1326-1329. Craik, C.S., Buchman, S.R., and Beychok, S. (1980), Proc. Natl. Acad. Sci. USA, 77, 1384-1388. Craik, C.S., Buchman, S.R., and Beychok, S. (1981), Nature, 291, 87-90. Cutler, R.L., Davies, A.M., Creighton, S., Warshel, A., Moore, G.R., Smith, M., and Mauk, A.G. (1989), Biochemistry, 28, 3188-3197. Daly, J.S.F., Little, J.M., Troxler, R.F., and Lester, R. (1967), Nature, 216, 1030-1031. Dautrevaux, M., Boulanger, Y., Han, K., and Biserte, G. (1969), Eur. J. Biochem., 11, 267-277. De Sanctis, G., Falcioni, G., Giardina, B., Ascoli, F., and Brunori, M. (1986), /. Mol. Biol., 188, 73-76. De Sanctis, G., Falcioni, G., Giardina, B., Ascoli, F., and Brunori, M. (1988), /. Mol. Biol., 200, 725-733. Dickerson, R.E. and Timkovich, R. (1970), The Enzymes, vol. 11, Chapter 7. Drabkin, D.L. (1945), /. Mol. Biol., 158, 721-722. Dutton, P.L. (1978), Methods in Enzymology, 54, Chapt. 23, Academic Press. Dziobak, M.P. and Medenhall, G.D. (1982), /. Magnetic Resonance, 50, 274-280. Edmundson, A.B. and Hirs, C.H. (1961), Nature, 190, 663-665. 122 Edmundson, A.B. (1965), Nature, 205, 883-887. Eley, C.G., Moore, G.R., Williams, G., and Williams, R.J.P. (1982), Eur. J. Biochem., 124, 295-303. Ellis, W.R. (1986), Ph.D. Dissertation, California Institute of Technology. Eltis, L.E., Mauk, A.G., Hazzard, J.T., Cusonavich, M.A., and Tollin, G. (1988), Biochemistry, 27, 5455-5460. English, A.M., Lum, V.R., DeLaive.D.E., and Gray, H.B. (1982), /. Amer. Chem. Soc, 104, 870-871. Evans, D.H., O'Connell, K.M., Petersen, R.A., and Kelly, M.J. (1983), /. Chem. Educ, 60, 290-293. Evans, S.V. and Brayer, G.D. (1988), /. Biol. Chem., 263, 4263-4268. Evans, S.V. and Brayer, G.D. (1990), /. Mol. Biol., in press. Feinberg, B.A. and Ryan, M.D. (1981), /. Inorg. Biochem., 15, 187-199. Frauenfelder, H., Petsko, G.A., and Tsernoglou, D. (1979), Nature, 280, 558-563. Frauenfelder, H. and Wolynes, P.G. (19S5),Science, 229, 337-345. Fuhrop, J. and Smith, K. (1975), Porphyrins and Metalloporphyrins, Chapter 19, edited by K. Smith, Elsevier Scientific Publishing Co.. Galaris, D., Cadenas, E., and Hochstein, P. (1989), Arch. Biochem Biophys., 273, 497-504. George, P. and Irvine D.H. (1952), Biochem. J., 52, 511-517. George, P. and Stratmann, C.J. (1952), Biochem. J., 51, 103-108. Gilbert, W. (1978), Nature, 271, 501. Goldkorn, T. and Schetjer, A. (1976), Arch. Biochem. Biophys., 177, 39-45. Goss, D.J., LaGow, J.B., and Parkhurst, L.J. (1982), Comp. Biochem. Physiol., 71B, 229-233. Gotoh, T. and Shikama, K. (1974), Arch. Biochem. Biophys., 163, 476-481. Gotoh, T. and Shikama, K. (1976), /. Biochem., 80, 397-399. 