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Mechanistic analysis of electron transfer in cytochrome b₅ Reid, Lorne Samuel 1984

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MECHANISTIC ANALYSIS O F ELECTRON TRANSFER IN CYTOCHROME b5 By LORNE SAMUEL REID B.Sc, The University of British Columbia, 1979 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 April 1984 ® Lome Samuel Reid, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Biochemistry  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 30, 1 9 8 4 ABSTRACT Cytochrome bs has a central role in many aspects of eukaryotic cellular metabolism. Its specific function is to transfer electrons from a NADH-fiavoprotein reductase to a variety of physiologically important oxidases. Since the late 1950rs, the chemical and phys-ical properties of cytochrome bi have been investigated extensively. Despite the importance of this protein in cellular metabolism and the availablilty of detailed structural information, relatively little attention has been directed toward characterization of the mechanism by which it changes oxidation state. The present work reports a characterization of the oxidation-reduction equilibrium and the reduction kinetics of cytochrome bs and two heme-substituted derivau'ves. Comparison of the functional properties of dimethyl ester heme-substituted cyto-chrome bi with those of the native protein reveals that esterification of the heme pro-pionates increases the reduction potential of the protein by 60 mV and significantly alters the electrostatics of the reduction kinetics with Fe(EDTA) 2" as reductant These results support a role for one of the heme propionates in stabilizing the oxidized form of the protein and provide direct evidence for electron transfer at the surface of the protein along the partially exposed heme edge. The functional properties of deuteroheme-substituted cytochrome b5 are shown to be very similar to those of the nadve protein. Consequendy, this derivative has been used as an optical probe for studying the electron transfer self-exchange rate of cytochrome bi in kinetic analysis of the reaction between the nadve and deuteroheme-subsdtuted proteins. The second-order rate constant for this reaction after correcdon to a driving force of zero is 3.8 x 10: M " 1 s_1 (u= 0.1 M , pH 7 (phosphate), 25 ° C ) and represents a prediction of the true self-exchange rate of the native protein. The relatively low magnitude of this rate is attributed to short-range electrostadc effects arising from the heme propionates at the site of electron transfer along the heme edge. This heme-subsdtudon strategy appears to be a generally useful technique for studying self-exchange rates of other hemeproteins ii with non-covalently bound heme groups. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix ABBREVIATIONS x ACKNOWLEDGEMENTS xi INTRODUCTION Overview 1 Historical Perspectives 2 Structure of Cytochrome bs Sequence Analysis 4 Crystallographic Analysis 4 Cleaved Forms of Cytochrome bs 11 Detergent Solubilized Form 12 Biosynthesis of Cytochrome bs 14 Physiological Roles of Cytochrome i 5 16 Electron Transfer Theory Overview 21 Marcus Theory 23 Electron Tunneling Theory 26 Electron Transfer in Cytochrome b<, 28 EXPERIMENTAL PROTOCOL General Procedures 31 Preparation of Trypsin-solubilized Cytochrome £>} 31 Microsome Preparation 32 iv Trypsin Solubilization 32 Purification of Solubilized Cytochrome bs 33 Reconstitution with Synthetic Porphyrins Preparation of Apo-cytochrome fc5 34 Preparation of Deuteroheme- substituted Cytochrome bs 36 Preparation of Protoheme IX Dimethyl Ester-substituted Cytochrome b5 36 pH Titration of Cytochrome b5 37 Spectroelectiochemical Measurements 37 Kinetics Measurements 40 Reaction of Cytochrome bs with Deuteroheme-substituted Cytochrome bs 45 Nuclear Magnetic Resonance Spectroscopy 47 RESULTS Electronic Spectra of Native and Heme-substituted Cytochrome bs 48 Proton Titration of Cytochrome bs 48 Oxidation-reduction Equilibrium of Native and Heme-substituted Cytochrome bs 48 Ionic Strength Dependence 54 pH Dependence 56 Temperature Dependence 58 Fe(EDTA)1-'2' Reduction Potential 60 Fe(EDTA)2" Reduction of Native and Heme-substituted Cytochrome bs 62 Ionic Strength Dependence 63 pH Dependence 67 Temperature Dependence 69 Cytochrome ij - Deuteroheme-substituted Cytochrome b5 Cross Reaction 71 Ionic Strength Dependence 72 Temperature Dependence 75 NMR Spectroscopy of Native and D M E - k 75 v DISCUSSION Oxidation-reduction Equilibria of Native and Heme-substituted Cytochrome b Genera! Considerations 80 Ionic Strength Dependence 82 pH Dependence 84 Temperature Dependence 85 Fe(EDTA)2- Reduction of Native and Heme-substituted Cytochrome b5 General Considerations 86 Cytochrome bi Ionic Strength Dependence 87 pH Dependence 89 Temperature Dependence 91 Theoretical Treatment Marcus Approach 91 Electron Tunneling Approach 92 Metalloprotein Electron Transfer Self-exchange Reactions General Considerations 95 Cytochrome bs Rationale for Experimental Design '. 96 Ionic Strength Dependence 96 Temperature Dependence 97 Theoretical Analysis of Reactions Rates 97 Significance of Results 99 Conclusions • 100 BIBLIOGRAPHY 101 APPENDICES A. Cytochrome 65 Midpoint Reduction Potentials 117 vi B. Fe(EDTA)2" Midpoint Reduction Potentials 1 2 2 C. Fe(EDTA)2" Reduction Kinetics: First-order Rate Constants 123 D. Derivation of Eq 19 • l ^ 5 E. Cross-reaction Reduction Kinetics: First-order Rate Constants 137 vii LIST OF TABLES I Proteins Containing the "Cytochrome bs fold" 10 II Cytochrome £>5 Metabolic Functions 17,18 III Amino Acid Content of Cytochrome b> Preparation 35 IV Cytochrome i } Electronic Absorption Spectrum Extinction Coefficients 53 V Extended Ion Size Analysis 56 VI pH Dependence of Midpoint Reduction Potentials 60 VII Electronic and Non-electronic Components of Thermodynamic Parameters 62 VIII Ionic Strength Dependence of Kinetics Data 67 IX pH Dependence of Kinetics Data 71 X Eyring Plot Parameters 71 XI Cross Reaction Ionic Strength Dependence 77 XII Marcus Theory Analysis 93 XIII Electron Tunneling Analysis 94 XIV Estimation of Protein-Protein Self-Exchange Rate - 98 viii LIST OF FIGURES 1 Amino Acid Sequence of Cytochrome £>5 5,6 2 3-D Stereo Representation of the Tryptic Fragment of Cytochrome bt 7 3 Structure of Ferriprotphorphyrin IX 30 4 Optically Transparent Thin Layer Electrode Cell 38,39 5 Stopped Flow Spectrophotometer Modifications 43,44 6 Electronic Absorption Spectra 49 7 Cytochrome bs pH Titration 50 8 Midpoint Reduction Spectra — 51,52 9 Ionic Strength Dependence of Midpoint Reduction Potentials 55 10 pH Dependence of Midpoint Reduction Potentials 57 11 Temperature Dependence of Midpoint Reduction Potentials 59 12 pH Dependence of Fe(EDTA)2" Midpoint Reduction Potentials 61 13 Ionic Strength Dependence of Kinetics Data 64 14 Residuals Plot 66 15 pH Dependence of Kinetics Data 68 16 Temperature Dependence of Kinetics Data ., 70 17 Cross Reaction Difference Spectra 73 18 Cross Reaction Ionic Strength Dependence 74 19 Cross Reaction Temperature Dependence 76 20 pD Titration of the Cytochrome bs and DME- bs Spectra 77 21 NMR Hyperfine Shifts 78 22 Compensation Plot 90 ix ABBREVIATIONS USED ATP Adenosine Triphoshate CD Circular Dichroism DME-&5 Dimethyl Ester Heme-substituted Cytochrome b5 deuteio- i 5 Deuteroheme-substituted Cytochrome bi EDTA Ethylenediaminotetraacetic acid ESR Electron Spin Resonance EPR Election Paramagnetic Resonance e.u. Entropy Unit (cal/mol ° C ) FAD Flavin Adenine Dinucleotide FMN Flavin Mononucleotide NADH-Cytochrome bs Reductase (E.C. NADPH-Cytochrome P 450 Reductase (E.C. MCD Magnetic Circular Dichroism NADH Nicotinamide Adenine Dinucleotide, Reduced NADPH Nicotinamide Adenine Dinucleotide, Phosphate, Reduced NaPi Sodium Phosphate Buffer NHE Normal Hydrogen Electrode NMR Nuclear Magnetic Resonance (Spectroscopy) OTTLE Optically Transparent Thin Layer Electrode SCE Saturated Calomel Electrode X ACKNOWLEDGEMENTS To the many people who have supported and encouraged me during the course of this work I extend a hearty thank you. In particular, full marks are due to Dr. Grant Mauk who has endeavored to teach me both an appreciation for careful research and the fine art of communicadon. This, combined with his willingness to let me "mess about" in the bowels of the computer has made him an ideal supervisor. I would also like to thank the other members of the lab who have washed the pipettes and only grumbled quietiy about some of my habits. Special recognition is due to Keith Withers for an endless stream of notes on paper towels concerning all manner of "computerabilia". Finally, the long-standing interest and support of my family has been truely ap-preciated during the course of my education. This is it Mom, I am no longer a student, but then again promises promises. Thanks must go to my sister for loaning me an inva-luable aid in the preparation of this thesis, her pencil sharpener which she saved from grade school. The one with the 3-D dancing cat on the front xi INTRODUCTION Overview Cytochrome b5 is a small, acidic microsomal hemeprotein thai has been found in several eukaryouc species (1). The heme group is located in a globular head region and is exposed to the cytoso!. A short hydrophobic tail anchors this heme-binding domain to the membrane surface (2). Cytochrome bt transfers electrons from NADH to many physio-logically important processes by accepting electrons from its reductase and transferring them to a variety of redox-active proteins located on the microsomal membrane (1,3). In parti-cular, cytochrome b<, participates in fatty acid elongation (4) and desaturation (5), chole-sterol biosynthesis (6,7) and cytochrome P-450 catalyzed hydroxylation of xenobiotics in the liver (8,9). A smaller, soluble form of this cytochrome occurs in erythrocytes (10,11) and is responsible for mediating electron transfer between methemoglobin reductase and methe-moglobin (12). The sequence and three dimensional structure of cytochrome b5 have been deter-mined by Strittrnatter, Ozols and Mathews (13,14), and many of its functional properties have been characterized by Strittrnatter and colleagues (1,15,16). However, relatively little attention has been directed to experimental analysis of the mechanism by which cyto-chrome bs changes oxidation state. For this reason, a systematic investigation of the oxidation-reduction equilibrium and reduction kinetics of this protein and two of its heme-substituted derivatives has been undertaken. This work has made exclusive use of the trypsin-solubilized protein because it can be purified in the large amounts required for this work with relative ease, it is soluble and monomeric in the absence of detergent, and several lines of evidence indicate its redox behavior is equivalent to that of the membrane-bound form (17). The results provide reasonably direct evidence for the in-volvement of the heme propionate groups both in determining the reduction potential of the protein and in affecting the electrostatic interaction of the protein with redox-active agents. 1 To place this work in a proper context, the following introductory comments review the discovery and characterization of cytochrome bf and related proteins. In addition, some theoretical possibilities for analysis of metalloprotein electron transfer reactions are consi-dered. Historical Perspectives In the late 19th century, MacMunn investigated the absorption spectra of tissue squashes from several organisms with the aid of a crude microspectroscope (18). He post-ulated that the spectrophotometric differences between tissues that he observed arose from pigments which were distinct from hemoglobin and its derivatives. This novel idea met with an unreceptive audience, and in 1889 Levy carefully repeated MacMunn's experimental protocol (19). He also extracted "modified myohaematin" from the muscles of mammals and birds, but he concluded, in agreement with the prevailing dogma, that no new com-pound was present. Although MacMunn attempted to defend his position, Hoppe-Sevier appended a short, editorial note to the surrebutal paper (20). He stated that MacMunn had not brought forth any fresh evidence and that further discussion of the matter was superfluous. Consequendy, the concept of additional pigments was abandoned until the 1920!s when Keilin, at the University of Cambridge, "rediscovered" these pigments in mammals, insects, bacteria, yeast, reptiles, batrachians (frogs) and fish (21). He coined the term cytochrome (from the Greek roots for cellular pigment) and identified three distin-ctive classes (a, b and c) on the basis of their relative spectral absorption lines. The original cytochrome b of Keilin was soon shown to be the prototype of a family of related proteins (3). Characterization of the family was based on reactions with redox-active agents (methylene blue, cytochrome c, flavoproteins and with oxygen) and spectrophotometric criteria. The b-typc cytochrome family consisted of Keilin's cytochrome b (21), a cytochrome bl isolated from liver (22) or bacteria (23), cytochrome fcj from pig kidney (24), cytochrome b7 which was inseparable from the enzymatic activity associated with yeast lactate dehydrogenase (25,26), cytochrome by from plants (broad beans) (27,28) and a cytochrome £>4 from halophilic bacteria (29). In 1950 Sanbarn and Williams isolated cytochrome x from cecropia silkworm larvae which was subsequently renamed cytochrome bi by Williams (31). Soon afterwards, Strittrnatter and Ball (32) identified cytochrome m from the microsomes of rat liver which Chance and Williams (33) observed to have an absorption spectrum identical to the cecropia cytochrome. These investigators renamed the protein cytochrome b5 based on the identity of its absorption spectrum with that of the cecropia protein. Cytochrome b$ was shown to be distinct from mitochondrial cytochrome b in 1956 when Strittrnatter and Velick demonstrated that treatment of bovine liver microsomal pre-parations with pancreatic lipase released a soluble, heme-containing protein with the cyto-chrome bi spectrum (34). The relative ease with which this protein could be isolated from various mammalian liver sources prompted numerous investigations into its chemical proper-ties (3). In the late 1960's, independent investigations by Sato in Japan (35) and Strittrnat-ter in the United States (36) established that detergent solubilization of the microsomal fraction yields a form of cytochrome b<, with an additional sequence of 40 amino acids at its carboxyl-terminus. This additional segment was found to have a very hydrophobic amino acid composition and apparently functioned to anchor the globular head region to the microsomal membrane. In contrast to most membrane-bound proteins, the amino-terminus was found to extend into the cytosol rather than the lumenal space (37). This unusual finding prompted investigation into the post- translational processing of the protein (38). Although the possible occurrence of a mitochondrial form of cytochrome b5 had been ackowledged previously in the literature (1,39), the existence and characterization of this form, named OM-cytochrome b for clarity, has only recently been established unequi-vocally (40,41). It has been shown that this cytochrome participates in the NADH-semidehydroascorbic acid reductase activity of the rat liver (42). It has also been suggested that this cytochrome may interact with a pool of cytochrome c free in the in-termembrane space (43). 3 A soluble form of cytochrome 6, has been isolated from mammalian erythrocytes. During maturation of the cell all subcellular organelles are degraded. In this process cyto-chrome bs is solubilized by an endogenase protease and remains in the cytosol. In avian erythrocytes, which maintain a complete subcellular organization no soluble cytochrome b< has been isolated. Structure of Cytochrome b<. Sequence Analysis The complete amino acid sequence of lipase and trypsin-solubilized and native cy-tochrome bi have been determined for several species. In addition, the cytochrome 6<-like domains of sulfite oxidase (44), yeast cytochrome b2 (45) and nitrate reductase (46) have been determined. A comparative sequence alignment, as discussed by Lederer and colleagues (46), between these proteins, cytochrome b from rat outer mitochrondrial membrane (46) and cytochrome bs from bovine (13), rat (47), and chicken (48) is presented in Fig 1. The empirical molecular weight of cytochrome bi solubilized from bovine liver microsomes with Triton X-100 is 15,233 daltons (including the heme). Solubilization of cytochrome bs with trypsin releases a fragment (residues 7 to 88) with a molecular weight of 10,082 da-ltons. Crystallographic Analysis The three dimensional structure of the lipase-solubilized protein residues 4-96) has been solved to 2.8 A resolution (49), and with real space refinement, it has been resolved to 2.0 A by Mathews and co-workers (50). A stereoscopic view of the oxidized protein is given in Fig 2. In this orientation, a "crown" of negative charges surrounds the heme edge and an hydrophobic patch of residues, on the surface, is to the back. The molecule can be approximated as a cylinder with a diameter of 31 and a height of 37 A. 4 Fig 1. Amino acid sequence alignment of cytochrome bs and proteins known to possess a similar heme crevice. 1 L T 10 1 BMC Ac - A l a - G l x - G l x - S e r - S e r - L y s - A l a - V a l - L y s - T y r 2 RMC Ac II G l u G i n i i A s p i t A s p II II 3 ROM H2N - Asp G l y G i n G l y II A s p P r o A s l It ' T h r l i 4 c h i c k e n H2N - G l y A r g l i 5 b2 H2N - A s n P r o L y s L e u A s p Met A s n L y s G i n L y s 6 SOX H2N - A l a P r o S e r T y r P r o A r g 7 b557 20 H2N - A s p 1 T y r - T h r - Leu - G l u - G i n - H e - G l u - L y s - H i s - Asn ' - A s n - S e r - L y s 2 M i t t t II G l u II II t i II L y s A s p It M 3 It A r g i t II G l u V a l A l a II A r g i t T h r A l a G l u 4 - II A r g t t G i n G l u V a l G l x II II i t II II G i n 5 H e S e r P r o A l a G l u V a l A l a i t II i t L y s P r o A s p 6 It A r g t t G l u V a l G l y A r g II A r g L y s P r o G l u 7 Leu G l u T y r t i H e L y s G i n T y r 30 II 7 7 i t 1 Ser = - T h r - T r p - L e u - H e - L e u - H i s - T y r - L y s - V a l - T y r - Asp 2 t l II " V a l II t t II H i s II t l 11 " 3 G l u - II " Met V a l H e t l G l y A r g 11 11 4 = II " H e II V a l II H i s A r g H e 11 " 5 A s p = Cys " V a l V a l H e A s n G l y T y r t l It 6 G i n A r g V a l V a l T h r H i s G l y T h r A s p 11 Phe " 7 ? t i t t 40 M I i B t l t i 7 * * 1 L e u - T h r - L y s - Phe - L e u - G l u - G l u - H i s - P r o - G l y - G l y - G l u - G l u 2 . " t i 11 t t II II II II II II II i t 3 H e A r g t t t t S e r II II t i II 11 i t 4 H e " i t It i t A s p II II II II II 11 tt 5 t t " A r g II i t P r o A s n II t l II II G i n A s p 6 V a l " -COOH 7 II 50 II II * 7 7 II 11 II 7 60 t t 1 V a l - L e u - A r g - G l u - G i n - A l a - G l y - G l y - A s p - A l a - T h r - G l u - Asp 2 II i t i t II II 11 i i II •t II II II Asn 3 It II Leu II II 11 i i A l a i i II II II S e r 4 t l II t i t t II 11 i i i t t i II II II A s n 5 II H e L y s Phe A s n II i i L y s i t V a l II A l a H e 7 -COOH 5 * B 70 1 9 Phe I I - G l u i i - Asp H - V a l it - G l y - H i s It - S e r I I - T h r - Asp It - A l a I I - A r g it - G l u -II Leu I I 3 I I ti I I I I •t I I it - P r o It tl I I II Met 4 I I I I I I tl II i i " I I I I tl I I A l a M 5 tl I I Pro Leu = I I A l a P r o Asn V a l H e A s p Lys 80 1 Ser - L y s - T h r - Phe - = - = - H e - H e - G l y - G l u - Leu - H i s - P r o 2 n I I II T y r - = II " II 11 ii II 11 3 Leu II G i n T y r = = T y r II II Asp V a l II n 4 it G l u II I I = = I I II II n II II it 5 T y r H e A l a P r o G l u L y s L y s L e u 11 P r o II G l u G l y T 90 E " L E 1 Asp - A s p - A r g - S e r - L y s - H e - T h r - L y s - P r o - Ser - G l u - S e r - H e 2 II I I II II A l a II 11 it tf T h r Leu 3 Asn M L e u L y s P r o L y s -COOH 4 II n L y s P r o A r g -COOH 5 Ser Met P r o P r o G l u L e u V a l C y s II P r o T y r A l a P r o 100 110 1 H e - T h r - T h r - H e - A s p - S e r - A s n - P r o - S e r - T r p - T r p - T h r - A s n 2 II II II V a l G l u II II S e r II 11 t l If II 5 G l y G l u L y s -COOH 120 1 T r p - L e u - H e - P r o - A l a - H e - S e r - A l a - L e u - Phe - V a l - A l a - Leu 2 11 • V a l I I II II II 11 II ti V a l II It II 130 1 H e - T y r - H i s - L e u - T y r - T h r - S e r - G l u - A s n -COOH 2 Met _ II A r g II Met A l a If A s p -COOH LEGEND: " r e s i d u e i s e q u i v a l e n t t o A l i g n m e n t #1 = no r e s i d u e , space a l l o w e d f o r a l i g n m e n t ? unknown r e s i d u e * r e s i d u e i n v o l v e d i n c h a r g e - p a i r i n t e r a c t i o n s B l i g a n d t o t h e heme i r o n E bond c l e a v e d i n e r y t h r o c y t e b5 L bond c l e a v e d by t h e p r o t e a s e i n l i p a s e p r e p a r a t i o n s T bond c l e a v e d by t r y p s i n BMC b o v i n e microsomes RMC r a t microsomes ROM r a t o u t e r m i t o c h r o n d r i a l membrane b2 l a c t a t e d e h y d r o g e n a s e ; c y t o c h r o m e b2 SOX s u l f i t e o x i d a s e b557 n i t r a t e r e d u c t a s e ; c y t o c h r o m e b557 6 Fie 2. Stereo drawings of the o-carbon chain of trypsin-solubilized cytochrome b>. The heme group and side chains of His 4 3 and His 6 ?. The residues involved in charge-pair interactions (Asp^, Glu 4 7 4 g and Glu 5 2) are indicated. 