123 Hagler, L., Coppes, R.L, and Herman, R.H. (1979), /. Biol. Chem., 254, 6505-6514. Hagler, L., Coppes, R.I., Askew, E.W., Heeler, A.L., and Herman, R.H. (1980), J. Lab. Clin. Med., 95, 222-230. Heineman, W.R., Anderson, C.W., Halsall, H.B., Hurst, M.M., Johnson, J.M., Kreishman, G.P., Norris, B.J., Simone, M.J., and Su, S. (1982), Electrochemical and Spectrochemical Studies of Biological Redox Components, Chapt. 1, edited by Kadish, K.M., American Chemical Society. Heineman, W.R. (1983), /. Chem. Educ, 60, 305-308. Hill, R. (1933), Nature, 132, 897-898. Hill, R. and Holden, H.F. (1926), Biochem. J., 20, 1326-1339. Hodges, L.H., Holwerda, R.A., and Gray, H.B. (1974), /. Amer. Chem. Soc, 96, 3132-3137. Hoffman, B.M. (1974), /. Amer. Chem. Soc, 97, 1688-1694. Hoffman, B.M. (1979), The Porphyrins, Chapter 9, Vol. VII part B, edited by Dolphin, D., Academic Press. Hoffman, B.M. and Bull, C. (1976), Proc Natl. Acad. Sci. USA, 73, 800-803. Hoffman, B.M. and Petering, D.H. (1970), Proc. Natl. Acad. Sci. USA, 67, 637-643. Hulquist, D.E., Sannes, L.J., and Juckett, D.A. (1984), Curr. Top. Cell. Regul., 24, 287-300. Huth, S.W., Kimberly, K.E., Piszkiewicz, D, and Fleischer, E.B. (1976), /. Amer. Chem. Soc, 98, 8467-8471. Kagen, L.J. (1973), Myoglobin; Biochemical, Physiological, and Clinical Aspects., Columbia University Press. Kendrew, J.C, Bodo, G., Dintzis, H.M., Parrish, R.G., and Wyckoff, H. (1958), Nature, 181, 662-666. 124 Kendrew, J.C, Dickerson, R.E., Strandberg, B.E., Hart, R.G., Davies, D.R., Phillips, D.C, and Shore, V.C (1960), Nature, 185, 422-427. Kolthoff, I.M. and Auerbach, C. (1952), /. Amer. Chem. Soc., 74, 1452-1456. Koppenol, W. H. and Margoliash, E. (1982), /. Biol. Chem., 257, 4426-4437. La Mar, G.N., Budd, D.L., Viscio, D.B., Smith, K.M., and Langry, K.C. (1978), Proc. Natl. Acad. Sci. USA, 75, 5755-5759. La Mar, G.N. and Walker(Jensen), F.A. (1979), The Porphyrins, Chapter 2, Vol. 4, part B, edited by D. Dolphin, Academic Press Inc. La Mar, G.N., Budd, D.L., Smith, K.M., and Langry, K.C (1980), /. Amer. Chem. Soc, 102, 1822-1827. La Mar, G.N., Davis, N.L., Parish, D.N., and Smith, K.M. (1983), J. Mol. Biol, 168, 887-896. La Mar, G.N., Toi, H., and Krishnamoorthi, R. (1984), /. Am. Chem. Soc, 106, 6395-6401. La Mar, G.N., Emerson, S.D., Lecomte, J.T.J., Pande, U., Smith, K.M., Craig, Wayne, G . , and Kehres, L.A. (1986), /. Amer. Chem. Soc, 108, 5568-5573. Lecomte, J.T.J, and La Mar, G.N. (1985), Biochemistry, 24, 7388-7395. Light, W.R., Rohlfs, R.J., Palmer, G., and Olson, J.S. (1987), /. Biol. Chem. 262, 46-52. Lim, A.R and Mauk, A.G. (1986), Biochem. J., 229, 765-769. Linder, R.E., Records, R., Barth, G., Bunnenberg, E., Djerassi, C, Hedlund, B.E., Rosenberg, A., Benson, E.S., Seamans, L., and Moscowitz, A. (1978), Anal. Biochem., 90, 474-480. Livingston, D.J., Davis, N.L., La Mar, G.N., and Brown, W.D. (1983), /. Amer. Chem. Soc, 106, 3025-3026. Livingston, D.J., McLachlan, S.J., La Mar, G.N., and Brown, W.D. (1985), /. Biol. Chem., 260, 15699-15707. 125 Longo, L.D., Koos, B.J., and Power, G.G. (1973), Amer. J. Physiol, 224, 1032-1036. McGourty, J.L., Blough, N.V., and Hoffman, B.M (1983), /. Amer. Chem. Soc, 105, 4470-4472. McLachlan S.J., La Mar, G.N., and Sletten, E. (1986), /. Amer. Chem. Soc, 108, 1285-1291. McLellan, T. (1984), Biochemical Genetics, 22, 181-200. McLendon, G. and Miller, J.R. (1985), /. Amer. Chem. Soc, 107, 7811-7816. MacGregor, R.B. and Weber, G. (1986), Nature, 319, 70-73. Maniatis, T., Fritsch, E.F., Lauer, J., and Lawn R.M. (1980), Ann. Rev. Genet., 14, 145-178. Marcus, R.A. and Sutin, N. (1985), Biochim. Biophys. Acta, 811, 265-322 Margalit, R. and Schejter, A. (1976), Eur. J. Biochem, 32, 492-499. Mathews, F.S. (1985), Prog. Biophys. Molec. Biol, 45, 1-56. Matthew, J.B. (1985), Ann. Rev. Biophys. Chem. 1985, Chapter 14, 387-417. Mauk, A.G., Coyle, C.L., Bordignon, E., and Gray, H.B. (1979), /. Amer. Chem. Soc, 101, 5054-5056. Mauk, M.R., Mauk, A.G., Weber, P.C. and Matthew, J.B. (1986), Biochemistry, 25, 7085-7091. Mayo, S.L., Ellis, W.R., Crutchley, R.J., and Gray, H.B. (1986), Science, 233, 948-952. Meites, L. and Meites, T. (1948), Anal. Chem., 20, 984-985. Miki, K., II, Y., Yukawa, M., Owatari, A., Hato, Y., Harada, S., Kai, Y., Kasai, N., Hata, Y., Tanaka, N., Kakudo, M., Katsube, Y., Kawabe, K., Yoshida, Z., and Ogoshi, H. (1986a), /. Biochem. (Tokyo), 100, 269-276. Miki, K., Harada, S., Hato, Y., Iba, S., Kai, Y., Kasai, N., Katsube, Y., Kawabe, K., Yoshida, Z., and Ogoshi, H. (1986b), /. Biochem. (Tokyo), 100, 277-284. 126 Moore, G.R., Harris, D.E., Leitch, F.A., and Pettigrew, G.W. (1984), Biochim. Biophys. Acta, 764, 331-342. Moore, G.R., Pettigrew, G.W., and Rogers, N.K. (1986), Proc. Natl. Acad. Sci. USA, 83, 4998-4999. Moore, J.W. and Pearson, R.G. (1981), Kinetics and Mechanism, 3 r d edition, John Wiley & Sons, Inc. Morner, K.A.H. (1897), Nord. Med. Arkiv., 30, 1. Newton, T.W. (1968), /. Chem. Educ., 45, 571-575. Nobbs, C.L. (1966), Hemes and Hemoproteins, p. 143-147, edited by Chance, B., Estabrook, R.W. and Yonetani, T., Academic Press. Nobbs, C.L., Watson, H.C., and Kendrew, J.C. (1966), Nature, 209, 339-341. Nocera, D.G., Winkler, J.R., Yocum, K.M., Bordignon, E., and Gray, H.B. (1984), /. Amer. Chem. Soc, 106, 5145-5150. Northrup, S.H., Reynolds, J.C.L., Miller, CM., Forrest, K.J., and Boles, J.O. (1986), /. Amer. Chem. Soc, 108, 8162-8170. Northrup, S.H., Boles, J.O., and Reynolds, J.C.L. (1988), Science, 241, 67-70. 