7 A direct comparison of the cytochrome b. heme-binding pocket with die equivalent folds in myoglobin and hemoglobin indicates several common structural features (14). The four o-helical segments which are in an antiparallel conformation (a stabilizing structure (51,52)) act as pincers to hold the prosthetic group in place. The floor of the crevice is composed of an antiparallel sheet. The interior of the crevice is comprised primarily of aromatic (20%) and other nonpolar (60%) residues (53). Differences between proteins with this same basic crevice occur in the binding of the heme prosthetic group. The heme of cytochrome bs is non-covalendy bound, the propionates are oriented towards the surface of the protein, and two histidine ligands are coordinated to the iron (His^ and His^. These characteristics are generally more reminiscent of myoglobin and hemoglobin (17) than c-type cytochromes (54). The three-dimensional homology of these structures has been analyzed in detail by Rossman and Argos (55). The orientation and relative solvent accessibility of the heme prosthetic group is believed to be critically important to the reactivity of heme proteins (53,54,56). After pu-blication of the crystal structure, NMR studies by LaMar and colleagues (57) prompted a reinvestigation of the orientation of the heme in the heme pocket Re-evaluation of the X-ray diffraction data by Mathews (58) subsequendy confirmed that the heme group in cytochrome b5 can exist in either one of two orientations that differ by a 180° rotation about the a-p-meso axis. In this analysis, Mathews found that the principal (80%) heme orientation present in the crystals studied by diffraction analysis differs from that originally reported by a 180° rotation about the o-p axis. Distortion of the heme structure by the crevice has made this assignment a difficult task. The possibility of functional differences between these two forms has not been evaluated owing to difficulty in isolating homo-geneous preparations of either form. The relatively rapid interconversion between these forms (t^2 0 3 3hr (57)) precludes convenient use of most standard techniques for detect-ing possible effects. At the protein surface, the heme is relatively exposed with 23% of its surface area accessible to the solvent (53). The accessible heme surface, however constitutes only a small (3.6%) percentage of the total surface area of the protein (53). One 8 propionate (pyrrole ring III) is extended out into solution while the other (pyrrole ring IV) bends back onto the surface of the protein and forms hydrogen bonds with a main chain amide and the hydroxyl oxygen of Ser^ g. The solvent accessiblity of this propionate is limited. One oxygen (402) of this hydrogen-bonded propionate is 6.4 A. from the heme iron, and its carbon backbone (atoms 4CM and 4CA) has a slight solvent accessibi-lity through the surface hydrophobic patch (17). The lower third of the cytochrome is a hydrophobic core thought to be important for structural stability. A "tunnel" lined with hydrophobic residues can be observed in this region. The function of this and certain other patches, which are conserved in all species, is unknown (14). Virtually no conformational difference was observed between ferri- and ferrocytochrome bs (59). The major differences observed on reduction of the protein are a slight shift (<0.12 A) of the iron atom into the plane of the heme, disruption of a Lys^ - Tyr^ hydrogen bond and the binding of a tetrahedrally coordinated monovalent cation near the buried propionate. The heme crevice of cytochrome bs is believed to be an evolutionarily ancient folding pattern (55). Through an analysis of crystal structures (17), the side chains of oth-er b cytochromes, which have the "bi fold", were placed on the backbone of the cyto-chrome bi structure (60). With minor exceptions it was found that most changes occurred on the surface of the protein and that environment around the heme was conserved. As a result, it is believed that the gene sequence coding for the "bi fold" was fused to the primitive enzymatic domain. A compilation of proteins known to possess the "cytochrome bs fold" is given in Table I. It will be of interest to determine the spatial relationship between the heme group and the enzymatic active site in these multidomain enzymes when crystallographic data become available. Crystallization of detergent solubilized cytochrome bs is hampered by strong hydro-phobic interactions of the tail segment It is thought that cytochrome b5 is joined to the tail segment by a flexible link. Chou-Fasman analysis (64) predicts the following secondary 9 Table I. Proteins Which are Known from Physical Properties or Analysis to be Related to Cytochrome bt Protein Tissue .Source Efif Cytochrome i>5 bovine microsomes 13 rat mitochondria 47 human red blood cell 11 Hemopexin blood 61 Intestinal Heme intestine 62 Binding Protein Lactate Dehydrogenase Saccharomyces cerevisia 60 (cytochrome b2) Multiple Heme-Binding blood 63 Plasma Protein Nitrate Reductase Neurospora crassa 46 (cytochrome bi51) Sulfite Oxidase liver 44 structure for the carboxyl terminal sequence of the protein binding segment as; residues 91-97 flexible link, random coil 98-102 e sheet 103-112 £ turn 10 115-125 a helix 126-130 e sheet Functional Properties Cleaved Forms of Cytochrome bs Analytical estimates of the molecular weight of the tryptic fragment of cytochrome bs differ somewhat from that determined from the amino acid sequence (10,082 daltons). In comparison, the molecular weight has been measured (16) at 11,500 by iron analysis, 11,000 by osmotic pressure and 10,600 from sedimentation equilibrium studies. In contrast, tryptic-cytochrome bs behaves anomolously on Sephadex G-25 gel chromatography and elutes slightly ahead of cytochrome c. A higher apparent molecular weight is also observed in sodium dodecyl sulfate (SDS) gel electrophoresis. This latter observation has been re-lated to a lower binding capacity for SDS than is commonly found for many proteins (0.7 vs 1.4 g SDS/g protein) (37). A wide range of spectroscopic techniques has been employed to characterize cyto-chrome bs. The electronic spectrum of cytochrome bs is unremarkable except for a split a-band in the reduced protein. The origin of this observation is uncertain, although one report notes that crystallized cytochrome b$ produces a species with a symmetrical a-band and a Soret band with significantly reduced intensity (65). No rationalization of this obser-vation has been suggested. Spectroscopic studies of the protein have been reported that employ CD (66), EPR (67,68), NMR (57,69,70), resonance Raman (71,72) and MCD (73) techniques. The heme-ring methyl substituents exhibit extensive downfield shifts in the proton NMR spectrum of oxidized cytochrome bs (69). The shifting of peaks, caused by contact and pseudo-contact (through space) interactions with the paramagnetic iron atom, are asymmetric and indicative of non-equivalent electronic distribution in the heme ring. Over-lap between the iron d orbitals and the heme TT orbitals, in conjuction with an asymmetric electron cloud may serve as a pathway for an electron from the heme periphery to the 11 iron center (69). The tryptic fragment of cytochrome b; is an acidic protein at neutral pH (pl = 4.3) (74). From the sequence data (assumed charge on His= 0.5+ e) lipase solubilized fer-ricytochrome bs has a net charge of -6.5 at pH 7 while the charge on the tryptic frag-ment is -7.5. The hexacoordinate iron is low spin and does not normally combine with CO, 0 ; , azide or CN". Millimolar concentrations or cyanide can slowly displace heme with the resultant formation of a dicyano-heme complex (67). Estimates of the reduction potential of cytochrome bs have ranged from 0-20 mV vs the normal hydrogen electrode (NHE) (75-78). Some early studies (75,79) reported the reduction potential of the me-mbrane bound form as -120 mV vs NHE, but subsequent investigation (77) attributed this effect to artifactual partitioning of the redox dye into the hydrophobic membrane layer rather than to an inherent difference between membrane-bound and free forms of the protein. Ferrocytochrome bs autoxidizes through the formation of a superoxide anion radical with a second-order rate constant of 5 x 10"3 M" 1 s_1 (80). The radius of cytochrome 65 is estimated as 17 A from the X-ray crystal stru-cture, 19.5 A from gel filtration (Stokes radius), 14.6 A from a minimum possible radius for a perfecdy spherical particle, as determined by gel filtration studies (16), and as 15.5 A from the previously discussed relationship R^=0.717 x (mol wt)1'3 (81). Detergent solubilized form Cytochrome bs can be solubilized from microsomes with Triton X-100. It is gener-ally believed that this protein is not glycosylated (17) although one report claims that the protein is glycosylated (82). The functional properties of the active site are not significantiy altered by the presence of the hydrophobic domain (1,3,16). One distinctive, physical change is aggregation of the protein to an octamer through hydrophobic interactions of the membrane binding segment (3,83). 12 A single layer (33 molecules) of slowly exchangeable lipid surrounds the hydro-phobic tail (84). Each molecule of cytochrome b5 will prevent only 14± 1 phospholipids from undergoing a phase transition (85,86). Macroscopic changes in the physical chemistry of a membrane occur at high concentrations of the cytochrome (86,87). This observation has been attributed to the increased proportion of bound to free lipid in the preparation. The proportion of cytochrome bs can be increased in microsomes from 2 to 20% of the bound protein by adding detergent solubilized cytochrome bs (39). The porphyrin prosthethic group imparts conformational stablilty to the cytochrome bs polypeptide (66). Resonance Raman (72) and optical (68,88) studies disclose that during thermal unfolding of the tryptic fragment, the iron changes from low spin six-coordinate to high spin five-coordinate and ultimately to a final high spin six-coordinate form with water as the distal ligand. These changes were partially reversible. This process occurs with a low stabilization energy <AG° r g 25° C= 2 5 ± 2 kJ/mol) which indicates a flexible mole-cular structure. A similarly low value for cytochrome c unfolding has prompted speculation that this may be a common feature of redox proteins (88). Detergent-solubilized cytochrome b$ exhibits a broader, more complex denaturation profile. Some evidence suggests that the hydrophilic and hydrophobic domains undergo thermal denaturation at different rates during the unfolding of the protein (80). The insta-bility of the detergent-solubilized form relative to the trypsin-cleaved form was attributed to reduced protein-water interactions between domains (72). Recent scanning calorimetry re-sults indicate that an inter-domain interaction may be responsible for reduced exposure of nonpolar amino acid residues to the solvent during the denaturation process (84). The re-lative importance of inter-domain interactions or of phospholipid headgroup effects on this complex process remains to be determined. 13 Biosynthesis of cytochrome b5 Cytochrome £>. has been found in the microsomes of several dssues; however, the concentration of the protein varies from source to source (1). In rabbits, the concentration of cytochrome bs ranges from a high of 0.88 nmole/mg protein in the liver to only 0.10 nmole/mg protein in the kidney. Cytochrome £5 has never been detected in skeletal or heart muscle. For many years, it was thought that synthesis of cytochrome bs was not in-ducible and that elevated levels occur only as part of the generalized increase in protein content caused by phenobarbital induction of cytochrome P-450 (89). However, recent work has clearly shown that microsomal cytochrome bi biosynthesis can be selectively induced without a concomitant increase in cytochrome P-450 by furylfuramide (90), griseofulvin (an oral and-fungal agent) (91), diisopropyl l,3-dithiol-2-ylidenemalonate (NK.K-105) (92), and ^-nitroanisole (93). Cytochrome b5 is distributed on many but not all cytosol-facing subcellular me-mbranes. This fact, in conjunction with the protein's reverse orientation (p 4), has led to considerable speculation on its mode of post-translational processing (94,95). After some inital confusion (96), it was clearly demonstrated (97,98) that mRNA coding for cytochrome bs is translated on free rather than membrane bound polysomes. It is believed that the apo-protein then migrates to membrane surfaces and binds through interaction of its hydrophobic tail with the membrane (39,98). Such a process would be expected to yield a subcellular distribution in accordance with the thermodynamic stability of the interaction of the tail with the particular membrane. Reconstitution experiments have shown that detergent solubilized cytochrome b5 will aggregate to form octamers until the lipid content reaches the critical micellular concentra-tion (37,83,99,100). Cytochrome bs then partitions into the lipid phase, but in contrast to isolated microsomes this association is not stable, and intervesicle exchange will occur even when vesicle fusion is clearly inhibited (101). This so called "tight" versus "loose" binding has been attributed to the placement of the hydrophilic C-terminus (102). 14 In one model for light binding observed in isolated microsomes and in artificial vesicles prepared by sonication, the protein is oriented in a transmembrane fashion with the hydrophobic tail spanning the membrane and the C-terminus on the surface opposite to that of the globular head. An alternative model of tight binding involves looping of the hydrophobic tail halfway into the bilayer of the membrane but retains the C-terminus on the same surface as the globular head. In this second model, loose binding is distin-guished from tight binding by postulating (103) that a different conformational arrangement inhibits certain stabilizing interactions of the C-terminus with the globular head region (102). The evidence with which one must decide between these models is indeterminanL The C-terminus of cytochrome fc5 is not cleaved when carboxypeptidase Y is reacted with isolated microsomes and this observation suggests a transmembrane orientation (102). Ho-wever, migration of the hydrophilic C-terminus (. . .Glu^ ^ s n]j3 COOH, bovine liver mi-crosome, Fig 2) across the membrane is a thermodynamically unfavorable process (104). Stabilizing interactions between the globular head and the C-terminus may also result in a blocking of the C-terminus, thus accounting for the negative result with the peptidase. Scanning calorimetry studies (86) and lipid binding (84,87,105) experiments with isolated tail fragments also support a looped tail model. However, other studies on isolated microsomal membranes have localized the C-terminus on the lumenal surface (106,107). In summary, the globular heme-binding domain is exposed to and folds in the cytosol during synthesis, and the 40 amino acid hydrophobic tail remains associated with the hydrophobic channel of the free ribosome (97,108). The apoprotein is released, and migrates to and becomes associated with membrane surfaces through hydrophobic interac-tions. If a specific, but as yet unidentified helper is present, the C-terminus will cross to the lumenal surface or undergo a conformational change which results in tight binding. Otherwise, the cytochrome bs will remain loosely associated with the membrane. The oc-curence of an exchange of newly synthesized cytochrome bs between subcellular organelles is uncertain (38,94,98). 15 Physiological Roles of Cytochrome 6< Initial studies of cytochrome b, revealed that this cellular pigment could be sele-ctively reduced upon addition of NADH to microsomal membranes isolated from the he-patocyte. However, no information was available regarding the significance of this observa-tion. Three metabolic roles for cytochrome bs were ultimately suggested in the early 1970's. Studies by Oshino and co-workers on intact microsomal membranes (109,110) and Wakil and Holloway's group on isolated enzymes (111) suggested a role for cytochrome 6> in the A 9 desaturation of long chain fatty acyl CoA. At the same time, it became ap-parent that the red blood cell methemoglobin reduction activity was probably mediated by cytochrome bi in vivo (112). Finally, it was suggested by Estabrook and co-workers that cytochrome bs may provide the second electron to cytochrome P-450 in its catalytic cycle (113,114). This synergistic function has now implicated cytochrome bs in a wide variety of metabolic processes (Table II). NADH cytochrome bb reductase (EC from calf contains an FAD prosthetic group, has a molecular weight of 43,000 daltons, a pi of 6.7 and is isolated as a membrane-bound protein (1,17). Following synthesis on free polysomes, the protein is in-corporated into the endoplasmic reticulum, Golgi, outer mitochondrial and plasma me-mbranes in a manner similar to that of cytochrome bi (115). As found for cytochrome bs, the N terminus is blocked (116). The catalytic cycle of NADH-cytochrome b< reductase (Fp )^ has been studied by Strittmatter and colleagues (17). A NADH is. bound near the flavin moiety through cy-steine and lysine residues. The flavin, in turn, also interacts with a tyrosine. With hydro-lyzed cytochrome bs as the electron acceptor, the rate limiting step is a stereospecific pro-ton transfer from NADH to FAD. The reductase catalyzes the transfer of two electrons from a molecule of NADH to two molecules of cytochrome bs, in sequential one-electron steps. This transfer is eliminated when a free sulfhydryl is blocked with 16 Table II. Metabolic Reactions Which Utilize Reducing Equivalents from Cytochrome b$ JLe^ cjion Cholesterol biosynthesis (4 demythylation) (A 5 desaturation) Ergosterol biosynthesis (4 demethylation) (A 5 desaturation) Fatty Acid Chain Elongation A 5 desaturation A 6 desaturation A 9 desaturation Hydroperoxide reduction Hydroxylamine reduction Methemoglobin reduction Plasmalogen biosynthesis Prostaglandin synthesis Sceciss Eel Rat Liver 5. cerevisae 117 118 Rat Liver 4,119 Rat Liver 120 Rat Liver 121 Rat Liver 5,122 Pig Liver 123 Rat Liver 124 Erythrocyte 78 Pig Spleen 125 Sheep Vesicular Gland 126 17 Table II. (cont) Cytochrome P 450 Isozvme Reaction Substrate PCB 448 H Hydroxylation aniline 127 P450LM2 Hydroxylation cumene 128 PB1 Hyroxylation metyrapone 129 PCB P448H O-dealkylation nitrobenzene/cumene 127 P450B1 O-demethylation p- Nitroanisole 130 P450 LM2 N-demethylation benzpetamine 131 ^-chloro-mercuribenzoate (PCMB) or N-ethyl maleimide (NEM) (132). Many mechanistic details of the catalytic cycle are still unkown. The reduction of cytochrome bs by Fp^ is dramatically increased when the two proteins are restricted to lateral diffusion in the plane of a membrane bilayer (133). Pre-sumably this indicates that the active sites are optimally positioned with respect to each other for electron transfer when the proteins are membrane-bound (134). Chemical modifi-cation studies implicate a number of charge-pair interactions near the heme edge of the cytochrome as critical for this reaction (135). Complementary interactions involve the side chain carboxyls of Glu^, Glu^g, Glu^. Asp^ a n d the single exposed heme propionate on cytochrome bs. However, the interaction domain may extend beyond this region. Cyto-chrome b2, which is thought to have a similar heme edge environment, reacts thirty-fold slower with Fp^. Cytochrome c, interacting only at the exposed heme edge of cytochrome bs and cytochrome b2, exhibits identical rates of electron transfer with each protein (136). Lack of an hydrophobic patch on the surface of cytochrome b2 may account for the slower interaction of this protein with Fp^ (60). NADPH will also catalyze the reduction of cytochrome bs with a lower efficiency in vitro. Another membrane-bound reductase (E.C. which has both FAD and FMN 18 prosthetic groups will react with only the membrane-bound form of cytochrome bs (133). As with Fp^, this reductase (Fp^ .) also requires the same complementary charge pair in-teractions at the exposed heme edge for activity (137). Since the primary acceptor of ele-ctrons from Fp is cytochrome P-450, the relative metabolic importance of this alternate pathway to cytochrome 65 reduction is uncertain (9). Desaturation at the C9-C10 position is an important step in the biosynthesis of long chain polyunsaturated fatty acids (120). This reaction is supported by NAD(P)H and ascorbate at high concentration. This finding led investigators to postulate a role for cyto-chrome bs, as this was the only microsomal component known to be reducible by all three electron donors (110,122). The terminal, cyanide-sensitive factor of the reaction, stearyl CoA desaturase, is now known to receive electrons from cytochrome b$ at its non-heme iron (5,138). The desaturase reacts with palmitoyl CoA and 0 2 to form the double bond at the C9-C10 position and a molecule of water (139). In addition, cytochrome bi is involved in a second fatty acid desaturation reaction at the A 6 position (121). This reaction is the primary regulatory step in the biosynthesis of polyunsaturated fatty acid (138). The reaction is similar to that catalyzed by the A 9 desaturase but is affected by a unique enzyme. The A 6 desaturase (66,000) daltons has a polarity of 49% (like that of cytochrome bs) and is solubilized at concentrations of Triton X-100 which do not solubilize the 53,000 dalton A 9 desaturase still buried in the me-mbrane (121). Microsomal fatty acid elongation is dependent on cytochrome bs (4, 119). The re-quirements for this reaction are a fatty acid-CoA thioester, two carbon equivalents from malonyl CoA, ATP and Mg 2 \ Cytochrome bs supplies electrons in the reductive steps; ke-toacyl CoA to hydroxy CoA and trans a,p-unsaturated acyl CoA to elongated acyl CoA. Reducing equivalents are supplied primarily from NADPH for de novo synthesis, but NADH can drive the reaction at 30% efficiency (119). 19 The liver is the principal site of detoxification of drugs and xenobiotics. Enzymatic hydroxylation of chemical species by cytochrome P-450 is often the first step in their metabolism (8,9,140). A broad spectrum of reactions in which cytochrome P-450 (a generic name which covers a family of related proteins) participates was screened for cytochrome bs involvement during the 1970rs (8,114,115,127.130.141-150). Results from reconstitution and immunological studies have ranged from obligatory, to insignificant, to even inhibitory ef-fects of cytochrome bs on the reaction rate. A recent report by Bosterling et al cautions, however, that the observed rate may be dependent on the cytochrome 6}:P-450 ratio (131). At a ratio of 1:1 used in many of the reconstitution experiments, reaction was in-hibited whereas at a ratio of 1:10 (65:P-450 mohmol), which reflects the situation in vivo, the reactions were stimulated. Many reactions of intermediary metabolism use reducing equivalents provided by NADH and NADPH. Cytochrome b<, appears to mediate electron transfer from these redu-ctants in many processes (Table II). However, the list of reactions in which cytochrome bt is known to participate continues to expand. Although most efforts in elucidating the metabolic roles of cytochrome bs have fo-cussed on hepatic processes, the importance of closely related processes in erythrocyte function is now well-established. The first component of this erythrocytic system to be discovered, methemoglobin reductase, was originally identified by its absence in blood of "blue babies" by Gibson (151). This enzyme was found to require NADH as a source of reducing equivalents and to have a relatively low degree of activity (12,78). Although Raw and coworkers demonstrated in 1959 that hepatic cytochrome bs could greatly stimulate this activity (152), it was not until twelve years later that Hultquist and Passon established that a soluble form of cytochrome bs occurs in erythrocytes and has a similar stimulatory ef-fect (153). Subsequent immunological studies showed that hepatic cytochrome bs reductase and erythrocytic methemoglobin reductase have similar antigenic properties and indicate that methemoglobin reductase is in fact a cytochrome bs reductase (154,155). As previously dis-cussed, sequence analysis has established the identity of the hepatic and erythrocytic 20 cytochromes (10). A direct correlation between cytochrome bs content and methemoglobin level has been observed in the aging red blood cell (156,157). Interactive computer graph-ics studies have now been used to generate a specific, hypothetical mode! for the interac-tion between cytochrome 65 and methemoglobin that occurs during electron transfer (158). As with Fp d > Fp^ and stearyl CoA desaturase, an important role was ascribed to comple-mentary charge-pair interactions at the exposed heme edge of the cytochrome. In addition to cytosolic activity,' recent studies have shown that a smaller level of reductase activity is associated with the erythrocyte plasma membrane (159,160). This activi-ty arises from residual cytochrome i 5 and reductase which are not cleaved during matura-tion of the cell (158). Although several proteases have been isolated from maturing red cells and reticulocytes, the nature of the protease(s) responsible for the release of the 96 amino acid fragment of cytochrome bf is still controversial (162-165). Recent work has suggested a potential role for calmodulin in the regulation of cathepsin D proteolytic acti-vity in the fetal and newborn infant's erythrocyte (165). The disease states caused by impaired reductase activity are classified as Types I and II (166). In the former, an abnormal reductase (gene DIA1, human chromosome 22) is localized to the red blood cell, and heterozygotes display cyanosis. Deficiency of the reductase throughout the body is associated with the Type II disease. Symptoms of this progressive disease (166,167) include methemoglobinemia, mental retardation, impaired fatty acid metabolism in the brain (168) and early death. As yet, no biochemical rationale for the neurological symptoms has been suggested. Electron Transfer Theory Overview Taube's seminal investigations concerning electron transfer reactions between simple metal complexes have made such processes one of the best understood categories of chemical reactions (169). In general, electron transfer between two coordination centers is 21 discussed in terms of two mechanisms: an inner-sphere mechanism which involves a bridging ligand or molecule and an outer-sphere mechanism in which no bridge between metal centers occurs (170). Outer-sphere electron transfer is one of the simplest chemical reactions possible because no bonds are made or broken. Consequently, it has been a rea-sonable starting point for development of reaction rate theory. Insofar as metalloproteins can be regarded as coordination complexes with very complex and extended ligands, it is reasonable to use the insight gained from analysis of small molecule reactions as a point of departure for studying protein behavior. A further simplification in analysis of this type is achieved if reactions between redox-active coor-dination complexes and metalloproteins are considered (at least initially) rather than reac-tions between proteins. In so doing, the coordination complex involved serves, in a sense, as a model for the active center of the second metalloprotein, the theoretical complexity is reduced by 50%, and the experimental challenge of following the reaction between two in-tensely colored chrbmophores is eliminated. Two general approaches have been developed in attempts to predict the rates of electron transfer reactions. Marcus theory employs what might be considered as a semi-empirical approach in which two known rate constants, the self-exchange rates for the two reactant species, are used to predict a third, the cross-reaction between the two complexes. An alternative approach, developed by Levich and Dogonadze (171), Hopfield (172), and Jortner (173), attempts to predict electron transfer rates from first principles. Excellent detailed reviews of these and related theoretical treatments have been published (174-176). For the present purpose, the highlights of the Marcus and Hopfield treatments will be outlined to provide the requisite backdrop for the analysis of the cytochrome b5 kinetics presented here. 22 Marcus Theory Electron transfer reactions can be distinguished according to their degree of ele-ctronic interacdon: 1) very weak (non-adiabadc), 2) weakly interacting (adiabatic) and 3) strong interactions (strongly adiabatic). an 'adiabatic electron transfer reaction' is defined as being one in which the probability of electron transfer occurring when the system reaches the activated com-plex, is unity. (177)" As discussed elsewhere (178) electrostatic theory is incapable of treating the strongly coupled case due to certain simplifying assumptions. Marcus theory applies when the amount of electronic overlap is sufficient to ensure adiabatic behavior but not so large that the overlap integral cannot be approximated to zero (179). The basic conclusion of Marcus theory is that the self-exchange rates of two redox-active complexes can be used to predict the rate of electron transfer for the cross-reaction between the two complexes. The self-exchange rate is the rate at which a reduced molecule can transfer an electron to its oxidized homologous counterpart: t A - + A <= = = > f A + A-According to Marcus theory, the rate of reaction between two reactants can be predicted from the self-exchange rate constants of the two reactants, if the equilibrium constant for the reaction is known and the reaction proceeds adiabatically through an outer-sphere mechanism. In this case k „ = (k„ k „ K / T J [1] where log f= [log(Kl2)p / [4 log (k„ k22/Z2)] 23 Z= collision frequency = 1 x 10n M ' 1 s"1 (or 1 x 10") kj,= self-exchange rate constant for reactant 1 k22= self-exchange rate constant for reactant 2 K n = equilibrium constant for cross reaction In the relative Marcus theory (180) the strict requirement of adiabatic transfer is replaced with the requirement that the reactants be at least uniformly non-adiabatic. Use of Eq 1 to describe electron transfer reactions is limited by certain considerations. When derived from general thermodynamic principles (181) the theory, as usually applied, requires only the following assumptions: (a) The activation process for each chemical species is assumed to be independent of its reacting partner. (b) The activated states are assumed to be the same for the symmetric electron exchange reactions and the asymmetric electron transfer reaction. Assumption (a) may break down in the case of large ligands or inner-sphere re-actions. In the case of' proteins their large bulk and high net charge will decrease their collision frequency. For electron transfer reaction to occur, the reacting species must be brought to-gether and the energy of the orbitals equalized in the activated complex. Within Marcus theory, the free energy of activation for electron transfer is given by (182): A G * = £ J * ( 0 ) [l + (AG/4 AG+(0))]3 [2] In Eq 2 A G = A G 0 + Wp - Wr, in which A G ° is the standard free energy change 24 (-ni*"AE°, F is the faraday); is the work required to bring the reactants together; Wp is the work required to bring the products together and A E° = standard potential difference beween the donor and the acceptor. The parameter AG^(o) is the intrinsic bar-rier related to the internal and solvent rearrangements that must occur prior to electron transfer when there is no free energy difference between reactants and products. The work terms are generally neglected when at least one of the reactants is uncharged and the re-action proceeds in a polar solvent This assumpton is not valid when describing protein-protein interactions which usually exhibit a strong electrostatic dependence. While application of Marcus theory to predict rates of electron transfer between substitutionally inert complexes has been highly successful, a different strategy has been required in applying it to reactions between metalloproteins and transition metal complexes. An alternate approach was required for analysis of such reactions because the self-exchange rates are not generally available for proteins. The approach developed by Wherland and Gray (174) was to use the measured protein/reagent cross-reaction rate constant, the reagent self-exchange rate constant, and the driving force of the reaction be-tween the protein/reagent pair to back-calculate the protein self-exchange rate constant By assuming that the reactants are spherical and have a totally symmetric charge distribution, Debye-Huckel theory may be used to produce an electrostatics-corrected protein self-exchange rate constant, k n c o r r . If a protein metal site obeys Marcus theory in its reactions with a variety of outer-sphere reagents, then the k n c o r r values calculated for these reactions will be constant (within an order of magnitude—the limits of theoretical and experimental error). The blue copper protein stellacyanin appears to demonstrate this type of behavior, indicating that the redox mechanism employed by this protein is more or less constant regardless of the na-corr ture of the reagent with which it is reacted. The k n values calculated for other pro-teins vary over several orders of magnitude, indicating that the mechanism employed by a given protein depends upon the nature of the redox reagent A general feature of these non-Marcus cases is that complexes with relatively hydrophilic ligand structure (e.g., 25 Fe(EDTA)2") give rise to low k u values while complexes with hydrophobic, TT-conducting ligands (e.g., Co(phen)3?*) produce relatively large values for k „ c o r r . Assuming thai the rate of electron transfer is approximately inversely proportional to the distance over which electron transfer occurs, it was reasoned that the more hydrophilic complexes are not able to interact with the non-Marcus metalloprotein sites as efficiently as the more hydrophobic reagents because they cannot penetrate into the relativley hydro-phobic protein environment as readily. Consequently, the more buried or hydrophobic a protein metal site is, the greater the range in k n c o r r values it will demonstrate. The failure of Marcus theory to account for the observed protein reactivity does not vitiate its usefulness in analyzing these reactions. Its value lies in its ability to correct the observed rate constants for factors such as thermodynamic driving force, reagent reacti-vity, and non-specific electrostatic interactions to yield rate constants that are more useful for mechanistic interpretation. In retrospect, it is not at all surprising that protein redox reactions do not generally conform to Marcus theory. By placing one of the reactant metal sites within a protein, it should be expected that reactant-reactant (ie., protein-reagent) in-teractions will occur that are not relevant to reactions between two inorganic complexes and that were consequently not accounted for by the theory. Electron Tunneling Theory While an outer-sphere mechanism requires contact of the coordination spheres of the donor and acceptor molecules, electron transfer in biological systems may often take place between redox sites buried within the protein matrix. The interaction of donor and acceptor orbitals at large distances is small, and the rate of electron transfer decreases ex-ponentially with increased separation. Consideration of these two alternatives in this way suggests that as the distance between metal centers increases, there is a gradual change in mechanism from outer-sphere to tunneling. In this sense, these two mechanisms are not so much opposites between which to select as they are closely related phenomena, the quan-titative relationship between which is a subject of some uncertainty. 26 The first indication that electron tunneling may be important in biological electron transfer processes was based on studies by DeVault and Chance that concerned photo-induced oxidadon of cytochrome in the photosynthetic bacterium Chromatium (183). This work stimulated the consideration of theoretical models to explain the phenomenon of a temperature independent rate constant over the range of 4.5 to 100 K (184). The theory of nonadiabatic multiphonon electron tunneling has been used to fit the experi-mental data successfully. At the high temperature limit, where the semi-classical approach of Hopfield (172) and the quantum mechanical treatment of Jortner (173) are identical, the following equations apply: k a b = 6.023 x 10-4 (2 TT / h) |Tab (r)|2 (4 TT ^ T )->'' x (2 TT x 3 r/R ) exp[-(Ea - E^ - A )2]/(4 k^  T A )] [3] with the activation parameters A H * = (E - Ek A ).V(4 A ) - 3RT/2 [4] and AS*= R ln[(2.38 x H r V l ^ T) (2 TT: X3 r/R ) (4TT ^ T"A)"V1 x [5] | Tab (r)|3]- 3RT/2 In these equations, the electron is initially localized at molecule a and is transferred to b, A is the vibronic coupling parameter, E g (E^) is the half-potential (oxidation scale) of a (b), is the Boltzman constant, T is temperature in degrees Kelvin, ra(rb) is the dis-tance of closest approach of a (b), r is the sum of ra and rb and Rp is the radius of the protein. The tunneling matrix element is estimated by 27 Tab (r) - 2.7 (NaNb)r; exp(-0.72 r) [6] where Na(Nb) is the number of electrons in a (b). The larger Tab becomes, the greater the overlap between donor and acceptor orbitals. and the closer the reaction centers be-come. Finally X, is the characteristic decay constant defined as: Within the Hopfield formulation, all rate constants are calculated at an infinite ionic strength to avoid complicating, electrostatic interactions between the reactants. Electron Transfer in Cytochrome b$ As outlined in the preceeding discussion, the sole physiological function of cyto-chrome b> is electron transfer (185). The present work examines some of the mechanistic features involved in the oxidation-reduction cycle of this heme protein. As described above, electron transfer from NADH cytochrome bs reductase (135) is thought to occur at the exposed heme edge. This same site has been implicated in complex formation with hemoglobin (186), stearyl CoA desaturase (137) and cytochrome c (187,188). Thus particular importance has been placed on understanding the role of this region of the protein in the function of the cytochrome. Inspection of the crystal structure of cytochrome bt reveals that one of the heme propionate groups is freely exposed to the solvent while the second is hydrogen-bonded to the surface of the protein. Removal of this second propionate from an aqueous environ-ment is energetically unfavorable and suggests that it may have chemical significance. Based on differences in the three-dimensional structure between the oxidized and reduced proteins Argos and Mathews suggested that in the oxidized state the buried propionate provides coulombic stabilization of the net positive charge on the heme iron (59). This stabilization 1/2(0.72)-' = 0.7 % [7] 28 of the oxidized protein results in a relatively low reduction potential for the protein. Upon reduction, this coulombic interaction is abolished as the net charge on the iron goes to zero. The protein does not undergo a conformational change to accomodate the buried ne-gative charge. Instead, the propionate appears to interact electrostatically with a cation, (Na" or K+) from the solvent To better define the oxidation-reduction properties of cytochrome bs in light of the structural information that is now available, the present investigation reports the characteri-zation of the electrochemical properties of cytochrome bs and two heme-substituted deriva-tives. The structure of ferriprotoporphyrin IX and the deutero- and dimethyl ester deriva-tives which were utilized is shown in Fig 3. In addition, the kinetics of reduction of each of these derivatives by Fe(EDTA)2- have been analyzed to evaluate the factors governing the dynamics of this redox interconversion. Fe(EDTA)2" was selected for this purpose as it has previously been shown to be a useful mechanistic probe in the study of a variety of other redox-active metalloproteins (174,189). Finally, the electron transfer reaction between native and deuteroheme-substituted cytochrome bs has been studied as a means of esti-mating the electron transfer self-exchange rate of the native protein. 