8170. Olivas, E., de Waal, D.J.A., and Wilkins, R.G. (1977), /. Biol. Chem., 252, 4038-4042. Olson, J.S., Mathews, A.J., Rohlfs, R.J., Springer, B.A., Egeberg, K.D., Sligar, S.G., Tame, J., Renaud, J-P., and Nagai, K. (1988), Nature, 336, 265-266. Ozols, J. and Strittmatter, P. (1963), J. Biol. Chem., 239, 1018-1023. Pain, R.H. (1983), Nature, 305, 581-582. Pain, R.H. (mi),Nature, 326, 247. Peters, J.B., Barnard, R.J., Edgerton, V.R., Gillespie, C.A., and Stempel, K.E. (1972), Biochemistry, 11, 2627-2633. Peterson-Kennedy, S.E., McGouryt, J.L., and Hoffman, B.M. (1984), /. Amer. Chem. Soc, 106, 5010-5012. 127 Phillips, S.E.V. (1978), Nature, 273, 247-248. Phillips, S.E.V. (1980), /. Mol. Biol., 142, 531-554. Ray, G.B. and Paff, G.H. (1930), Amer. J. Physiol., 94, 521-528. Reid, L.S. (1984), Ph.D. Dissertation, University of British Columbia. Reid, L.S. and Mauk, A.G. (1982), /. Amer. Chem. Soc, 104, 841-845. Reid, L.S., Taniguchi, V.T., Gray, H.B. and Mauk, A.G. (1982), /. Amer. Chem. Soc, 104, 7516-7519. Reid, L.S., Lim, A.R. and Mauk, A.G. (1986), /. Amer. Chem. Soc, 108, 8197-8201. Reinhammar, B. and Malmstrom, B.G. (1981), Copper Proteins, Chapter 3, edited by T.G. Spiro, John Wiley and Sons. Ringe, D., Petsko, G.A., Kerr, D.E., and Oritz de Montellano, P. (1984), Biochemistry, 23, 2-4. Romero-Herrera, A.E. and Lehmann, H. (1974), Biochim. Biophys. Acta, 336, 318-323. Romero-Herrera, A.E., Lehmann, H., Joysey, K.A., and Friday, A.E. (1978), Phil. Trans. Roy. Soc London, Ser. B, 283, 61-163. Rosenberg, R.C, Wherland, S, Holwerda, R.A., and Gray, H.B. (1976), /. Amer. Chem. Soc, 98, 6364-6369. Rossi-Fanelli, A., Antonini, E. (1957), Arch. Biochem. Biophys., 72, 243-246. Salemme, F.R. (1976), /. Mol. Biol., 102, 563-568. Satoh, Y. and Shikama, K. (1981), /. Biol. Chem., 256, 10272-10275. Saunders, B.C. (1973), Inorganic Chemistry, Vol. 2, Chapter 28, edited by Eichorn, G.L., Elsevier Scientific Publishing Co., New York. Schwarzenbach, G. and Heller, J. (1951), Helvetica Chimica Acta, 34, 576-591. Scott, R.A., Mauk, A.G., and Gray, H.B. (1985), /. Chem. Ed., 62, 932-938. Segal, M.G. and Sykes, A.G. (1977), /. Amer. Chem Soc, 100, 4585-4592. Sherwood, C, Mauk, and A.G., Brayer, G.D. (1987), /. Mol. Biol., 193, 227. 128 Shikama, K. (1985), Experientia, 41, 701-836. Shire, S.J., Hanania, G.I.H., and Gurd, F.R.N. (1974), Biochemistry, 13, 2974-2979. Simolo, K.P., McLendon, G.L., Mauk, M.R., and Mauk, A.G. (1984), /. Amer. Chem. Soc, 106, 5012-5013. Smulevich, G., Mauro, J.M., Fishel, L.A., English, A.M., Kraut, J., and Spiro, T.G. (1988a), Biochemistry, 27, 5477-5485. Smulevich, G., Mauro, J.M., Fishel, L.A., English, A.M., Kraut, J., and Spiro, T.G. (1988b), Biochemistry, 27, 5486-5492. Springer, B.A. and Sligar S.G. (1987), Proc. Natl. Acad. Sci. USA, 84, 8961-8965. Stellwagen, E. (1978), Nature, 275, 73-74. Stellwagen, E. and Cass