29 Fig 3. Structure of heme: Ferriprotoporphyrin IX Rl = -CH = CH2. R2= H; Deuteroheme R l = H, R2= H; Dimethylester heme Rl= -CH = CH2, R2= -CH3. 30 EXPERIMENTAL PROTOCOL General Procedures Glass distilled water further purified to a resistivity of 17-18 M 9. by passage through a Barnstead NANOpure water purification system was used throughout A Ra-diometer Model PHM 84 meter equipped with a Radiometer Type GK 2321 C combina-tion electrode was used for all pH measurements. Conductivity readings were taken with a Markson Model 10 conductivity meter. Reagent grade chemicals were used throughout ex-cept as noted. Concentration of protein solutions was achieved by ultrafiltration in stirred cells (Amicon-12 and -52) fitted with Amicon YM-5 (5,000 molecular weight cutoff) me-mbranes. The concentration of ferricytochrome bs was determined from its electronic ab-sorption spectrum based on the published extinction coefficient (E412.5= 117,000 cnr1 M"1) (186). Electronic absorption spectra were obtained with a Cary 219 spectrophotometer equipped with a thermal isolation accessory and a rear beam attenuator. The spectra were digitized by a programmable interface accessory on the Cary 219 and presented in BCD format at an external port of a North Star Horizon microcomputer system designed by OLIS (On Line Instrument Systems, Jefferson, GA). This computer was programmed to ac-cept and manipulate the digitized data and to display the results on a high-resolution graphics terminal (Micro Angelo, Scion Corp, Vienna, VA). Preparation of Trypsin-solubilized Cytochrome bi The tryptic fragment of bovine liver cytochrome 65 was prepared by a combination of the methods of Strittrnatter (190) and Omura and Takesue (191) as follows. All opera-tions were carried out at 4 ° C . 31 Microsome Preparation Fresh steer liver was freed from connective tissue and run through a meat grinder. The ground liver was mixed 1:2 (v:v) with 0.25 M sucrose, ImM EDTA pH 7.8 (mea-sured at 25 ° C ) and homogenized for 30 sec (medium speed) in a Waring blendor. The resulting homogenate was centrifuged at 4,000 x g for 10 min to precipitate cellular de-bris. The turbid supernatant fluid was filtered through cheese cloth. Care was taken to avoid carry-over of pelleted material. The supernatant fluid was taken to 45% saturation in ammonium sulfate by adding 875 ml of saturated ammonium sulfate (pH 7.4 at 25 ° C ) . to each liter of supernatant fluid. This mixture was stirred with an overhead stirrer for at least one hour. The solution was then centrifuged at 16,000 x g for 25 min to yield a clear, red supernatant fluid and a firm, buff-colored pellet The pellet was placed in cleaned, 3" diameter dialysis tubing and dialyzed against cold, distilled water. The water was changed several times until the conductivity of the microsomal preparation was about 1 mmho. The final total protein concentration of these preparations was approximately 100 mg/ml as determined by the dye binding assay of Bradford (192) marketed by Bio-Rad. In these assays, the effect of lipid was corrected by saponification prior to analysis. Approximately 6-7 liters of microsomes were obtained from a normal (5-6 kg) liver. Mi-crosomes were stored at -70 ° C . Trypsin Solubilization In a typical preparation, about 3 liters of the frozen microsomes were used. Subje-ctively, solubilization appeared to be more efficient if the microsomes were frozen prior to addition of protease. A preparation containing 5 g of trypsin (Sigma Type II, #T-8253 or Millipore Type 3704 TRL, 190 units/mg), 0.15 g of CaCl2-2H30, and 0.07 g of L-l-(tosylamido)-2-phenylethyl chloromethyl ketone (TPCK, Sigma # T-4376) was mixed in 15 ml of 1 mM HQ. This trypsin slurry was added to the microsomes (15 ml per liter of microsomes), and the pH was adusted to 7.7. The suspension was stirred at 4 ° C for 24 hours. The pH was maintained at 7.7 throughout the course of the digestion by 32 periodic addition of dilute NaOH. The digestion mixture was then taken to 46% saturation in ammonium sulfate by addition of 267 g of solid (NH,)2S04 per liter of solution while maintaining the pH at 7.2 - 7.3. This mixture was stirred at 4 ° C for 45 min to ensure complete precipitation of microsomal membranes. Centrifugation at 16,000 x g produced a clear, red supernatant fluid and a firm buff-brown pellet. An aqueous suspension of the trypsin inhibitor ^-nitrophenyl-^'-guanidinobenzoate-HCl (NPGB, mol wt= 336.7, Sigma #N-0822) was added to a final concentration of 3 molar equivalents (210 mg NPGB/5g trypsin). The solution was then dialyzed in cleaned, 3". diameter dialysis tubing against cold, distilled water until the conductivity of the protein solution was less than 2 mmho. The solution was adjusted to pH 7.2, and the conductivity was adjusted to 1.8 - 2.1 mmho with water or 0.15 M Na phosphate (NaPi) buffer pH 7.2 at 4 ° C . The solution was clarified by filtration through fast flow filter paper (Schleicher & Schuell #588) Purification of Solubilized Cytochrome bs The protein solution was loaded onto a 7.5x5.0 cm column of DEAE-cellulose (Whatman DE-52®) that was equilibrated with 20 mM NaPi, pH 7.2 at 4 ° C . Under these conditions, cytochrome bs binds as a red-purple band near the top of the column, and the principal contaminant, hemoglobin, does not bind. The column was washed with the equilibration buffer until the eluant fluid was nearly colourless. The .buffer was then changed to 0.15 M NaPi, pH 7.2 at 4 ° C , and fractions were collected. All fractions with ^ ^412 5:j^280 r a ^ ° ^ v a l ue) greater than 2.5 were pooled for further purification. This protein was dialyzed against cold, distilled water until the conductivity of the protein solu-tion was 2.1 mmho or less. The protein solution was then rechromatographed over a se-cond DE-52 column employing the same conditions. Fractions with an R value greater than 3.5 were concentrated to a volume of 2-3 ml. The concentrate was eluted over a 70x2.5 cm column of Sephadex® G-75 (superfine) that was equilibrated with 20 mM NaPi, pH 7.2 at 4 ° C . Fractions with an 5:A280 r a t * ° °^ 0 r 8 r e a t e r w e r e pooled, concentrated (as above) and stored in liquid nitrogen. Fractions with a ratio 33 between 4.0 and 5.8 were pooled, concentrated and rechromatographed over the gel filtra-tion column as described above. Approximately 60-70 mg of cytochrome b, with a R va-lue > 5.8 were obtained for each liter of microsomal suspension. The maximum purity ratio observed during the course of this work was 6.05. The amino acid content of the purified cytochrome was analyzed with a Dionex Model D-500 by Mr. D. MacKenzie of this department. The results of this analysis (Table III) confirm that homogeneous cytochrome bi is obtained with this procedure. Preparation of Apo-cytochrome b5 Heme was removed from cytochrome bi by the method of Teale (193) with minor modifications as follows. The protein (15-100 mg) was dialyzed against cold, distilled water until the conductivity was about 4 mho. This protein (12 mg/ml) was put into a conical test tube fitted with a ground glass stopper, and the pH was carefully adjusted to 1.5 with 1 N HQ. The solution changed from red to brown at pH 4 as the heme started to dissociate from the protein. Heme was then extracted by addition of 1 volume of 2-butanone (Burdick and Jackson) and gende inversion of the conical tube. The brown ketone layer was carefully pipetted off, and the extraction was repeated once or twice as necessary. Heme extraction was complete when no significant color was present in the or-ganic layer. The apo-protein was then dialyzed (Spectra-Por® tubing) against a liter of 0.6 mM NaHC03 containing ImM EDTA and then against a similar volume of 0.6 mM NaHCOj. At this point, the apo-protein precipitated, as it had reached its isoelectric point The apo-protein went back into solution on continued dialysis against 20 mM sodium phosphate, pH 7.2 at 4 ° C , and then back into 0.6 mM NaHC03. The recovered protein was centrifuged for 3 min in a Silencer model H-25F1 microcentrifuge (12,000 rpm) to remove a small amount of material which did not redissolve. The concentration of apo-cytochrome bs was > 0.5 mM (E 2 ! 0= 10,500 mM"1 cm"1 (186)). All manipulations of apo-cytochrome bs during its preparation or subsequent reconstitution with modified pro-sthetic groups were conducted at 4 ° C . 34 Table III. Amino Acid Content of Purified Cytochrome b5. Amino Acid Theoretical! Hydro! ysate HC1 methane sulfonic acid NH4 7.70 7.68 ALA 4 3.96 4.07 ARG 3 3.00 3.06 ASX 9 9.04 9.04 CYS 0 <.01 <.01 GLX 13 13.16 13.43 HIS 5 4.77 4.75 ILE 4 3.50 3.30 LEU 8 7.78 7.83 LYS 6 5.93 5.92 MET 0 <.01 <.01 PHE 3 3.11 2.94 PRO 2 2.34 2.44 SER 4 2.59 3.38 THR 6 4.93 5.57 TRP 1 nd nd TYR 4 3.68 3.85 VAL 4 3.99 4.06 t Theoretical values from the sequence analysis (Fig 1) ± Residues are normalized to a value of 9.0 for ASX. n.d.= not determined. 35 Preparation of Deuteroheme-substituted Cytochrome bs Deuteroprotoporphyrin IX (Porphyrin Products Lot 319) was dissolved in a minimal volume of 0.1 N NaOH, centrifuged to remove any insoluble material, and diluted with water to give a 1 mM stock solution (E383 = 55+ 5 mM"1 cm"1 (186)). The porphyrin was added to cold apo-cytochrome i 5 (30-100 mg) at a 1.1 molar ratio, and the absorbance at 402 nm was monitored until no further increase was detectable. In general, reconstitu-tion was complete in 1 hour. The protein solution was adjusted to pH 7.2 and concen-trated to a volume of 1 ml by ultrafiltration. The reconstituted protein was applied to a 70x2.5 cm column of Sephadex® G-75 (superfine) equilibrated in 20 mM NaPi, pH 7.2 at 4 ° C , and eluted with the same buffer. A brown band of unbound heme eluted after the bright, red protein. Fractions with an ^402'^280 r a t * ° ~ ^ w e r e P ° ° ' e ^ a n c * c o n ~ centrated. The deuteroheme-substituted derivative was recovered with a yield of 80-85% (E,03= 122,000 M" 1 cm"1 (186)) and stored in liquid nitrogen. Preparation of Protoheme IX Dimethyl Ester-substituted Cytochrome bs Dimethyl ester protoporphyrin IX was obtained commercially (Porphyrin Products Lot 90181) and was checked for purity by thin layer chromatography (Gelman Instant TLC® system) (194). A single species was observed both in solvent I (2,6-lutidine:H20 20:1) with R f= 0.92 and in solvent II (hexane:CHC13:methanol 1:1:0.2) with R f= 0.72. A 1.1 molar equivalent of DME-heme to apo-cytochrome bt (30-100 mg) was dissolved in redistilled, dry dimethyl sulfoxide (Me2SO) (stored over molecular sieve 4A) to a final concentration of 2 mg DME-heme/ml. This solution was added dropwise to a slowly stirring solution of apo-cytochrome bs maintained on ice. When the addition was completed, the solution was placed in a refrigerator for 24 hours. An additional 0.15 equivalents of DME-heme was then added, and the solution was returned to the refri-gerator for another 24 hours. The material was then concentrated by ultrafiltration. At this point, the A. . . . :A . , s r . ratio was usually 3.5 to 3.9. The mixture was centrifuged to remove 36 any insoluble material and then eluted over a 70x2.5 cm column of Sephadex® G-75 (su-perfine) that was equilibrated with 20 mM NaPi, pH 7.2 at 4 ° C . Excess porphyrin eluted as a green band ahead of the reconstituted protein. The A^^A^gn r a u 0 °^ u n e reconstituted protein was > 6.0 with a yield of 80-85%. The extinction coefficient of this derivative in the Soret was assumed to be identical to that of the native protein (117,000 M" 1 cm"1). Upon re-extraction of the heme into a minimal volume of Me2SO, pH 5, no change was observed in migration patterns in the thin layer chromatography systems de-scribed above. Any ester hydrolysis should be readily detected under these conditions. The protein was concentrated as required and stored in liquid nitrogen. pH Titration of Cytochrome fc5 Cytochrome b5 was extensively dialyzed against cold, distilled water. The solution was centrifuged at 12,000 rpm in the bench-top centrifuge for 4 min to precipitate any insoluble material, and the concentration of cytochrome bs in the supernatant fluid was determined. Cytochrome solution (1.25 mM) was placed in a water-jacketed beaker (20 ml) equipped with a small magnetic spin bar. The pH was monitored as 0.01 N NaOH (Dilut-it®, Baker) was added from a Manostat digital pipet (Digipet model 71-633-00). Accuracy of the addition was ± 0.2 1. The pH was measured with and without the magnetic stirring engaged. The temperature was controlled at 25.0± 0.1 ° C by an external (Lauda RM-3) water bath. Spectroelectrochemical Measurements Midpoint reduction potentials were determined with an optically transparent thin layer electrode (OTTLE) cell (195). An exploded diagram of the cell is shown in Figure 4. The body of this cell was machined from lucite stock. Optical surfaces were constructed from 2x2x0.3 cm quartz plates (Wilmad, Buena, NJ), the windows were separated by spac-ers cut from 2 mil Teflon® tape (Type DF-1200, Dilectrix Corp, New York, NY), and a working electrode was constructed from 500 line/inch electroformed gold mesh (Buckbee 37 Fig 4. Optically transparent thin layer electrode A. Schematic diagram of cell body, machined from lucite. All dimensions are in inches. A port for the subminiature thermocouple is indicated at the left hand reference ele-ctrode opening. B. Exploded view of observation cell: Q - quartz window; T - Teflon spacer; G - gold working electrode. C. Assembled OTTLE cell mounted on the thermostatable holder. Hose bibs are connected to an external circulating water bath. The cell block is mounted on a single cell support assembly, Cary Type 00-952780-00. The SCE reference electrode is shown mounted in the water-jacketed, salt bridge (left electrode opening) and the platinum counter electrode is in the right electrode opening. 38 Mears Co., Minneapolis, MN). Windows, spacers, and working electrode were sealed to-gether onto the lucite base with epoxy cement. The OTTLE was mounted in the spectrophotometer on a specially designed alu-minum, water-jacketed cell holder, and the temperature was monitored to ± 0.2 ° C with a Euke Model 2175 A digital thermometer equipped with a copper-constantan subminiature thermocouple (Omega Eng. Inc. Stamford, CT). The reference electrode was maintained at 25 ± 0.1 ° C with a water-jacketed salt bridge connected to a refrigerated, circulating water bath (Lauda RM-3). This bridge was Tilled with a saturated KCI solution, and electrical contact with the sample solution was achieved with a platinum wire. A Ra-diometer Type K 4112 saturated calomel electrode (SCE) was employed as a reference. A platinum wire counter electrode was isolated from the test solution with an adaptor filled with the test solution buffer. This adaptor was in contact with the test solution through a glass frit. The potential of the minigrid electrode was controlled with a Princeton Applied Research Model 173 potentiostat and measured to ± 0.1 mV with a Keithley Model 177 digital microvoltmeter. A 680 ohm resistor was placed in series with the working electrode to ensure that a sufficient resistance was present to prevent overloading the potentiostat. Electronic absorption spectra were obtained with a Cary 219 spectrophotometer described above. Temperatures could be reliably controlled over the range of 5-40 ° C . Figure 4C illustrates the positioning of the OTTLE cell on the cell holder. In a typical experiment, about 0.5 ml of protein solution (120 uM) containing trip-ly recrystallized (196) Ru(NH3)6Cl3 (12 u M) (Alfa) to facilitate equilibration of the protein with the working electrode was introduced into the OTTLE cell. After temperature equili-bration, the potential was slowly lowered to -600 mV vs SCE and maintained for at least 15 min to achieve electrochemical deoxygenation. The potential was then raised to +100 mV and lowered once again to effect one redox cycle. The electronic absorption spectrum of the fully reduced protein was then recorded. The absorbance at the a-band of the reduced protein was monitored while the potential was slowly increased. Al the first sign of a change in intensity of the absorbance, the increase in potential was halted, and the 40 absorbance was monitored until it was stable (typically 15-30 min). The electronic spectrum was then recorded. The potential was increased in a stepwise fashion (15-30 mV), the absorbance was monitored until equilibration was achieved, and the electronic spectrum was recorded again. This procedure was repeated (6-8 times) until the protein was fully oxi-dized. Potentials measured against the SCE were converted to the hydrogen scale by ad-ding the factor 244.4 mV (25 °C) (197). Kinetics Measurements The reduction of cytochrome bs and its heme-substituted derivatives by Fe(EDTA)2" was studied with a stopped flow spectrophotometer. Buffered reductant solution was pre-pared anaerobically at controlled pH and ionic strength (198). Humidified, oxygen-free ni-trogen gas was prepared from commercial bottled stock by passing the gas through a train of two vanadous bubblers (199) and one photo-reduced methyl viologen bubbler (200). The latter bubbler contained 50 yM proflavine, 0.5 mM methylviologen, 50 mM EDTA and u = 0.1 M, pH 7 NaPi. Gas was distributed through a multiple outlet manifold and flexible tubing to 3" stainless steel needles (20 G) which penetrated the cleaned, rubber serum stoppers sealing 50 or 250 ml serum bottles. All solutions were degassed by vigor-ous bubbling with the nitrogen for at least 20 min. At this point, the needle was raised, and gas continued to flush over the surface of the protein solutions. Anaerobic dilution of stock Fe(EDTA)2" with the appropriate amount of degassed buffer was achieved with nitrogen-flushed gas-tight Hamilton syringes. The stopped-flow spectrophotometer was based on the design of Gibson and Milnes (201) as marketed by Dionex (Model D-103). Several modifications were employed to achieve greater anaerobicity in reactant transfer into the drive syringes. The transfer line from the serum bottles containing the nitrogen-flushed reactants was composed of a nylon luer fitting connected with compression O ring seals to small bore Saran® tubing (0.125" o.d., 0.031" wall, Pyramid Plastics, Hope, AR). A split-ring retainer . (202) was machined from stainless steel to secure the threaded nylon end piece to the Kel-F® valve block in 41 the stopped flow apparatus. Design specifications are illustrated in Fig 5A. In addition, the ceramic-tipped, drive-syringe plungers were replaced with Viton® O-ring sealed nylon tips. This change allowed for a continuous adjustment of the pressure exerted by the O-ring against the wall of the drive syringe, as illustrated in Fig 5B. This feature not only improved the anaerobicity but it also permitted compensation for the contraction and expansion of the drive syringe assembly that occurred in the variable te-mperature studies. Attempts to replace the Teflon® valve tips with nylon were abandoned owing to the inability of nylon to form an effective seal with the Kel-F® valve block. The remainder of the stopped-flow spectrophotometer system was designed by On Line Instrument Systems. The optical system was comprised of a 100 watt tungsten-halogen lamp operated at 5 amps by an OLIS XL-150 variable amperage power supply, a YSL Type 1200 uv-vis single beam, double monochromator (accurate to ± 1.0 nm) at a 2 mm slit width, and a 2.0 cm observation cell. The monochromator was equipped with a computer-driven stepping motor to permit acquistion of spectra of the contents of the stopped-flow cuvette over the range of 200 to 800 nm. Signals from an EMI-Gencome Model RFI/QL-30F photomultiplier tube were sent by coaxial cable to an OLIS Model HVA-SF amplifier equipped with a 0.05 to 20 msec adjustable time constant Analog signals were collected in single ended mode and digitized by a 12 bit (1 in 4096 maxi-mum resolution) successive-approximation analog to digital converter (Analog Devices Type AD572) housed in the OLIS 3620 interface. All data manipulation and control signals were performed by a NOVA 2/10 16 bit minicomputer (Data General Corp, Westboro, MA) equipped with two 8" floppy diskette drives. Output was distributed between the master console, a Tektronix Type 620 oscilloscope monitor or as hardcopy on a Hewlett Packard Type 7010B X-Y recorder. Data collection software originally written by OLIS permitted signal averaging of input decays and control of data input timing. Subsequent software modifications to allow control of the monochromator stepping motor and collection of spe-ctra were written in collaboration with OLIS. 42 Fig 5. Schematic drawings of modifications to the stopped-flow spectrophotometer. All di-mensions in inches. A. Transfer line:machined parts composed of nylon; brass nuts; viton O-rings. B. Drive Syringe:brass adjustable rod;stainless steel sleeve;nylon end pieces; viton O-ring. 43 .07 5 ^ri— -; 166 _ J -j.325 1.70 •1.06 B .150 .575 H h . 1 90 ".006 O ring 2.25 \ . 3 8 5 - i r 3/8' PTE .240 • — 1.20 .063 .330 .2 85 r 3.76 .325 .315 .128 I 2/32' PTE i 3.35 - .2 12 .137 .262 I .325 f-.078 .3 1 7 385 Temperature was controlled to ± 0.1 C with a Haake Model F3 refrigerated water bath. Reactant solutions were allowed to equilibrate for 25 min at 25 ° C and for 45 min at all other temperatures. Reactions were run under pseudo- first-order conditions with Fe(EDTA)2- in at least 30- fold excess over protein. Reduction of the protein was monitored at the reduced Soret band (423 nm for cytochrome fc5 or DME-ij and at 413 nm for deutero-fc5) with a typical change in absorbance of 0.15 OD. At least five tracings were averaged for each first-order rate constant determined. First-order rate constants were obtained from weighted linear least-squares analyses; identical results were obtained from nonlinear regression analyses. Second-order rate con-stants were obtained from weighted linear least-squares analyses of concentration depen-dences. Activation parameters were obtained from weighted linear least-squares analyses of Eyring plots. Reaction of Cytochrome fc5 with Deuteroheme-substituted Cytochrome fc5 The oxidation of deutero-fc5 by the native cytochrome was studied with the stopped-flow system described above. The extensive overlap of the spectra of the two proteins made careful selection of the wavelength to be monitored critically important in allowing optimal sensitivity and in reducing the complexity of the observed decays. Conse-quently, the choice of this wavelength was based on computer-assisted optical difference spectroscopy. The electronic spectra of native and deutero-£5 were measured in the oxi-dized and reduced (Na2S204) states with a Cary 219 spectrophotometer. The samples were maintained at 25.0 ± 0.1 ° C in water-jacketed cuvette holders with a circulating refri-gerated water bath (Lauda Type RM-3). In the cross-reaction, oxidized native cytochrome bs (1 uM) was mixed with re-duced deutero-fcj (10-30 uM) to assure psuedo-first-order conditions. Deutero-65 autoxi-dizes readily in the presence of dioxygen, so precautions were required to assure its 45 complete reduction and anaerobic transfer into the observation cell. Higher concentrations of deutero-iij could not be used owing to saturation of the photomultiplier response. Lower concentrations of native cytochrome did not allow adequate changes in absorbance. Two methods were used to prepare reduced deutero-63 free of excess reductant. In the first method, the cytochrome was reduced by H : in the presence of platinum (203). Platinum black (Alfa) was activated by washing with 10% HN0 3 and then rinsed three times with distilled water. After each wash, the platinum was collected by centrifugation at 2,000 x g for 5 min. After the final rinse, the platinum was placed in a vacuum desic-cator and dried for 48 hours before use. Activated platinum black was stored in vacuo. A 5 P M protein solution (10 ml) was degassed with oxygen-free nitrogen for 20 min in a stoppered serum bottle. The serum stopper was briefly removed, and a small amount (fine spatula tip) of the activated platinum black was added. Water-saturated hydrogen gas was directed into the protein solution with a concurrent nitrogen flush. The bottle was swirled gently to help suspend the platinum. Within 10 min, the solution underwent, a considerable color change, indicating reduction of the cytochrome. The solution was allowed to bubble for an additional 15 min with the H 2 . Anaerobic removal of the platinum was achieved by an inline filter as the solution was pulled into the drive syringe. The transfer line described above (Fig 5A) was inter-rupted with a 25 mm O-ring sealed stainless steel filter (Gelman Type 1209) equipped with Swagelock Vac Torr fittings (Type 2-UT-1-2). The fine platinum particles were effi-ciently retained by a single, thick glass-fiber filter (Gelman Type 66075). To ensure an-aerobic conditions, all transfer lines and stopped-flow surfaces were flushed with 3 mM Na2Sj04 and several rinses of degassed water prior to introduction of protein solutions. The second method of protein reduction used photochemically reduced methyl vio-logen (204,205). Specifically, a solution of protein (5 u M), 10 mM EDTA, 1.0 uM acridine orange and 1 u M methyl viologen was thoroughly degassed and transferred to the upper syringe barrel of the stopped flow unit A 200 watt light bulb was placed about 15 cm 46 from the barrel, and the solution was irradiated for 25 min. The drive syringes were maintained at 25 ° C during this process. Nuclear Magnetic Resonance Spectroscopy The proton NMR spectra of native and DME-b5 were recorded in collaboration with Dr. Colin Tilcock on a Brucker WP-200 NMR spectrophotometer operating at 200.13 MHz for 'H. Spectra were accumulated (up to 500 transients) with a spinning sample, a 4 fi s 90 r.f. pulse, 5 kHz sweep width and a 1 sec interpulse pulse delay in the presence of homonuclear gated decoupling centered upon the ! H resonance of water. An exponential multiplication corresponding to 1 Hz linebroadening was applied to spectra before the Fourier transformation. Protein solutions were exchanged into 0.1 M pD = 7.0 (pD=pH + 0.403 (206)) deuterated sodium phosphate buffer by three successive exchanges in an ultrafiltration cell. A calibration curve for the pD titration experiment was established by titrating a 2.0 ml sample of the stock buffer with 0.1 N NaOD. It was assummed that the presence of the protein would have a negligible effect on the observed pD. A 2.0 ml sample of the con-centrated protein solution (about 2mM) was introduced into a 5 mm NMR tube (Type 526 PP, Wilmad). The proton NMR spectrum was collected, and the volume of 0.1 N NaOD required to achieve a desired pD was added (Pipettman Model P200). Successive additions of NaOD to the protein solution enabled an entire pD titration curve to be de-termined with a single protein sample. 47 RESULTS Electronic Spectra of Native and Hcme-substituted Cytochrome b5 The spectra of the oxidiz.ed and reduced forms of native cytochrome bs and the heme-substituted derivatives studied here are shown in Fig 6. The spectra of the native protein and the deuteroheme-substituted cytochrome are identical to those reported previ-ously by Strittmatter and Ozols (186). The absorption spectra of reduced and oxidized DME-fcj are virtually identical to those of the native protein. The principal changes in the DME- bs spectra are a small shift in the Soret maximum of the oxidized proten from 412.5 to 412 nm and a more pronounced splitting of the a-band in the reduced form of the protein. Extinction coefficients for the principal peaks in the oxidized and reduced forms of each protein are given in Table IV. The spectra of cytochrome bs and its deri-vatives were found to be invariant from pH 5.5 to 8.0. In each case the Soret/^gQ nm absorbance ratio is approximately 6.0. This value is consistent with that reported for related heme proteins (207) and argues strongly against the presence of contaminating species. Proton Titration of Cytochrome bs The titration curve of native cytochrome bs is shown in Fig 7. One unit of net charge was assumed to arise from the deprotonation of one functionality by one equivalent of OH" (208). A net charge of -5.3 was estimated at pH 7.0 (at an ionic strength ap-proaching zero). Oxidation-reduction Equilibrium of Native and Heme-substituted Cytochrome bs Representative families of spectra collected from potentiometric titrations of each protein studied are illustrated in Fig 8. Equilibration to the applied potential was moni-tored at either the Soret or a-band and typically was achieved within 20 min. Isosbestic points were observed in these measurements at 353, 415, 438, 513, 534, 544 and 566 nm 48 Fig 6. Electronic absorption spectra in the oxidized and reduced (Na,S:0,) states A. Cyto-chrome bs B. Deutero-ij C. D M E - i 5 Fig 7. Titration of Cytochrome b:. with 0.1 N NaOH [25 ° C , dialyzed into water] 50 Fig 8. Representative families of thin-layer spectra obtained at different values of applied potential, Eapp (mV vs NHE). Protein at 120 yM, Ru(NH3)6 2* ' 3 + at 12 yM, pH 7 phosphate, y = 0.1M A. Cytochrome b$ (25 C): (a) -255.6,(b) -55.6,(c) -35.6,(d) -15.6,(e) 4.4, (f) 24.4,(g) 44.4,(h) 64.4,(i) 84.4,0) 244.4. B. Deutero-^ (20 ° C ) : (a) -205.6,(b) -95.6,(c) -75.6,(d) -55.6,(e) -35.6,(0 -15.6,(g) 4.4,(h) 24.4,(i) 244.4. C. DME- b5 (20 °C):(a) -205.6,(b) -5.4,(c) 24.2,(d) 54.6,(e) 74.7,(f) 95.0, (g) 114.9,(h) 144.3,(0 344.1. 51 ABSORBANCE ABSORBANCE ABSORBANCE Table IV. Extinction Coefficients from Electron Absorption Spectra (Fig 6)t Soret £band a band Protein oxidized reduced reduced reduced Cytochrome b5 412.5(117) 423.0(176.3) 527(13.5) 555(25.6) Deutero- bs 402.5(122) 409.5(156.8) 516.5(13.3 543.5(19.3) DME- bs t 412.0(117) 422.5(175.6) 526(13.1 5544(21.8) t values are reported as wavelength {nm}(extinction coefficient) {M_1 cm-1}. ± values normalized to be equivalent to the native cytochrome bs oxidized Soret peak. with the native protein, at 345, 406, 429, 501, 525, 536 and 552 nm with deutero- bs and at 350, 414, 436, 513, 535, 543.5 and 566 nm with DME-65. All derivatives were well behaved and could be cycled repeatedly between oxidized and reduced states with no de-tectable change in behavior. The Nernst plot (E applied vs log([0]/[R])) obtained from each experiment was fitted with a nonweighted, linear least-squares line. To allow for ex-perimental error, only those plots with a slope of 60 ± 2 mV (n = l , theoretical slope = 59.7 mV at 25 ° C ) were retained. A listing of the midpoint reduction potential and asso-ciated slope measured at each solution condition for each cytochrome derivative is pre-sented in Appendix A. As cytochrome bs has a net negative electrostatic charge and the mediator is posi-tively charged, the effect of mediator concentration on midpoint reduction potential was evaluated to determine whether the use of Ru(NH3)6 2 4 ' 3* perturbed the behavior of the protein. Variation of the [bs] :[Ru(NH3)6 2+'3*] ratio over several values in the range from 0.1 to 15 had no effect on the observed reduction potential [25 ° C , pH 7.0 (phosphate), u= 0.1 M]. This result also indicates that aquation of Ru(NH3)6 2 < , 3 \ which is known to have no effect on the reduction potential of the complex (209), had no adverse effect on the measurements. 53 Ionic Strength Dependence The dependence of the reducdon potential on ionic strength for each derivative is illustrated in Figure 9. These data have been analyzed in terms a Debye-HUckel type ex-pression (210): E ° , .= E° - RTA/F (q n 2 - q 2) f( y ) [8] obsd ox M red p where is the reduction potential observed at a given ionic strength, E the stan-dard reduction potential extrapolated to an ionic strength of zero, A is the Debye-Huckle constant (0.5115), and q Q x and q^ are the net electrostatic charges of the oxidized and reduced protein respectively (Qox = clrec} + Various forms of the function f( y ) were evaluated as previously suggested (210-212). The difference between these functions is equivalent to an evaluation of the "extended ion size", R , on the calculated protein charge. In each case, weighted non-linear regression analysis was applied with approximately equivalent Fits to the data. The resulting output parameters from these analyses are set out in Table V. The solid line in Figure 9 is the fit of the data to Eq 8 in which the form of f(y ) = y 1 'V(l + (R 0.329 y1'2) has been used with the value R p = 15.5 A. The reduction potential usu-ally could not be measured at ionic strengths less than 0.05 M as the electrical conducti-vity decreased substantially, and equilibration times increased drastically. 54 Fig 9. Variation of reduction potential with ionic strength [pH 7.0, phosphate, 25 The solid line is a fit of the data to Eq 8. A. Cytochrome fc5 B. Deutero-i3 C DME-k Table V. Parameters Calculated from Analysis of the Ionic Strength of the Reduction Po-tential. Different "Extended Ion Size" Radii Incorporated into the Function y ' 'V (1 + (Rp .329)y''2).t Protein Parameter R =17.0$ P R =15.5 P R =6.08 P R =3.04 P R =0 P Cyt-fcj- E y = 0, mV -39.2 -36.8 -22.1 -17.7 -10.6 E y = inf, mV 20.5 20.8 24.0 27.1 34.3 q Mox -5.95 -5.24 -1.76 -0.97 -.24 Deutero- 6S E y=0,' mV -95.2 -98.7 -76.3 -70.9' -63.4 E y = inf, mV -29.6 -25.9 -24.3 -20.2 -13.6 q Mox -6.6 -6.5 -2.1 -1.2 -.032 DME- ^ Ey=0, mV 3.77 8.26 3.63 4.55 5.52 E y = inf, mV 88.6 88.7 90.6 92.3 97.1 qox -8.6 -7.5 -2.2 -1.0 -0.19 t Infinity point estimated from fit at y= 1.0 M $ values in A pH Dependence The effect of pH on the midpoint potential is shown in Fig 10. Following the approach developed by Clark (213) and Ricard et al (214), these results may be analyzed in terms of a redox-linked functional group that undergoes a change in pKa as the pro-tein changes oxidation state. 56 Fig 10. Dependence of the reduction potential with pH. The solid line is the theoretical fit of the data to Eq 9. Cytochrome bt (n) y = 0.5 M, (•) y= 0.1 M; ( A ) Deutero-b> y= 0.1 M; (0) DME-ij y= 0.1 M. Error bars are contained within the limits of the symbol used. CD 57 The dependence of the reduction potential on pH in this case is described by the follow-ing equation: E = E + R T In Kred + [ H * ] [9] m 2.3nF K + [Hi ox The data shown in Fig 10 were fitted to Eq 9 by nonlinear, least-squares regres-sion analysis to produce the lines displayed in the figure. Parameters calculated from this analysis are given in Table VI. Temperature Dependence The temperature dependences of the reduction potentials are shown in Fig 11. The thermodynamic parameters (215) obtained from these data have been analyzed in the man-ner of Schejter et al (216). As shown above, cytochrome bs is a highly charged molecule, and as a direct consequence of this electrostatic factor, the reduction potential exhibits a strong ionic strength dependence. As analyzed by Schjeter, the free energy difference be-tween the oxidized and reduced states of the protein can be divided into two terms at zero ionic strength: an electrostatic term, AG° j , corresponding to the free energy change caused by adding one electron to the oxidized protein, and a non-electrostatic term A G ° ,, that involves formation of metal-liaand bonds, lieand-field stabilization for calcu-nonel lation of the electrostatic components are: K r e ta0x - 1 } 1 ( R P D ) [ 1 0 ] A G ° , = e2 N (1-q )/(R D) = AH° - T A S° [11] el v Moxy v p ' 1 J ASel= e2 N £ (qQx - 1) / (R D2) [12] AHel = e2 N / R (1 - qox) (a- 2f> T)/D2 [13] 58 •FlEp 3in 01 UJ S3IBbS-lSB3[ 'IB3UIJ . 'pSiqgtSM 8 3IB S3UI] piJOS -JA{ TO =rt 5<7-3Wa (0) :ro = " ^-ojsansa ( v ) -Vi ?'0 = r t (nj -Vi VO = r t (•) s'<? auioiqo - O I / Q :(3ieqdsoqd) QV Hd IB [Bnusiod uoryonpsi sqi jo souspusdsp siniEisdiusj. 'II 3 i J Table VI. Parameters Calculated from an Analysis of the pH Dependence of the Reduc-tion Potential (25 °C) . Protein Ionic Strength pK pK , E c y ox y red (A/) (mV vs NHE) Cytochrome b, 0.1 5.8 6.2 11 Cytochrome Z>, 0.5 5.7 5.9 25 Deutero- bs 0.1 5.9 6.2 -13 DUE-b, .0.1 5.6 5.8 81 The variables used in these equations are: N= Avogadro's number, e= electronic charge, R = distance of closest approach, D = dielectric constant of water, p= DebYe-Huckel con-P " stant (0.4), and a = the constant 196.9. The contributions of these electrostatic and none-lectrostatic components to the measured thermodynamic parameters as described by this formalism are shown in Table VII. Fe(EDTA)1'2- Reduction Potential The reduction potential of Fe(EDTA)>"'2" has previously been estimated to be +120 mV vs NHE (217,218). This value is valid over pH 4-6 at 0.1 M ferrous sulfate. In a more alkaline range, the potential begins to decrease. Spectroelectrochemistry was employed to measure the potential of Fe(EDTA)1"'3" at u= 0.5 M (0.25 M contributed by Nal) and 25 ° C over the pH range 5.5 to 8.0. These results are illustrated in Fig 12. Electro-chemical equilibration required about 45 min at each intermediate potential. In the absence of Nal the equilibration time increased to about 2 hr per point. Attempts to measure re-duction potentials at lower ionic strengths by this technique were prevented by extremely slow electrochemical equilibration. A listing of the midpoint reduction potential and 60 Fig 12. Dependence of Fe(EDTA)2- reduction potential cn pH [u= 0.25 M phosphate, + 0.25 M Nal, pH 7] .The solid line has no theoretical significance. Table VII. Partitioning of the Reduction Potential and Thermody into Electrical and Non-Electrical Components (pH 7.0, phosphate) Rp= 15.5 A). Protein Component E m y=0.1 M y = 0 M Cytochrome bs Obsd -37 mV Elec -80 Nonelec 43 Deutero-65 Obsd -99 Elec -82 Nonelec -17 DME- bs Obsd 8.3 Elec -116 Nonelec 124 E ° AS° AH° m y=0.1 M y=0.1 M u=0.1 M 5.1 mV -37 e.u. -11 kcal/mol -80 -5.1 -2.5 85 -32 -8.5 -44 -45 -12 -82 -5.2 -2.6 38 -40 -9.4 70 -31 -11 -116 -7.3 -3.6 184 -24 -7.4 associated slope measured at each pH value tested is given in Appendix B. The solid line shown in Fig 12 is a fit of the data to a spline type smoothing equation (Tellagraf pro-gram of Issco Graphics Software, San Diego, CA) and has no theoretical significance. A value of 92.5 mV vs NHE was obtained at the reference conditions (pH 7.0, y= 0.5 M, 25 °C) . Fe(EDTA)2" Reduction of Native and Heme-substituted Cytochrome bs First order plots of protein reduction by Fe(EDTA)2" were linear for at least 90% of the reaction in most cases. At Fe(EDTA)2- concentrations less than 2 mM for 62 cytochrome b} and 6 mM for deutero-these plots were not linear. In all cases, the DME-65 plots were linear, presumably because the reduction potential of this derivative is significantly higher than that of the other two forms of the protein. In those cases where first-order plots were not linear, examination of the spectrum of the reaction mixture after mixing in the stopped-flow apparatus revealed the presence of both oxidized and reduced protein. This finding suggested that under these conditions the reaction did not proceed to completion. Consequently, the absorbance decay data were analyzed by a non-linear regres-sion fit to a pseudo-first-order reversible rate equation (219,220): f(t) = Ajjbj {(Keq + 1) ffKeq + n + expfk, O tfl -1)} Keq [1 + (Keq + 1) exp(k2 Q t)] [14] where Q = 1 + 2/Keq and, A obs is the observed change in absorbance at time t and Keq = k2/k.2 (vide infra). The linearity and zero intercept of the plot of first-order rate constants derived by this analysis demonstrate that the assumptions are quantitatively consistent with the experimental values. The absorbance change on complete reduction of the protein in these cases was determined by adding a few crystals of Na 2 S 2 0« to the stock protein solution and record-ing (Cary 219 spectrophotometer) the absorbance change at the wavelength used in moni-toring the kinetic experiment First-order rate constants are tabulated in Appendix C for all conditions studied. The dependence of observed rates on Fe(EDTA)2" concentration was linear in all cases examined. Ionic Strength Dependence The variation in the rate of the reduction reaction with ionic strength is illustrated in Fig 13. , 63 Fig 13. Dependence of second-order rate constants for reduction of cytochrome by Fe(EDTA)'" on ionic strength [pH 7.0 (phosphate), 25 ° C ] : (n) Cytochrome i s ; (A) Deutero-MO) DME-fc, {rate x 103}. The solid lines are Tits of die data to Eq 15. These results have been analyzed by fining the data (solid lines Fig 13) to the Wherland-Gray adaptation of the Marcus ionic strength relationship (223): In k= In k - Z,Z, e; {exp(- K R3) + exp(- K RQ1 [15] • 4eeo:t fkTR 2 -- 1 + I c R l 1 + k R ^ where the pre-exponential constant evaluates to 3.576 at 25 ° C , y= 0.329 P !'