R.D. (1975), /. Biol. Chem., 250, 2095-2098. Strittmatter, P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B., and Redline, R. (1974), Proc. Natl. Acad. Sci. USA, 71, 4565-4569. Sugawara, Y. and Shikama, K. (1979), Sci. Rep. Tohoku Univ. Ser.IV (Biol), 37, 253-262. Sugawara, Y. and Shikama, K. (1980), Eur. J. Biochem., 110, 241-246. Sundberg, R.J., Bryan, R.F., Taylor, I.F., and Taube, H. (1974), /. Amer. Chem. Soc, 96, 381-392. Sweetser, P.B. (1969), Anal. Chem., 39, 979-982. Tajima, G. and Shikama, K. (1987), /. Biol. Chem., 262, 12603-12606. Takano, T. (1977a), /. Mol. Biol, 110, 537-568. Takano, T. (1977b), /. Mol. Biol., 110, 569-584. Tamura, M., Asakura, T. and Yonetani, T. (1973a), Biochim. Biophys. Acta, 295, 467-479. Tamura, M., Woodrow III, G.V. and Yonetani, T. (1973b), Biochim. Biophys. Acta, 317, 34-49. Tanford, C. (1961), Physical Chemistry of Macromolecules, Chapters 20 & 26, John Wiley and Sons, New York. 129 Taniguchi, V.T., Sailasuta-Scott, N., Anson, F.C, and Gray, H.B. (1980), Pure & Appl. Chem., 52, 2275-2281. Taube, H. (1977), Advances in Chemistry Series, No. 162, Bioinorganic Chemistry II, 128-144. Taube, H. (1984), Science, 226, 1028-1036. Taylor, J.F. and Morgan, V.E. (1942), /. Biol. Chem., 144, 15-19. Teale, F.W.J. (1959), Biochim. Biophys. Acta, 35, 543. Tew. D. and Ortiz de Montellano, P.R. (1988), /. Biol. Chem., 263, 17880-17886. Theorell, H. (1932), Biochem. Z., 252, p. 1 Theorell, H. (1934), Biochem. Z., 268, p. 46. Tipler, T.D., Edwards, Y.H. and Hopkinson, D.A. (1978), Ann. Hum. Genet., London, 41, 409-418. Tollin, G., Meyer, T.E., and Cusonavich, M.A. (1986), Biochim. Biophys. Acta, 853, 29-41. Tomoda, A., Takizawa, A., Tsuji, A., and Yoneyama, Y. (1981), Biochem. J., 193, 181-185. Tsukahara, K., Okazawa, T., Takahashi, H., and Yamamoto, Y. (1986), Inorg. Chem., 25, 4756-4760. Underwood, L.E. and Williams, R.S. (1987), Amer. J. Physiol, 252, C450-C453. Uyeda, M. and Peisach, J. (1981), Biochemistry, 20, 2028-2035. Varadarajan, R., Szabo, A., and Boxer, S.G. (1985), Proc. Natl. Acad. Sci. USA, 82, 5681-5684. Varadarajan, R., Zewert, T.F., Gray, H.B., and Boxer, S.G. (1989), Science, 243, 69-72. Weller, P., Jeffreys, A.J., Wilson, V. and Blanchetot, A. (1984), EMBO J., 3, 439-446. Weller, P.A., Price, M., Isenberg, H., Edwards, Y.H., and Jeffreys, A.J. (1986), Mol. Cell. Biol, 6, 4539-4547. 130 Wherland, S., Holwerda, R.A., Rosenberg, R.C, and Gray, H.B. (1975), /. Amer. Chem. Soc., 97, 5260-5262. Wherland, S. and Gray, H.B. (1976), Proc. Natl. Acad. Sci. USA, 73, 2950-2954. Whitburn, K.D. (1987), Arch. Biochem. Biophys., 253, 419-430. Wilson, G.S. (1978), Methods in Enzymology, 54, 396-410. Wittenberg, B.A. and Wittenberg, J.B. (1987), Proc. Natl. Acad. Sci USA, 84, 7503-7507. Wittenberg, J.B. (1965), /. Biol. Chem., 241, 104-114. Wittenberg, J.B. (1970), Physiol. Rev., 50, 559-636. Wood, D., Blanchetot, A. and Jeffreys, A.J. (1982), Nucleic Acids Res., 10, 7133-7144. Wyman, J. (1965), /. Biol. Chem., 241, 115-121. Yusa, K. and Shikama, K. (1987), Biochemistry, 26, 6684-6688. 