-\ Ri is the protein radius, R2 is the radius of Fe(EDTA)3- (4 A.), Z2 is the net electrostatic charge on Fe(EDTA)1- (-2), R = R) + R2, and Zx is the net electrostatic charge on the protein. Values estimated for k and Zt from the non-linear least squares fit of the data to Eq 15 are presented in Table VIII. < Two other equations have been reported to describe the ionic strength dependence. Generally, the following Debye- Huckel expression (221) is applied at low ionic strengths: In k = In ko - £ K _ {111 + Z2i - (Zl + Z2);? [16] 4eeo 2kTu 1 + R ^ 1 + R 2 K 1 + R K where the constant has a value of 1.174 at 25 ° C , and the variables have the same meaning as in equation 15. Or, more recently, an equation which incorporates a factor to compensate for the dipole interaction between a protein and a small molecule has been described (222): In k = In ko + JL&L (Zi. - P,ros9 K Jk) [17] 4 e eo7T2kT ( I + K R,) eR where ?i cos 0 describes the effect of a protein dipole Pi on molecule 2 (P2 = 0) at an angle 8 to the site of interaction. Parameters estimated by a non-linear least-squares fit of the present data to Eq 16 and Eq 17 are also given in Table VIII. To illustrate the quality of the fit to each equation, a residuals plot is given in Fig 14 for the data obtained for native cytochrome A similar plot of the residuals can be shown for each of the other two proteins. 65 Fig 14. Residuals of theoretical fits to the cytochrome £><. ionic strength data for reduction by Fe(EDTA)-1- [pH 7.0. phosphate, 25 °Q. The lines are included for visual purposes onlv: ( + ) Debve-Huckel (Eq 14); (o) Wherland-Grav (Eq 15); (X) van Lecuwen (Eq 17). t o d [t-sL-kN] zi>\ ivnais3cj 66 Table VIII. Parameters Calculated from the Electrostatic Analysis of the Reduction Kinetics (pH 7.0, phosphate, 25 °C) . Debye-Huckel (Eq 16) Z, Wherland-Grav (Eq 15) van Leuween (Eq 17) k „ kn Z, kn R = 15.5 A P dipole (Debye) Cyt-fc -7.3 0.41 -13.5(8) 0.25 420(20) -12.8 0.11 266 212 Deutero- bs -7.2 0.13 -12.2(2) 0.17 139.5(2) -9.6 0.19 f 110 102 DME- bs -4.3 45 -6.9(2) 97 4310(66) -3.9 200 inf -10.2 R = 17.0 A P • Cyt-fc, -8.3 0.30 -15.6(8) 0.14 410(20) -14.3 0.08 266 235 Deutero- bs -8.4 0.09 -14.5 0.09 138(2) -10.8 0.14 111 112 DME-b5 -5.1 35 -8.0(3) 73 4270(70) -4.3 180 inf -25.2 t rate constants expressed in M - 1 s1 pH Dependence The pH dependence of the rate of reduction of all three cytochrome bs derivatives by Fe(EDTA)2" has been measured over the range pH 5.5 to 8.0. The pH dependence is reminiscent of the reduction potential pH dependence curve (Fig 15). Since Fe(EDTA)2" does not have an ionizable group with a pK that lies within the pH range studied cL (217,218), this rate dependence can be attributed to an ionizable group on cytochrome bs that is functionally linked to the redox activity of the protein. With this assumption, the following reactions may be considered: k Fe(EDTA)2- + cyt-k-FP ----> products Fe(EDTA)2" + cyt-6, > products 67 Fis 15. pH dependence of cytochrome reduction rate [25 ° C , phosphate]: (n) cytochrome 6,W(V= 0.5 M); ft) Deutero-bs (y= 0.1 M); (0) DME-fcj (u= 0.1 M) (rate * 10'5). C L 68 cyt-fc-H* <====> cyt-fc5 + H* In this case, the following relationship can be derived (81): k13 = k. [Hj + K K [18] " a "lrT]—+"^ir*~ a The parameters in Eq 18 were evaluated by a nonlinear regression fit of the data in Fig 15 to this reladonship (Table IX). The effect of increased driving force on the rate of the observed reduction reaction can be compensated for by Eq 19: k,1MJ= 6.21 x 10" exp (In k12 - 38.95 (AE) -29.45) M " 1 s'1 [19] where A E= A ^ ^ (Fe(EDTA)2") - A E ^ (protein). This relationship produces a rate constant, k*^ , that has been adjusted to a thermodynamic driving force (AE^) of zero. A deriva-tion of Eq 19 is presented in Appendix D. Significantiy, the second-order rate constants corrected in this manner were found to be independent of pH. Second-order constants (+SD, n>5) at a zero driving force, 25 ° C , were ( M - 1 s"1) 1.69 x 101 ± 0.26 for na-tive cytochrome bs (u= 0.5 M ) , 1.1 x 10° ± 0.4 for deutero- b5 (u= 0.1 M ) and 7.98 x 102 ± 0.49 for DME-b5 (u= 0.1 M ) . Temperature Dependence The activation parameters for reduction of the various cytochrome derivatives were determined from Eyring plots (Fig 16) of the rate constants observed at different tempera-tures (pH 7.0). In the case of native cytochrome bs, the temperature dependence of the reaction has been studied as a function of pH. Values calculated from these analyses are presented in Table X. 69 Fig 16. Eyring plots of the reduction rates of cytochrome by Fe(EDTA)2" (pH 7.0,^  phosphate): (n) Cytochrome b, (y = 0.5 M); (A) Deutero-fc5 (u= 0.1M); (0) DME-1 5 (u= 0.1 M). Table IX. Parameters Calculated from a Fit of the Reduction Kinetics Data to Eq 18 (25 ° C , phosphate buffer). proton Ionic Strength pK y a k a kb (M) (protonated (unprotonated form) form) (M"1 s-1) (M-1 s"1 Cytochrome bs 0.5 5.85 720 250 Deutero- bs 0.1 5.84 390 87 DME-65 0.1 5.74 4400 1800 Table X. Activation Parameters for the Reduction of the Cytochrome Derivatives in Phosphate Buffer. Protein Ionic Strength pH A H * AS* (M) (kcal/mol) (e.u.) Cytochrome bs 0.5 5.5 7.1 (2) -22.1 (8) 6.0 6.5 (1) -24.3 (5) 6.5 5.7 (2) -27.8 (8) 7.0 5.4 (2) -29.2 (8) 8.0 4.9 (2) -31.1 (5) Deutero- bs 0.5 7.0 16.8 (1) -26.5 (4) DME- bi 0.1 7.0 4.1 (1) -29.9 (2) Cytochrome bs/Deuteroheme- substituted Cytochrome bi Cross Reaction The kinetics of cytochrome bs reduction by deutero- bs (red) were studied under pseudo-first-order conditions with the reductant in excess. The electronic spectra of oxi-dized and reduced deutero- bs were recorded, manipulated by the North Star computer system described above, and the ordinate axis was converted to an extinction coefficient 71 scale. The difference spectra (reduced - oxidized) were computer-generated at three dif-ferent protein concentrations (10, 20 and 30 y M) and are shown in Fig 17. In a similar manner, the simulated difference spectrum for cytochrome 6, at 1 y M is also shown as the dashed line in Fig 17. Analysis of this figure indicates that the optimal wavelength at which to monitor the reaction is 429 nm. This wavelength is an isosbestic point for deutero-6S. Also, the signal strength from a change in the oxidation state of cytochrome bi against the background absorption is sufficient to detect even when the reductant is in 30 fold excess. Ionic Strength Dependence The variation of the second-order rate constant for the reaction between native cy-tochrome bs and deutero- bs with ionic strength is shown in Fig 18. The data were fitted to the Wherland-Gray relationship (Eq 15) and also to the relationship recently described by van Leeuwen (Eq 20) (223) to fit kinetic data collected at a given ionic strength for the reaction between two charged proteins each with a net dipole moment: In k = In k°° - (Z,Z2 + (ZP)(1 + K R) + (PP)(1+KR)2) q2 f(k) [20] x q 2/(4TTe eo k T R) / (u) where ZP = (Zi P2 cos 6 2 + Z2P,cos 6 ,) PP = P, P2 cos 6 ,cos 6 :/(q R)2 f(k) = (1- exp(-2KR2))/(2 < R2(l+< R0) and, q is the elementary charge, P (in Debye) is the dipole moment of the protein which interacts at the angle and all other variables are the same as previously defined. The dashed line of Fig 18 is the non-linear regression fit of the data to Eq 20 where 72 Fig 17. Computer simulation of difference spectra of deutero-63 and cytochrome b$ (re-duced - oxidized). Cytochrome bs, 0.1 M (- - -);Deutero-i5 at 10, 20 and 30 uM ( ). The extinction coefficient for the changes in absorbance at the deutero- b5 isosb-estic points are 5.0 M" 1 cm"1 (405 nm) and 70 M" 1 cm-1 (429nm). A 4 0 4 . 5 4 0 5 . 5 4 2 7 4 2 9 4 3 1 WAVELENGTH (nm) 73 Fig 18. Dependence of the reduction rate of cytochrome bs (ox) by deutero- b$ (red) ionic strength (pH 7.0, 25 ° C ) . Theoretical fits as described in text: (- - -) Wherland-Gray.Eq 15; (— - —). van Leeuwen ,Eq 17. the variables have the following values; Zi= -6.2, Z2 = -6.5, R i = R2 = 15.5 %. and k = 5015 M ' 1 s"1. With this analysis, P,cose,= 109(14) Debye and P2cose2 = 67(56) Debye. ^Results of the analyses based on Eq 15 and Eq 20 are collected in Table XI. Temperature Dependence The temperature dependence of this cross-reaction is shown in Figure 19. The thermodynamic parameters calculated from a weighted least-squares analysis indicates that ± ± AH - 1 6 + 1 kcal mol"1 and AS — 11 + 4 e.u. Autoxidation of cytochrome bs fol-lowing its reduction was not found to be a problem as the observed decay could be fitted quantitatively to a first-order process. The plots were linear for at least 90% of the reaction. Dependence of apparent first-order rates on the concentration of ferricytochrome bi was linear and extended through the origin with no evidence of rate saturation. A compilation of all first-order rate constants is given in Appendix E. All of these data were obtained with deutero- bs reduced by the photochemical technique. With the platinum black technique, a substantial proportion of ferrodeutero- bs underwent autoxidation during the 25 min thermal equilibration period despite the precau-tions described previously. The photoreduction method was successful in maintaining the deutero- bs in the reduced state regardless of the length of time it remained in the syringe barrel. In control experiments, it was found that deutero- bi would not reduce in the absence of acridine orange and that at least 10 mM EDTA was required for complete reduction in 15 min. NMR Spectroscopy of Native and DME-ft5 The pH dependence of the proton NMR spectrum of oxidized cytochrome bs is illustrated in Fig 20A. A similar analysis of DME-fc5 is illustrated in Fig 10B. The de-pendence of heme-ring hyperfine shifts on pH is illustrated graphically in Fig 21. 75 76 Table XI. Alternate Analyses Tor Electrostatic Correction of the Reaction Between Native Cytochrome 6} and Deutero- bs (pH 7.0, phosphate, 25 ° C ) . Debve-Huckel (Eq 17) Wherland-Grav (Eq 15) van Leeuwen (Eq 20) z , t z . z , t z 2 k 1 2 ± (M-« s-z , t ') z , t Dipole 1 (Debye) Dipole (Debye) R =15.5 A P -12.2 -1.71 -12.2 -3.2(0.6) 5015 -12.2 -3.2 143(108) 59(7) -6.9 -2.0 -6.5 -6.2(1.1) 5015 -6.5 -6.2 67(56) 109(14) -3.3 -3.3 -5.3 -7.8(1.4) 5015 -5.3 -7.8 47(46) 137(19) R =17.0 A P -3.3 -4.0 -14.5 -3.4(0.7) 4889 -14.5 -3.4 174(128) 64(7) -3.7 -3.7 -6.6 -7.9(1.5) 4889 -6.6 -7.9 59(58) 147(18) -5.3 -10(2) 4889 -5.3 -10 43(46) 183(24) t a fixed variable in the analysis X at infinite ionic strength 77 Fig 20. NMR spectra of the low-field portion (-9 to -30 ppm) of cytochrome b< (\) 6 53 (c) noid)BUl(t) Ul = M ' d e m e r a l C d P h 0 S P h a t e ^ : P D values arc (a) 5.74.(b) CO E Q a S a a. 78 Fig 21. Hyperfine shifts, of selected peaks in the low-field NMR spectra of cytocrome bs (A) and DME-fc, (B). Conditions as in Fig 20. 79 D I S C U S S I O N Oxidation-Reduction Equilibria of Native and Heme-Substituted Cytochrome bt General Considerations The structural characteristics of the apo-protein environment thai control the oxidation-reduction equilibrium of the heme group have been the subject of extensive in-vestigation for many years. Several critical factors have been identified, many of which have been summarized by Moore and Williams (224). These factors arc as follows: 1. Dielectric constant of the heme pocket (Kassner (225)) / heme exposure (Stel-lwagen (53)) 2. Electrostatic charge on axial ligand 3. TT -Acceptor power of axial ligand 4. Spin-state change on redox interconversion 5. Steric factors favoring oxidized vs reduced form 6. Special electrostatic interactions (eg heme propionate stabilization) The magnitude of each parameter in the control of the reduction potential in a particular case is difficult to quantify. It is not yet possible to partition the para-meters, vary each independentiy and determine the relative effect of each factor. Spectroscopic and crystallographic analyses of cytochrome bs permit a qualita-tive examination of the factors which control the reduction potential in this heme-protein. The two histidine ligands are fixed in a nearly coplanar orientation by hy-drogen bonds to the polypeptide backbone. This alignment of ligand planes is consi-stent with a preferential derealization of unpaired electron spin from the oxidized iron into the TT orbital system of the porphyrin ring (227). This stabilization contri-butes to a lower reduction potential. The coordinated histidine also interacts electron-ically with the' iron atom. As the strength of the hydrogen bond between the histi-dine N 6 1 nitrogen atom and the polypeptide backbone increases (equivalent to de-protonation of the imidazole ring) the electron density at the coordinating Ne 1 ni-trogen atom also increases to provide stabilization of the ferric state relative to the 80 ferrous (226,228-230). It had been suggested, based on ESR data, that the histidine of cytochrome bi is fully deprotonated (231). However, X-ray crystallography, MCD spectroscopy (232) and NMR spectroscopy (N5l-hydrogen, bond) are consistent with the presence of an off-axis hydrogen bond (233). With fully deprotonated histidine, a strong bond between the N61 atom and the iron is associated with a low spin state on the iron (234). The postulated weak hydrogen bond in cytochrome 65 is consistent with the observation that cytochrome bs is low spin in both the oxidized and reduced states (pH 7, 25 ° C ) (68,80). Kassner proposed that the reduction potential of a heme group would be modulated by the dielectric constant of the surounding solvent (225,229). However, the content of hydrophobic and aromatic acids lining the surface of the heme cre-vice of cytochrome bi was found to be nearly identical to that of other c-type cy-tochromes, hemoglobin and myoglobin (53). A wide range (5-320 mV) of reduction potentials is exhibited by this group of hemeproteins. Stellwagen has attributed the low reduction potential of cytochrome bs to the relative great exposure of its heme group to the solvent (53). These findings are indicative of an heterogenous dielectric constant surrounding the heme group in cytochrome b$. Kassner's model was deve-loped to explain the redox behavior of heme in an ideal, homogeneous shell with a low dielectric constant and consequently, it is inadequate to accurately predict the reduction potential of cytochrome bs. In addition to "the general effect exerted by the hydrophobic environment of the heme crevice on the reduction potential, other specific interactions may be of importance. Amino acid side chains within van der Waals contact distance may in-fluence the electronic distribution in the heme ring. In cytochrome bs, many aromatic and nonpolar residues line the crevice and interactions from them are difficult to evaluate either qualitatively or quantitatively. Long- range electrostatic interactions can occur in a material of low dielectric constant, such as the heme crevice (52). In particular, as discussed previously, Mathews has suggested that the heme propionate 81 is important in coulombic stabilization of the oxidized iron (59). Cytochrome b<. • An increase in reduction potential by 60 mV between cytochrome b; and DME-iu was observed under standard conditions. This is taken as evidence in sup-port of the Argos-Mathews model; as a consequence of esterificatipn of the heme propionate, the proposed coulombic stabilization of the ferric protein would be abo-lished and the reduction potential would increase. A similar shift"-in reduction poten-tial ( + 47 to +65 mV) which is observed in certain c-type cytochromes has recently been ascribed to the protonation of heme propionate 7 (236). Electron derealization from the iron to the heme TT orbital system is greater when electron withdrawing substituents are present on the heme periphery. In aque-ous solution, the replacement of the electron-withdrawing vinyl groups on the heme with protons (deutero-heme) is accompagnied by a 50 mV increase in potential to -190 mV (protophorphyrin IX E^= -240 mV) (237). However, when deutero-heme is incorporated into myoglobin (237) or cytochrome bs the potential difference is both reduced in magnitude and different in trend (-25 mV difference in myoglobin). The 40 mV decrease in potential observed in deutero- bs relative to the native pro-tein dramatically illustrates the extent to which potentials are dictated by complex forces within die protein structure rather than by the heme moiety alone. Ionic Strength Dependence The observed change in reduction potential with ionic strength arises from two factors. The first, is a general electrostatic effect on the charged redox center and the second, is due to the specific binding of a cation to the reduced form of the protein. The failure to demonstrate a dependence of the reduction potential on Ru(NH 3y'' J* presumably results from, the inability of the redox-linked site to 82 accomodaie cations of this size. A non-specific increase in reduction potential by the mediator was not observed because the change in ionic strength caused by the me-diator was negligible in comparison to that of the supporting electrolyte. The net charges estimated for cytochrome bs and for deutero- bs from the ionic strength dependences of their reduction potentials (Table V) are consistent with other estimates only when the full protein radius is used in Eq 8. In this regard, these results agree with previous findings for horse heart cytochrome c and are at variance with those reported for cytochrome c from Euglena (238,210). One empirical observation may be of relevance. An over-estimate of the net 'charge results when the full protein radius is used in the ionic strength function for either Euglena cy-tochrome c or DME-ij- A trial and error approach indicates that a value equivalent to the net charge estimated from the sequence analysis can be calculated when smaller radii are employed. However, the difference between the protein radius and the value used for the calculation is not equivalent for the two examples. Uncer-tainty in this approach arises from simplifying assumptions required when estimating the net charge from the amino acid sequence. In both Euglena cytochrome c and DME- bi the binding of a cation is not part of the reductive cycle. In addition, the protein microenvironment may significantly alter pK values of the amino acid side a chains (239,240). Analysis of the effect of the microscopic environment on the proton titration curves has only recently been reported for lysozyme (241). The experiments reported here were performed by necessity at ionic strengths outside the range normally regarded as valid. A rigorous Monte-Carlo evaluation of experimental results obtained with monovalent electrolytes suggests that the Debye-Huckle equation may be appropriate, under certain conditions, up to 1.5 M (242,243). The ability of Eq 10 to fit the current data supports its application in this case. However, a better understanding of the role of the ionic radius in this equation is clearly needed. 83 pH Dependence The poieniiometric daia indicate that protonation of cytochrome £>< produces a slight increase in its reduction potential. From the poieniiometric data for the native protein alone it might appear that the heme propionate, which hydrogen bonds io the surface of the protein, would be responsible for this effecL An examination of the proton NMR titration data (Fig 22) indicates that the protonation status of the propionate does affect the hyperfine shifts in the native proiein. The complete ab-sence of such shifts in the DME-6 5 derivative is strong evidence for the electrostatic interaction of the propionate group with the heme iron. However, the demonstration that DME-is exhibits an identical dependence of E ^ on pH to that of the native protein argues strongly against a role for the propionate in the observed pH depen-dence of the reduction potential. Studies on horseradish peroxidase and c-type cytochromes suggest a mechan-ism by which this observation may be accounted for. The reduction potential of the heme iron is very sensitive to both the coplanarity of the axial ligands and the strength of the hydrogen bond (vide supra). NMR studies of horseradish peroxidase indicate that titration of a group other than the heme propionate affects the axial ligand field strength and consequently, the reduction potential in this hemeprotein (230). In c-type cytochromes recent NMR studies have identified two types of redox pH dependences (229,244,245). The first involves protonation of the heme propionate and is accompanied by an increase in potential of some 60 mV. The second class exhibits a smaller' change in reduction potential ( + 23 mV) upon the protonation of a surface histidine. It is unknown if an alteration in a long-range electrostatic in-teraction between the histidine and the iron is sufficient to account for this observa-tion. An alteration in the axial ligand field strength may also be involved. The change in reduction potential observed with cytochrome bs and its derivatives is +20 mV. By analogy to the studies on c-type cytochromes His.^, located on the surface of cytochrome bi near the heme crevice, may be responsible for this effect An 84 examination of the pH dependence of ihe reduction potential of cytochrome b: or sulfite oxidase where the homologous surface histidine has been substituted by Asn and Gly respectively, might be informative. Temperature Dependence The thermodynamic parameters for the cytochrome bs oxidation-reduction equilibrium permit a more detailed analysis of the mechanism by which this protein changes oxidation state. By restricting the comparison to data obtained for six-coordinate, low spin hemeproteins using a similar experimental method, the dis-cussion is limited to a few measurements of c-type cytochromes (215). The most striking feature of these data is that A H° for cytochrome bt is 4-5 kcal/mol more positive than that observed for cytochrome c while A S° is approximately 6 e.u. more negative. A direct comparison in this manner has been criticised for its failure to account for the electrostatic component inherent in the observed thermodynamic parameters (216). In horse heart cytochrome c, the non-electrostatic components (cal-culated at q = 5.1, pH 7.0) are A S° .= -33.5 e.u. and A H ° .= -14.3 o^x y ' nonel nonel kcal/mol. Given this approach (Table VI) the difference in enthalpy is 6 kcal/mol whereas the entropy difference is substantially reduced. A consistent non-electrostatic enthalpy value for cytochrome bs and its derivatives would argue that the observed difference with cytochrome c is due to redox linked conformational changes in the latter protein, and not to a coulombic stabilization of cytochrome bs by the heme propionate. Significandy, the non-electrostatic entropy component is different for each de-rivative. Relative to native cytochrome bi, the deutero-65 derivative exhibits reduced standard entropy. With this derivative, a degree of "free space" in the interior of the protein may accompany the substitution of vinyl groups on the heme ring with hydrogen atoms. A "tightening" - of the protein's conformation upon reduction could account for the observed difference in entropy. Notably, the nonelectrical entropic 85 componeni of temperature dependence is 8 e.u. less negative for DME-6.< than that for the native protein. This observation can be explained by the inability of reduced DME-k to bind a cation at the heme propionate as presumably occurs in the na-tive protein. Fe(EDTA)!" Reduction of Native Cytochrome bs General Considerations Electron transfer between a donor (D-) and an acceptor (A) can be de-scribed in simplest terms by the following series of steps (246): -ki k, k3 A + D- < = = >. AD- < = = > - A D <==> - A + D k-s k.3 With cytochromes, this reaction is studied by following changes in the electronic ab-sorption spectrum of the protein, which occurs during the electron transfer step (k2/k.j). Under pseudo-first order conditions ([A]<[D]), a linear dependence of kobsd on [D] indicates that electron transfer is the rate determining step. "Saturation kinetics", in which the observed rate levels off at high concentrations of D-, can arise by several mechanisms (247). The formation (kj/Li) or dissociation (k3/k_3) of the activated complex may not be rapid steps, or alternatively, a nonproductive "dead end complex" may be formed between the reactants and lead to complex reduction kinetics. In the case where the driving force for the forward reaction is low, k_2 may be important in determining the overall reaction rate (so called pseudo-first-order reversible kinetics). The reduction kinetics of many metalloproteins have been studied with Fe(EDTA)1" as the reductant (174,248). As discussed in the introduction the rate of protein metal center reduction with this reagent reflects the degree of exposure of the protein's active site to the solvent. Comparative experiments with several different 86 proteins (248) have now established this reaction as a means by which to estimate the "kinetic accessibility" of a given protein (174). Cytochrome b$ Ionic Strength Dependence Interaction of a protein with a charged small molecule as a function of ionic strength is governed by multiple effects (248,249). At very low ionic strengths (<10mM) and in aqueous media, the electrostatic field generated by the charged re-sidues on the surface of the protein is spherical. With increased electrolyte concen-tration, the electrostatic field is reduced as counter-ions interact with the exposed charges. The electrostatic field thus assumes a shape based on the non-homogeneous distribution of these charged residues. At an ionic strength greater than 0.1 M, the electrostatic field associated with the protein net charge (the monopole) is greatly re-duced, and the dipole moment and higher-order moments will predominate (250). At infinite ionic strength, all electrostatic contributions are eliminated, and the reaction rate is dictated by the reduction potential and steric constraints on the interaction of the reductant with the active site (248,249). A careful analysis of the dipole moment and the electrostatic field surround-ing cytochrome c has been reported (249). From this analysis, electrostatic forces ap-pear to be such that a negatively charged oxidant or reductant is drawn towards the heme edge at physiological ionic strengths (ca 0.1 M). Neutralization of the positive residues surrounding the heme crevice by chemical modification shifts the dipole away from the heme edge and results in a reduced reaction rate with negatively charged molecules (249). Similar calculations have not been reported for cytochrome but from an examination of the crystal structure suggests that the constellation of negative charges near the exposed heme should have a similar, though opposite ef-fect 87 A quantitative analysis of the ionic strength dependence data was performed as described above. A fit of die data to the full Debye-H'ucke! equation was found to diverge rapidly at an ionic strength greater than 0.1 M. A successful fit of the data above 0.1 M ionic strength (Fig 14) was achieved only with the Wherland-Gray approach (Eq 15) or the method of van Leeuwen (Eq 17). The net charge values estimated for cytochrome bs and deutero- b} by either the Wherland-Gray or van Leeuwen equations are in serious conflict with estimates from the ionic strength dependence of their reduction potentials (Table V) or from the pH titration (Fig 7). In contrast, the discrepancy with the DME~6j derivative is si-gnificantly reduced. In the DME-Z^ derivative, the charge at the heme edge is de-creased by two, and the discrepancy between the calculated charge and the sequence charge is decreased presumably because of reduced electrostatic interactions. These re-sults provide reasonably direct evidence that electron transfer occurs through the ex-posed heme edge of cytochrome 65. The cytochrome 65 dipole moment (Picos Q ) calculated by the fit of these data to Eq 17 (212 Debye) is smaller than previously-estimated (570 debye) (223). In addition, the dipole moments of the derivatives are reduced. The accuracy of these estimations will be clarified ultimately when the di-pole moment is calculated after the manner of Koppenol et al (249,251). It may be argued with some validity that use of the Wherland-Gray rela-tionship to estimate the net electrostatic charge on a protein from small reagent re-dox kinetics data is an overextension of its originally intended use. In fact, the changes determined from this type of analysis were not initially claimed to have any physical signifiance. Rather, they were simply means by which the contribution of electrostatic interactions between proteins and small molecule redox reagents to the rate of reaction could be evaluated quantitatively. The success of the equation in fitting a wide body of data of this type (174,248), attests to its usefulness in this regard. 88 pH Dependence The mechanism of reduction with Fe(EDTA);~ at the heme edge is unaf-fected by titratable groups over the range pH 5.5-8.0. This conclusion is based on the following observations. An isokinetic relationship (Fig 22) exists for the activation parameters {vide infra) associated with the cytochrome bs reduction reaction over the pH range 5.5-8.0. This relationship is linear in either the A H + - A S T plane or in i ± the A H • - A G + plane (not shown) (252,253) and suggests that the structure of the transition-state of this reaction is invariant with pH. Therefore, the origin of the observed reaction rate dependence on pH lies in the variation of reactant reduction potentials with pH, in the variation of protein-reagent electrostatic interactions with pH or both. The thermodynamic driving force of the reduction reaction is the dif-ference between the reduction potentials of the reactants. Both Fe(EDTA)2" (Fig 12) and cytochrome bs (Fig 10) exhibit a dependence in their reduction potentials with pH. As seen previously the observed pH dependence of the reduction reaction rate can' be accounted for quantitatively by correcting for differences in the reactant re-duction potentials. Titration of the heme propionate group, estimated from NMR data to occur with a pK of 5 (Fig 21), would be expected to change the electrostatic environment at the active site and thus influence the reduction rate. However, this cannot be experimentally confirmed owing to the instability of the protein below pH 5.5. As reported above, the second-order rate constant adjusted to a zero driving force increases substantially for the DME-6 5 derivative over that of the native pro-tein or deutero-65. Enhanced orbital overlap between Fe(EDTA)2- and DME-fc5 can be attributed to three factors: decreased electrostatic repulsion; loss of hydrogen bonds which results in disorder and a greater exposure of the prosthetic group to the solvent; and a "gap filling" role of the methyl group, which decreases the dis-tance of electron transfer and thus raises the adiabatic nature of the reaction (254). The relative contribution of each factor to the increased reaction rate cannot be 89 Fie 22. Compensation plot for the reduction of cytochrome b, by Fe(EDTA)3' (y- 0.5 M!phosDhatc):(o)pH 8.0;(A) pH 7.0:(n) pH 6.5;C) pH 6.0;(A) pH 5.5. In each case [Fe(EDTA);-] was 1.25 x 10':M. The solid line is a linear weighted least-squares analysis of die data. T I 1 1 1 1 I I LQ O LQ O LO O K K <S <5 ( | O U J / | D 3>l)jHV 90 determined at this time. Temperature Dependence The activation parameters measured from the temperature dependence of the reduction reaction may also be regarded as composites of electrostatic and non-electrostatic components (255). However, no method of partitioning these com-ponents has been reported. The relative consistency between activation parameters for cytochrome b<, may indicate relatively similar processes are involved in the formation of the activated complex. Analysis by electron tunneling theory (vide infra) supports this position. Compared with similar data for the Fe(EDTA)2" / cytochrome c reac-tion, the activation entropy for cytochrome b$ is relatively large and negative. This result alone suggests that this observation could arise from the binding of a cation to the cytochrome bs heme propionate. This explanation appears to be incorrect for three reasons. First, DME-bs has the same activation entropy as the native protein. Second, the cytochrome c parameters were not measured under identical solution conditions, so the electrostatic component may account for some of the observed dif-ference. Finally, analysis of the electron transfer data by electron tunneling theory (vide infra) predicts activation parameters which are close to those observed. Theoretical Analysis of Reaction Rates Marcus Theory The apparent protein self-exchange rate, k n c o r r , estimated by an application of the Marcus relationship (Eq 1) to the reduction rate data determined with Fe(EDTA)2-, is thought to yield mechanistic information about the interaction of the reductant with the protein (174). As described in the introduction, the calculated self-exchange rates, after correction for ionic strength effects, should agree to within an order of magnitude if equivalent mechanisms are operative. A compilation of the parameters associated with this calculation for cytochrome i 5 and its derivatives is 91 presented in Table XII. A comparison between k n values, in which compensation is made for all electrostatic efects, calculated from the reaction of other hemeproteins with Fe(EDTA) ;\ is (M _ 1 s"') 1.2 x 10' for cytochrome c and 2.3 x 10! for cyto-chrome C J J I . The method of Mauk, Scott and Gray (256) has been used to estimate the distance from the point of electron transfer within the protein to the protein surface from the reduction kinetic rate at pH 7.0 and 25 ° C (Table XII). A comparative analysis for the proteins listed above is 3.4 A. for cytochrome c and 4.0 for the cytochrome c3 j l. In the current work the increase in reaction rate with DME-6 5 can be correlated with a decrease in the distance (by 0.7 A.) across which electron transfer occurs. Electron Tunneling Theory Analysis of the Fe(EDTA)3" reduction reaction data by the multiphonon ele-ctron tunneling relationship is shown in Table XIII. All parameters are calculated at infinite ionic strength. Consequently, values for the reduction potentials extrapolated to infinite ionic strength were employed. The vibronic coupling parameter for Fe(EDTA)2" was taken as 0.5 eV as previously described (257). The average vibronic coupling parameter for cytochrome bs and its derivatives was estimated as 0.45 eV from its activation enthalpy (Eq 4). In keeping with a previous analysis of cyto-chrome c the vibronic coupling parameter was fixed at 0.5 eV (258). As a first ap-proximation, the distance of electron transfer in the hemeprotein was taken to be that calculated by the Mauk, Scott and Gray approach. An average value of Rp = 2.8 A for the native and deutero- bs protein predicts values for the rate constant and activation parameters which are in remarkably close agreement with the experimental data. This finding argues strongly for an equivalent active site between these two proteins. With DME-fc5, a shorter electron transfer distance is accomodated within the theory. 92 Table XII. Marcus Analysis of Fe(EDTA);"reduction rate data INPUT k„ El Z, Units mV vs NHE Cytochrome bi Deutero- b 113 5.1 -13.5 36.4 -44 -12.3 OUTPUT . corr kn \vl2 w21 w l l A ' G i | C O r r A G 0 f factor K eq oo K „ 00 oo A G n E ( *») m v ' M" 1 s"1 kcal/mol kcal/mol kcal/mol kcal/mol kcal/mol M" M" kcal/mol mV vi- NHE 3.7 1.15 0.618 2.04 16.7 2.02 0.018 0.0033 116 790 14.6 -64.3 3.85 1.05 0.567 1.70 16.6 3.15 0.028 0.0049 67.5 213 14.9 -115.6 rpt A 2.7 2.9 t protein radius calculated as described in text (ref 256). 93 Table XIII. Electron Tunneling Analysis Reaction pair (molecule a {oxidized} / molecule b {reduced}) (A) (A) (B) (B) (Q (C) (D>- (E) AHobsd (a) 5.4 5.4 6.8 6.8 4.05 4.5 n.d. 14.5 R P (b) 15.5 15.5 15.5 15.5 15.5 15.5 17 15.5 E inf * a (c) 92.5 92.5 92.5 92.5 92.5 92.5 21 -30 E, inf b (c) 21 21 -30 -30 89 89 260 21 Na 6 6 6 6 6 6 20 20 Nb 20 20 20 20 20 20 20 20 Va est (d) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Vb est' (d) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ra est (b) 2.7 2.8 2.9 2.8 2.1 2.8 2.0 2.8 rb est (b) 4 4 4 4 4 4 2.8 2.8 V est (d) 0.925 0.925 1.05 1.05 0.86 0.86 n.d. 2.65 V used (d) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 | Tabj2 (d) 3.9E-6 3.4E-6 2.9E-6 3.4E-6 9.3E-6 3.4E-6 1.8E-5 5.7E-6 kinf calc (e) 484 426 125 142 4363 1776 3.9E5 6953 k obsd (e) 420 420 140 140 4301 4301 3.1E5 5015 A H * calc (a) 5.8 5.8 6.5 6.5 4.93 4.93 2.28 4.27 AH*obsd (a) 5.4 5.4 6.8 6.8 4.05 4.05 n.d. 14.5 As*calc (0 -26.9 -27.1 -27.1 -25.3 -27.1 -27.1 -24.5 -26.5 AS*obsd (0 -29.2 -29.2 -27.9 -27.9 -25.2 -25.2 n.d. 6.3 + data of Millet et al. Ref 266. *' potential estimated at infinite ionic strength estimated value, see text (a) Kcal/mol;(b) A;(c) mV vs NHE;(d) eV;(e) M _ I s-';(f) e.u. (A) cyt-VFe(EDTA)2-;(B) deutero- VFe(EDTA)2-;(C) DME- bs /Fe(EDTA)2-; (D)cyt- c/cyt- bs;(E) cyt- bb/deutero- b}. 94 Metalloprotein Electron Transfer Self-exchange Reactions General Considerations Direct measurement of metalloprotein electron transfer self-exchange rates is not possible with optical techniques because the spectra of the reactants and products are idendcal. Consequentiy, the only meaurements of this type available for metalloproteins have involved NMR techniques such as paramagendc line broadening (cytochrome cS51 (259)) or saturation transfer (cytochrome c (260-262)). The self-exchange rate in protein-protein systems has been most extensively inve-stigated for the case of cytochrome c (260,262). At low (<50 uM) ionic strength the rate, as determined by NMR studies, was maximal (ca 2 x 104 M" 1 s_1) at pH 10, the isoe-lectric point of the protein. This rate was very dependent on pH and ionic strength which indicates that a significant electrostatic factor is operative during the formation of the complex between cytochrome c molecules. Optical methods have also been employed to measure the rate of electron transfer between cytochrome c type molecules from different species (189,263). These results are consistent with the previous estimates from NMR stu-dies. The present work develops an alternative approach in which the cross-reaction be-tween two derivatives of a protein is studied as an approximation to the self-exchange case. In this approach, it is critical that the modified protein be as functionally similar to the native protein as possible. Consequentiy, the reaction of cobalt-substituted cytochrome c with native cytochrome c as studied by Chien (258) is not a reasonable simulation of the cytochrome c self-exchange reaction because the Frank-Condon activation barriers for the cobalt-substituted protein will be much greater than that for the native protein. 