131 Appendix A Midpoint reduction potentials (vs. NHE) Potentials were measured in sodium phosphate or boric acid: sodium hydroxide buffers as noted (*). Midpoint reduction potentials were determined from an unweighted linear least-squares fitting program using the Nernst equation (19). The parameter / is ionic strength and [med.] is mediator concentration. The mediators for native Mb and DME-Mb are [Ru(NH 3) 6]Cl 3 and [Ru(NH3)5Im]Cl3 respectively. Myoglobin pH temp. / [med.] slope midpoint (°C) (M) 0*M) potential (mV 6.0 25 0.1 4 58.9 ± 0.2 64.3 ± 0.1 6.5 5 59.2 ± 0.2 63.1 ± 0.1 7.0 10 0.1 7 55.3 ± 0.1 75.0 ± 0.1 15 7 57.9 ± 0.2 70.6 ± 0.1 20 4 58.0 ±0.1 65.3 ± 0.1 25 4 59.6 ± 0.2 60.9 ±0.1. 30 3 59.4 ± 0.1 55.8 ± 0.1 35 0.5 60.6 ± 0.7 52.4 ± 0.4 7.0 25 0.05 5 59.1 ±0.2 79.4 ± 0.1 0.15 3.5 59.7 ± 0.4 53.3 ± 0.2 0.2 3 58.8 ± 0.1 49.9 ± 0.1 0.3 3 59.4 ± 0.1 45.4 ± 0.1 0.4 2.5 59.6 ± 0.3 42.2 ± 0.2 0.5 2 58.6 ± 0.2 39.0 ± 0.1 7.5 25 0.1 7 59.1 ± 0.1 55.9 ± 0.1 8.0 9 59.4 ± 0.2 44.5 ± 0.1 132 pH temp. I (°C) (M) *8 .5 25 0.1 * 9 . 0 *9 .5 Dimethyl-ester heme Myoglobin pH temp. / (°C) (M) 5.5 25 0.1 6 .0 6.5 7 .0 10 0.1 15 2 0 25 3 0 35 7.0 2 5 0 .05 0 . 1 5 0.2 0 .3 0 .4 0.5 7.5 25 0.1 8 .0 [med.] slope (MM) 9 5 9 . 8 ± 0 .4 16 5 9 . 6 ± 0 .3 2 0 59 .2 ± 0 .2 [med.] slope 0*M) 1 6 0 . 0 ± 0 .6 0 .75 60 .8 ± 0 .3 0 .75 5 9 . 6 ± 0 .6 0 .5 5 8 . 0 ± 0 .4 0.5 5 8 . 0 ± 0 .3 0 .5 57 .8 ± 0 . 2 0.5 59 .3 ± 0 .4 0 .4 60.3 ± 0 . 1 0 .25 61 .9 ± 0 . 4 0.5 60.1 ± 0 .5 0.5 60 .0 ± 0 .3 0 .4 6 0 . 6 ± 0 .4 1 60.2 ± 0 .2 1 59 .5 ± 0 .3 1 58 .8 ± 0 .3 2 .5 59 .3 ± 0 .5 6 60.8 ± 0 . 3 midpoint potential (mV) 33 .9 ± 0 .2 2 5 . 8 ± 0 .2 2 2 . 6 ± 0.1 midpoint potential (mV) 131.0 ± 0 .3 126.4 ± 0 .2 115.1 ± 0 .3 124.6 ± 0 .2 114.8 ± 0 .3 109 .0 ± 0.1 100.2 ± 0 .2 9 3 . 4 ± 0.1 86 .3 ± 0 .2 130.7 ± 0 .3 9 0 . 9 ± 0 .2 82.8 ± 0 .2 75.2 ± 0.1 70 .3 ± 0 .2 6 5 . 6 ± 0 .2 7 6 . 0 ± 0 .3 6 3 . 6 ± 0 .2 133 pH temp. I (°C) (M) *8.0 25 0.1 *8.5 *9.0 [med.] slope (uM) 6 59.1 ± 0.4 5 59.5 ± 0.5 3 59.7 ± 0.4 midpoint potential (mV) 64.4 ± 0.2 56.6 ± 0.3 52.9 ± 0.2 134 Appendix B Second order rate constants The second order rate constants reported below are for the reduction of native metMb and metDME-Mb by Fe(EDTA)2". Reactions were performed in sodium phosphate buffer. Second order rate constants were calculated from the slopes of first order plots of reduction rate versus [Fe(EDTA)2"] by a weighted linear least squares program. The data for native Mb has been published previously (Lim and Mauk, 1985) and is presented here solely for reference purposes. Myoglobin pH temp. I k\2 (°C) (M) (M-V1) 6.0 25 0.5 28.7 ± 0.7 6.5 16.9 ± 0.3 7.0 25 0.05 23.4 ± 0.8 0.1 22.5 ±0.5 0.2 14.8 ± 0.6 0.3 14.1 ±0.2 0.4 13.2 ± 0.3 0.5 12.6 ± 0.2 7.5 25 0.5 9.7 ±0.1 8.0 8.6 ±0.1 135 Dimethyl-ester heme Myoglobin pH temp. I * i 2 ( °C) . (M) ( M-V 1) 6.0 25 0.1 1891 ± 33 6.5 1630 ± 26 7.0 25 0.05 2475 ± 58 0.1 1341 ± 45 0.2 751 ± 19 0.3 501 ±21 0.4 299 ± 11 0.5 258 ± 6 7.0 10 0.1 580 ± 18 15 724 ± 25 20 1011 ± 29 30 1803 ± 82 35 2376 ± 28 7.5 25 0.1 861 ± 42 8.0 350 ± 19 136 Appendix C Second order rate constants adjusted for driving force The second order rate constants for the reduction of metDME-Mb and metMb by Fe(EDTA)2" were adjusted for the driving force. The difference in midpoint reduction potential (AE) between the reactants under the same conditions of pH, ionic strength (J) and temperature was defined as the driving force. The adjusted reaction rate, fcj2J» was defined from Equation 29. DME-Mb pH Em Fe(EDTA)2"£m AE *12 k® (mV) (mV) (V) (M'V 1) (M"V 6.0 126.4s 115.5b —0.0109 1891a 1529 6.5 115.1 108.9 —0.0062 1630 1444 7.0 100.2 96.6 —0.0036 1341 1250 7.5 76.0 84.4 +0.0084 861 1014 8.0 63.6 66.6 +0.0030 350 371 native Mb 6.0 64.3C 6.5 63.1 7.0 60.9 7.5 55.9 8.0 44.5 104. l d +0.0512 98.4 +0.0458 95.0 +0.0357 86.8 +0.0285 86.9 +0.0221 28.7e 77.8 16.9 41.2 12.6 25.2 9.7 16.9 8.6 13.2 at conditions of 7=0.1 M and 25°C " 7=0.1 M and 25°C from Kolthoff and Auerbach (1951) " 7=0.5 M and 25°C " 7=0.5 M and 25°C from Reid (1984) " 7=0.5 M and 25°C from Lim and Mauk (1986) 137 0 Appendix D Derivation of Equation 17 Equation 17 and its derivation applies to the analyses of both native and DME-Mb autoxidation experiments. Equations: By definition [Mb] = [oxyMb] + [metMb] By the Beer-Lambert Law: A = E C L Where A is the absorbance at a particular wavelength, E is the extinction coefficient at that wavelength, C is the concentration of chromophobe, and L is the pathlength of absorption. In a mixture of oxyMb and metMb, the observed absorptions at the o (581 nm) and p (543 nm) bands, A a and A b respectively, can be defined as follows (assuming a pathlength of 1 cm): = £ f y ([oxyMb] + (EfeX / ) [metMb]) = ^ ([oxyMb] + (£g"et / JSg"?) [metMb]) Let the constants Cx and C2 represent the ratios Ef^/E0*? and U m e t / £ b ) x y respectively. K = £^xy[oxyMb] + ^et[metMb] A b = ^[oxyMb] + ^'[metMb] ^ a = ^ x y ( [oxyMb] + Cj [metMb]) A b = £g x y( [oxyMb] + C2[metMb]) 138 Let the ratio of the of absorbances at the a and P bands (A a/A b) be represented by A(a/p). A { a / p ) = £Ty([oxyMb] + Ci[metMb]) £gxy([oxyMb] + C2[metMb]) Let R represent the ratio £a>x>'/£gxy. A ( a / / 3 ) = R [oxyMb] + Ci ([Mb] - [oxyMb]) [oxyMb] + C 2 ([Mb] - [oxyMb]) (1 _ d ) [oxyMb] + CjfMb] ( 1 - C 2) [oxyMb] + C2[Mb] The term [oxyMb]/[Mb] is isolated. A(aip)R-1 (1 - Cx) [oxyMb] + A(a/p)R-1C2[}Ab] = (1 - Ci) [oxyMb] - Ata/Z^/T'C^Mb] [oxy] ( A ( a / / ? ) ( l - C 2 ) J R - 1 - ( l _ d ) ) = W>\(Cx-A{aip)C2R-x) [oxyMb] = Cx-A(alp)C2R-1 [Mb] C j - 1 + ( l - C 2 ) A ( a / / ? ) Substituting DME-Mb for Mb gives Equation 17. Using data from the reference spectra and the MINSQ least squares analysis the following values for the parameters were calculated: native Mb 1.0525 0.19529 0.38058 P DME-Mb 1.0588 0.24435 0.42068 139 Appendix E Marcus Theory Calculation of A ^ i r r References: Wherland and Gray (1976); Marcus and Sutin (1985) 1. Calculate the free energy change of the cross-reaction (AG*2) from the electrochemical reduction potential difference (AE) between the reactants. AG*2 = - n F(AE) The terms n and F are the stoichiometry of the electron transfer reaction and the Faraday constant respectively. 2. Calculate the Marcus theory activation energies, AG*2 and AG22, from the first equation and the conversion between transition state (Eyring) and Marcus formulism. KT (—AG*/RT) k 2 = e AG* =AG*+ RT In ^ KT The term K is Boltzmann's constant; T is temperature (Kelvin); h is Planck's constant; Z is the collision frequency. 140 3. Calculate the electrostatic work terms w12, w2i, wxl, and w22 from: -BRhs/l -BRas/l It * a b 1 + BRay/l 1 + BRbs/l This equation describes the work required to bring together two reactants (a and with charges of Z a and and radii of Ra and Rb respectively. The charges are dependent on the ionic strength /. The term t is the dielectric constant of the solvent, water; Rab is the sum of Ra and Rb; is the electron charge. 4. Calculate the electrostatics-independent free energy of activation terms, AG* 2 and A G 2 2 from: 6. Calculate the electrostatics-independent free energy of activation, AGjJ, by solving the quadratic: A G j = A G j — Wji 5. Calculate the electrostatics-independent free energy change, AG°, from: AG° = AGf 2 — wl2 + w2l A(AG\\)2 + B(AG\\) + C = 0 141 where A = 4 B = 8AG*2 + 4AGR° — SAG** and C = 4AG*2* (AG22 + AGR° - 2AG*2) + (AG°) 2 7. Calculate the free energy of activation corrected for the electrostatics-work, AG*j o r r, from: A G * r = AG** + H.U 8. Calculate the corrected self-exchange rate, k\[n from Equation 9: h Equation 9, which is derived from transition-state theory, is used with a Marcus equation derived parameter (AG*\OTr) because the transition-state in a self-exchange reaction is considered similar to a mononuclear reaction, which can be analyzed by Equation 9. 142 

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