95 Cytochrome b$ Rationale for Experimental Design The deutero- 65/cytochrome bi couple is ideally suited for a determination of the cytochrome 65 self-exchange rate for the following reasons. First, the exposed heme edge is equivalent in each protein: the vinyl groups of protoporphyrin IX which are modified in the deutero-£5 derivadve are located in the interior away from the surace of the pro-tein. Reduction by Fe(EDTA)3" at the active site uses the same mechanism in reaction with each protein, as indicated by the equivalence of the k u 0 r T values calculated for each protein. Second, the thermodynamic driving force difference between the proteins is small (ca 20 mV) under all solution conditions. Finally, a significant shift (10 nm) in the ele-ctronic absorption spectrum of deutero- b5 relative to that of the native protein allows the reaction between the two proteins to be monitored optically. Ionic Strength Dependence The rate of electron transfer between native cytochrome b5 and deutero- bs increases significandy with ionic strength as expected for a reaction between two large, similarly charged proteins. The van Leeuwen equation (Eq 20), which describes the ionic strength dependence of two proteins with significant dipole moments would not converge if more than two variables were estimated. Consequentiy, the net charge of the reacting species was estimated at -5.3 from a plot (not shown) of the cross-reaction rate at low ionic strength as discussed by van Leeuwen. This estimate of the net charge is consistent with previous measurements {vide supra). The dipole moments calculated from a fit of Eq 20 to the protein-protein cross-reaction data are in reasonable agreement to the estimates from the protein-small molecule cross-reaction (Tables IX and XI). 96 Temperature Dependence The activation parameters for the protein-protein cross-reaction are significantly more positive than values determined for the protein-small molecule cross- reaction (Tables VII and X). The model of Clothia and Janin (264) for protein-protein complexes ascribes the stability of protein association to an extensive change in solvent structure accompanying the reduction of protein surface area exposed to solvent that occurs during an interaction between the proteins. In this case, other factors such as electrostatic charge-pair interac-tions, hydrogen bond formation and conformational rearrangements are not expected to sta-bilize the complex (265). Theoretical Analysis of Reaction Rates The cross-reaction rate between cytochrome bs and deutero- b5 after correction to infinite ionic strength (Eq 15) and zero driving force (Eq 19) is 7.0 x 102 ± 0.4 M" 1 S' 1 . From the reaction with Fe(EDTA)2", k n = 1.4 x 102 M" 1 s'1. At 0.1 M ionic strength the discrepancy is larger: 3.8 x 102 M" 1 s~' for the cross-reaction rate and 3.7 x corr 10° M" 1 s"1 for k n . A similar discrepancy has been observed with other redox active proteins (189). However, the self-exchange rates estimated from a variety of protein-protein reactions were consistent, as opposed to values estimated for the cross-reactions of the protein with a variety of small molecule reductants. This finding has been interpreted to mean that each protein employs only a single mechanism during electron transfer to another protein redox active center. The protein-protein based self-exchange rate of cyto-chrome bi has been estimated at 6.0 x 102 M" 1 s"1 (u = 0.1M, pH 7, 25 ° C ) by Mar-cus theory (Table XIV). In this calculation it was assumed that both deutero-i5 and the native protein have identical heme edges and hence identical protein-protein self-exchange rates (kn = k22). The consistent self-exchange rate constants estimated for other hemeproteins under similar conditions are (189); k n (M - 1 s~') = 2.8 x 10s for cytochrome da, 4.6 x 107 for cytochrome c5SU and 1.5 x 102 for cytochrome c. It will be necessary to measure the cross-reactivity of cytochrome bi in other systems to determine if it also exhibits a 97 Table XIV. Marcus Theory Analysis of the Cross-Reaction Rate Data (u= 0.1 M). Values from Trial and Error Analysis Previous Analysis to Estimate Self-Exchange Rate INPUT z, -6.2 -6.2 z, -6.2 -13.5 -6.4 -5.3 -5.2 -7.3 -13.5 z 3 -7.5 -13.2 z 2 -7.5 -13.2 -7.4 -6.3 -7.5 -8.2 -13.2 1545 1545 kn 0.65 0.65 600 600 600 600 600 k 2 2 4.31 4.31 k 2 2 4.31 4.31 600 600 600 600 600 OUTPUT wl2 .487 .487 wl2 .487 1.87 .496 .350 .411 .627 1.87 w21 .490 .490 w21 .490 1.85 .496 .350 .425 .626 1.85 wll .467 .467 wll .467 2.05 .496 .350 .342 .634 2.05 w22 .510 .510 w22 .510 1.69 .496 .350 .510 .618 1.69 kn 8.5E4 4.5E4 k» 4.32 4.38 1542 1543 1565 1542 1563 kn 1.9E5 9.9E4 k I 3 3515 6566 k 2 2 10.2 74.3 AG° ° 10.3 10.6 A G 1 2 16.6 16.6 13.1 13.1 13.1 13.1 13.1 OO A E 48.8 46.2 A G „ 10.7 11.1 f -.01 -.008 -.008 -.008' -.01 -.01 -.01 -.01 -.01 Note: units are the same as in Table XII. 98 single cross- reaction rate. The cross-reaction rate has also been analyzed by electron tunneling theory. For comparadve purposes the interacdon of cytochrome c (ox) with cytochrome bs (red), as reported by Millet and co-workers (266), has been analyzed in the same manner and the results are also reported in Table XIII. The close agreement between the calculated and observed reaction rates for these protein-protein cross reactions confirms that the correct values for the vibronic coupling parameter and the electron transfer distance of cytochrome bi have been determined. The failure of electron tunneling theory to accurately predict the thermodynamic parameters of the cross-reaction presumably arises from factors not accounted for by the theory such as solvent reorganization which are important in the formation of the activated complex between two proteins (262). Finally, the close correlation between observed and calculated reaction rates for the deutero- bs/cytochrome bt electron transfer is taken as evi-dence that the presence of the photo-activated reducing system did not influence the re-action mechanism. Significance of Results Calculation of the degree of heme exposure in cytochrome bs by Stellwagen indi-cates that the heme moiety is very accessible to the solvent (23%). A relatively large self-exchange rate would be predicted on this basis as compared to cytochrome c which has a relatively low degree of heme exposure (4%). The results presented here are incon-sistent with such a prediction. Presumably the slowness of the cytochrome bs self-exchange arises from the concentration of negative charges near the exposed heme edge. Electrostatic repulsion between these sites probably accounts for the slow self-exchange rate. That the reaction does occur attests to the importance of hydrophobic interactions and solvent rear-rangements which occur at the interface of the protein-protein surfaces. These factors are poorly understood (264). 99 Conclusions Rapid electron transfer between cytochrome bs and its physiological partners is de-pendent upon chemical and structural variables. As clearly shown by this work the heme propionates can funcdon to control electron transfer, the significance of this finding has not been fully recognized before this work. First, the propionate which hydrogen bonds back to the protein surface appears to modulate the reducdon potendal by some 60 mV which is equivalent to a relative decrease in the stability of ferricytochrome bi by ca 1.5 kcal/mol. Consequentiy, the thermodynamic driving force for reactions in which this cyto-chrome participates is regulated by the presence of this propionate. The heme group also affects the distance over (or, the mechanism by) which electron transfer can occur. With negatively charged reagents the electrostatic repulsion afforded by the exposed heme prop-ionate increases the transfer distance by 0.7 A and hence lowers the reaction rate. And finally, the deuteroheme substituion technique has proven very successful in measuring the self-exchange of cytochrome bi. The ease of operation, speed of analysis, and general ac-cessibility of this technique should generate widespread interest in determinations of the self-exchange rate of heme-exchangeable proteins. The sensitivity of the optical method, as compared to NMR techniques is also of interest for determinations in which only limited amounts of material are available. 100 BIBLIOGRAPHY 1. Oshino, N. (1978) Pharmacol. Ther. A. 13, 477-515. 2. Ozols, J. (1974) Biochemistry 13, 426-434. 3. Waino, W.W. (1978) Pharmacol. Ther. A. 2, 359-372. 4. Keves, S.R., Alfano, J.A., Jansson, I. and Cinti, D. 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(1971) Cold Spring Harbor Symp. Quant. Bid. 36, 405-411. 115 261. Gupta, R.K. (1973) Biochim. Biophys. Acta. 292, 291-295. 262. Chien, J.C.W., Gibson, H.L. and Dickinson, L.C. (1978) Biochemistry 17, 2579-2584. 263. Chothia, C. and Janin, J. (1975) Nature 256, 705-708. 264. Ross, P.D. and Subramanian, S. (1981) Biochemistry 20, 3096-3102. 265. Stonehuemer, J. Williams, J.B. and Millet, F. (1979) Biochemistry 18, 5422-5427. 266. van Holdee, K.E. (1971) Physical Biochemistry, Prentice-Hall, Englewood Cliffs, N.J., p55. ' 267. Wherland, S. and Gray, H.B. (1977) in Biological Aspects of Inorganic Chemistry, (Addison, A.W., Cullen, W.R., Dolphin, D. and James, B.R. Eds.) John Wiley and Sons, Inc., New York, pp289-368. 116 APPENDIX A Midpoint r e d u c t i o n p o t e n t i a l s (vs N . H . E . ) were measured in dium phosphate b u f f e r . Parameters were o b t a i n e d from a weighted l i n e a r l e a s t - s q u a r e s , f i t t i n g of the data to the Nernst e q u a t i o n . S t andard d e v i a t i o n s are i n d i c a t e d CYTOCHROME b5 PH Temp I Med Po tent i a l s lope 5.50 25 .0 0.1 0. 1 13.3 ± 0. 1 59.0 + 1 .0 5.67 0.5 28.2 0.4 58.7 0 . 5 5.87 28.7 0.6 59. 4 0.7 5.92 28.0 0.5 58.7 0 . 5 6.00 0.1 9.7 0.9 60. 1 0. 1 6.00 0.5 26.2 0.3 60. 9 0.3 23.0 0.5 58.4 0 . 6 6.15 22.0 0.4 62. 1 0.5 6.50 15.7 0.4 60.8 0 . 5 6.51 0. 1 5. 1 0.4 58.3 0 . 6 7.00 10.0 0.1 15.5 0.3 56. 3 0.4 15.0 13.2 0.3 •56. 5 0.3 20 .0 6.4 0.3 58.2 0.4 25 .0 5. 1 0.6 59.7 0.4 30 . 0 -3 . 5 0.5 60.4 0.6 35 .0 - 6 . 6 0.8 60. 0 0 . 1 40 .0 - 1 1 . 8 0.2 63. 1 0.2 7 .00 10.0 0.5 28 . 3 0.5 60.3 0 . 6 15.0 26.4 0.5 59.6 0 . 6 20 . 0 19.1 0.5 60.9 0.6 1 1 7 25.0 30.0 35.0 7.00 25.0 7.00 25.0 7.50 25.0 8.00 25 .0 0.03 0.05 0.2 0.3 0.4 0.1 0.1 0.5 1 .0 2.0 2.5 3.5 3.5 15.0 0.1 0.1 0.5 0. 1 0.5 15.5 0.4 16.6 0.5 13.6 0.4 5.4 0.4 - 4 . 5 0.4 0.0 0.8 - 2 . 3 0.6 9.2 0.3 14.0 0.2 17.8 0,3 4.4 0.5 5 .9 0.7 6.1 0.3 5.7 0.4 4.2 0.3 4.7 0.3 4 .7 0.3 5 .3 0.2 6.7 0.4 4.0 0.1 15.1 0.2 3.4- 0.5 14.1 0.9 60.6 0.5 58.3 0.6 60.7 0.5 62.6 0.5 59.6 0 .6 60.0 0.1 59.6 0.7 58.2 0.4 59.5 0.3 59.2 0.4 62.6 0 .6 59.3 0.8 60.1 0.3 59.6 0.5 59.7 0.4 59.9 0.4 58.9 0.4 59.9 0.5 60.3 0.5 60.0 0.2 59.9 0.3 63.1 0.7 57.0 1.0 DEUTEROHEME-SUBSTITUTED CYTOCHROME bs ' pH Temp I Med 5.56 25.0 0.5 0.1 5.67 5.76 5.87 6.13 6.47 6.80 7.00 10.0 0.1 15.0 .20.0 7.00 25.0 0.01 0.05 0. 1 1.0 0. 1 0 . 2 0.3 0.4 Potent i a l s lope - 1 8 . 2 0 . 9 60.0 2.0 -18 .7 0.2 58 . 0 0.3 - 1 9 . 5 0 . 2 57.7 0.3 - 2 1 . 9 0. 1 57 . 1 0.2 -24 .8 0.4 58 . 1 0.5 - 2 7 . 6 0.4 59'-. 1 0.5 -30 .2 0.2 59.3 0.3 -27 .9 0.2 56.7 0.3 - 2 6 . 5 0.9 56.0 1 .0 - 2 6 . 6 0.3 54.1 0.4 - 3 0 . 3 0.5 56.9 0.7 - 3 5 . 3 0.7 58.0 1 .0 -39 .2 0.4 59.8 0.6 -38 . 1 0.4 58.2 0.7 -37 . 1 0.3 58 .6 0.4 -62 .6 0.7 64 .0 1 .0 - 5 2 . 3 0.2 59.0 0.3 - 4 8 . 8 0. 1 58.9 0.2 - 3 9 . 5 0. 1 58.0 0. 1 -49 .4 0.2 58.9 0.3 - 3 9 . 5 0. 1 58 .4 0.3 - 4 1 . 2 0.2 59.6 0.2 -38 .2 0 . 3 60.2 0.6 -25 . 1 0.2 59. 3 0.2 - 3 0 . 3 0.6 59.6 0.9 - 3 1 . 8 0.3 60.5 0.4 1 1 9 0.5 -29 .0 0.6 60.4 0.9 -33.1 0.3 60.6 0.5 7.00 30.0 0.1 - 50 .9 0.2 60.5 0.2 35.0 -56.1 0.2 60.0 0.3 7.55 25.0 0.1 -34 .2 0.4 62.7 0.6 8.09 -33 .4 0.2 59.6 0.3 1 20 D I M E T H Y L E S T E R H E M E - S U B S T I T U T E D pH Temp I Med 5.46 25.0 0.1 0.1 5.66 5.76 5.97 6.27 6.50 7 .0.0 8 . 4 0.1 0 .1 10.0 11.0 15.0 20 . 0 20.8 7.00 25.0 0.1 0.2 0.3 0.4 0.5 7.00 30.0 0.1 33 . 4 35.0 7.52 25.0 0.1 7.70 7.97 CYTOCHROME b5 P o t e n t i a l S l o p e 77 . 4 0. 1 58 . 3 0.2 75. 1 0.3 58 . 2 0.2 75. 1 0.6 58.8 0.2 73.0 0.1 59.5 0.5 71.8 0.3 58 . 5 0.9 69. 7 0.7 59.0 1 .0 84.9 0.6 58.4 1 . 6 79.3 0.3 54 . 1 0.7 74.2 0.6 57.8 1 . 2 76.8 0.5 54.0 1 .0 74.9 0.4 58 .6 0.4 75.0 0.6 59.6 0.7 68.0 1 .0 61 .0 1 .0 69.6 0.4 58.8 ' 0.8 73.4 1 .3 61.7 0.7 77.8 0.9 58.0 1 .0 82.5 1 .2 57.6 0.3 84.6 0.3 59.2 0.6 69. 3 0. 1 60.0 0.2 69. 1 0.3 61.3 0. 1 64 . 3 0.9 63.0 1 . 0 61 .7 0.9 62.. 3 0.7 68.4 1 .0 58.0 2.0 68 . 2 0.6 59.4 0.5 65.8 0.3 54.6 0. 1 1 2 1 APPENDIX B Midpoint r e d u c t i o n p o t e n t i a l s (vs N.H.E.) for Fe(EDTA) 2" i n sodium phosphate b u f f e r . I o n i c s t r e n g t h i s 0.5 M with 0.25 M c o n t r i b u t e d from N a l . A l l measurements are at 25.0 °C. Para-meters were obtained from a weighted l i n e a r l e a s t - s q u a r e s f i t to the Nernst e q u a t i o n . Standard d e v i a t i o n s are i n d i c a t e d (n > 6) . pH Reduction Slope P o t e n t i a l (mV) (mV) 5.50 115.7 ± 0.7 60.8 ± 0.8 6.00 104.1 0.7 64.0 0.9 6.50 98.4 0.5 58.9 0.7 7.00 95.0 1.0 73.0 2.0 7.50 86.8 0.9 73.0 1.0 8.00 86.9 0.8 59.0 1.0 1 22 APPENDIX C F i r s t - o r d e r rate chromes l i s t e d by co n s t a n t s Fe(EDTA) 2 for the r e d u c t i o n of the " in phosphate b u f f e r . cy t o -CYTOCHROME bs pH Temp I Fe kobs + 5.50 34.4 0.5 0.0125 10.6 0.3 29.8 8.7 0.2 25.0 7.0 0. 1 20.0 5.68. 0.08 15.5 5.07 0.07 5.50 25.0 0.5 0.0125 7 .34 .0.05 0.01 5.75 0.05 0.0075 4.42 0.02 0.005 2.96 0.03 0.0025 1 .55 0.04 0.00125 0.82 0.03 6.00 34.5 0.5 0.0125 9.38 0.03 29.8 7.80 0.03 25.2 6.43 0.05 20.3 5.29 0.05 16.1 4.58 0.05 6.00 25.0 0.5 0.0125 0.01 0.0075 0.005 0.0025 6.65 5.06 3.457 2.280 1 . 1 67 0. 08 0.08 0.004 0.003 0. 003 1 23 0.00125 0.501 0.005 6.5 40.0 0.5 0.125 7.50 0.02 35.2 6.45 0.03 30.5 5.36 0.02 25.9 4.60 0.01 21.1 4.10 0.04 16.4 3.55 0.04 6.5 25.0 0.5 0.0125 4.06 0.05 0.01 3.20 0.02 0.0075 2.386 0.006 0.005 1.58 0.01 0.0025 0.817 0.004 0.00125 0.395 0.006 7.0 40.2 0.5 0.0125 6.25 0.05 35.4 5.2 0.1 30.7 4.43 '0.04 25.1 3.61 0.02 20.1 3.12 0.~03 15.4 2.70 0.02 7.0 25.0 0.5 0.02 6.07 0.02 0.016 4.591 0.009 0.012 3.352 0.007 0.008 2.214 0.004 0.004 1.151 0.002 0.0016 0.464 0.003 124 7.0 25.0 0.4 0.01 0. 008 0 . 006 0.004 0.002 0.001 7.0 25.0 0.3 0.01 0.008 0.006 0.004 0.002 0 . 001 2.70 1 . 97 1 .49 0.969 0.491 0.221 2.081 1 .686 1.142 0.841 0.456 0.251 0.06 0.04 0.03 0.007 0. 003 0.003 0.004 0.004 0.004 0.002 0.005 0.005 7.0 25.0 0.2 0.005 0.921 0.006 0.004 0.71 0.04 0.003 0.51 0.03 0.002 0.35 0.02 0.001 0.19 0.02 .0.0005 0.08 0.02 7.0 25.0 0.1 0.0020 0.223 0.005 0.0015 0.159 0.003 0.001 0.122 0.002 0.0005 0.056 0.002 0.00025 0.0203 0.0001 7.0 25.0 0.05 0.0012 0.053 0.001 125 0 .0009 0.040 0.001 0 .0006 0 .025 0.001 0 .0003 0 .0145 0.0001 0 .00015 0 .0078 0.0001 8 .0 40.1 0 . 5 0 .0125 5.54 0 .04 34 .4 4 .56 0 .02 29 .8 3 .92 0 .02 2 5 . 0 3.37- 0 .04 20.1 2 .985 0 .008 15.5 2 .53 0.01 8 .0 25 .0 0 .0125 3.28 0 .02 0.01 2 .548 0 .005 0 . 0 0 7 5 1.89 0.01 0 . 0 0 5 1.24 0.01 0 .0025 0,638 0 .004 0 . 0 0 1 2 5 0.291 0 .005 126 DEUTEROHEME-SUBSTITUTED CYTOCHROME bs pH Temp I Fe kobs ± 5.47 25.0 0'. 5 0.002 0.427 0.009 0.004 0.532 0.002 0.008 2.209 0.044 0.012 3.477 0.079 0.016 4.61 0.115 0.020 7.47 0.14 5.90 25.0 0.5 0.008 1.72 0.03 0.012 2.10 0.04 0.016 3.22 0.04 0.020 4.33 0.04 6.14 25.0 0.5 0.0015 0.189 0.005 0.003 0'.355 0.005 0.006 0.955 0.005 0.009 1.745 0.042 0.012 2.086 0.052 0.015 2.849 0.07 6.5 25.0 0.5 0.002 0.004 0.008 0.012 0.016 0 . 020 0.348 0.510 1 .270 1 .75 2 . 32 2.86 0.005 0.007 0.021 0.04 0.04 0.04 7.0 25.0 0.05 0.000125 0.0048 0.003 1 27 0.00025 0.0094 0.002 0.0005 0.0178 0.008 0.00075 0.093 0.001 0.001 0.0353 0.0001 0.00125 0.0344 0.002 7.0 9.8 0.5 0.015 0.804 0.0084 13.5 0.922 0.021 19.4 1.299 0.038 25.0 1.575 0.034 29.5 1.899 0.023 34.4 2.268 0.029 7.0 25.0 0.1 0.00025 0.0005 0.001 0.0015 0.002 0.0025 0.0139 0.0272 0.0468 0.0600 0.081 0.093 0.0005 0.0005 0.0005 0.005 0.001 0.002 7.0 25.0 0.2 0.0005 0.001 0.002 0 . 003 0.004 0.005 0.035 0.060 0. 126 0. 189 0.2526 0.327 0.003 0.006 0.005 0.005 0.0001 0.001 7.0 25.0 0.3 0.001 0 . 002 0.074 0.155 0.005 0.005 1 28 0.004 0.006 0.008 0.01 7.0 25.0 0.4 0.001 0. 002 0.004 0.006 0.008 0.01 0.315 0.479 0.609 0.804 0.095 0. 19 0.36 0.566 0.719 0.980 0.005 0.003 0.007 0.013 0.005 0.01 0.01 0.018 0.005 0.024 7.0 25.0 0.5 0. 002 0.004 0.008 0.012 0.016 0.02 0.276 0.518 0.914 1 .30 1 .66 1 .96 0.003 0. 003 0.007 0.03 0.03 0.02 7.5 25.0 0.5 0.002 0.004 0.008 0.012 0.016 0.02 0.21 0.391 0.804 1.18 1 .49 1 .92 0.002 0.003 0.006 0.012 0.025 0.035 8.0 25.0 0.5 0. 002 0.004 0.008 0 . 1 55 0.314 0.692 0.003 0.003 0.007 1 29 0.012 1 .04 0.01 0.016 1.41 0.02 0.02 1.81 0.04 1 30 DIMETHYLESTERHEME-SUBSTITUED CYTOCHROME bs pH Temp I Fe kobs ± 5 .45 ' 25.0 0.1 0.00025 0.956 0.002 0.005 1.896 0.019 0.001 3.950 0.014 0.0015 5.460 0.019 0.002 7.180 0.024 0.0025 8.788 0.031 5.65 0.0025 0.967 0.005 0.0005 1.690 0.014 0.001 3.420 0.016 0.0015 5.000 0.014 0.002 6.515 0.002 0.0025 8.162 0.035 5.74 0.00025 0.826 0.009 0.0005 1.550 0.011 0.001 3.030 0.02 0.0015 4.560 0.02 0.002 5.920 0.017 0.0025 7.327 0.038 5.94 0.00025 0.784 0.007 0.0005 1.410 0.009 0.001 2.820 0.016 0.0015 4.330 0.016 0.002 5.570 0.021 1 3 1 0.0025 6.930 0.02 28 0.00025 0.654 0.009 0.0005 1.270 0.009 0.001 2.480 0.014 0.0015 3.730 0.019 0.002 5.010 0.02 0.0025 6.140 0.022 54 0.00025 0.618 0.006 0.0005 1.190 0.008 0.001 2.410 0.014 0.0015 3.500 0.019 0.002 4.780 0.02 0.0025 6.135 0.068 00 0.05 0.000125 0.202 0.0002 0.00025 0.383 0.002 0.0005 0.736 0.008 0.00075 1.090 0.04 0.001 1.450 0.096 0.00125 1.770 0.014 00 5.95 0.1 0.0025 2.84 0.355 15.5 3.837 0.35 19.9 4.287 0.344 24.8 4.941 0.342 1 32 25.0 0.00025 0.515 0.004 0.0005 0.962 0.009 0.001 1.281 0.015 0.0015 2.417 0.019 0.002 3.929 0.02 0.0025 5.047 0.026 7.00 29.5 0.1 0.0025 5.626 0.338 34.4 6.31'6 0.041 39.1 7.003 0.041 7.00 25.0 0.2 0.0005 1.319 0.016 0.001 2.540 0.015 0.002 5.304 0.014 0.003 7.986 0.022 0.004 10.42 0.04 0.005 12.94 0.02 7.00 25.0 0.3 0.00075 2.360 0.015 0.0015 4.660 0.014 0.003 9.700 0.031 0.0045 14.20 0.04 0.006 18.77 0.04 0.0075 23.20 0.04 7.0 25.0 0.4 0.001 3.479 0.027 0.002 7.000 0.035 0.004 13.56 0.08 0.006 19.07 0.05 133 0.008 26.70 0.02 0.010 33.01 0.02 7.0 25.0 0.5 0.00125 5.060 0.016 0.0025 9.580 0.044 0.005 18.88 0.1 0.0075 27.62 0.02 0.010 36.58 0.16 0.0125 44.73 0.17 7.54 25.0 0.1 0.00025 0.466 0.002 0.0005 0.900 0.011 0.001 1 .800 0.017 0.0015 2.730 0.019 0.002 3.700 0.021 0.0025 4.650 0.023 8.03 25.0 0.1 0.00025 0.435 0.002 0.0005 0.849 0.016 0.001 1.712 0.017 0.0015 2.590 0.016 0.002 3.410 0.013 0.0025 4.376 0.016 1 34 APPENDIX D D e r i v a t i o n of Eq 19. From the s tandard r e l a t i o n s h i p for f ree energy AG = -n F (AE) [1] which a f t e r s u b s t i t u t i o n fo r the c o n s t a n t s i s ( p o t e n t i a l in v o l t s ) (Ref 267) AG=-23.06 (AE) kca l /mole [2] From t r a n s i t i o n s t a t e t h e o r y at 25 ° C k , 2 = (kB T ) / h e x p ( - G 1 2 / R T ) , [3] where kB = B o l t z m a n ' s c o n s t a n t ^ 3.298 x 1 0 " 2 U c a l / d e g h = P l a n c k ' s cons tant= 1.584 x 1 0 " 3 " c a l / s e c R = Gas c o n s t a n t = 1.9872 c a l / d e g mol T = Temperature (K) = 298 k 1 2 = r a te c o n s t a n t (M~ 1 s " 1 ) r e a r r a n g i n g 3 to - I n (h k , 2 / k B T) = A G 1 2 * / R T or l n ( k B / h ) - l n ( k , 2 / T ) = A G 1 2 * / R T [4] and s u b s t i t u t i n g the c o n s t a n t s A G 1 2 * / R T = 23.76 - ( -5 .697) - l n ( k 1 2 ) = 29.46 - l n ( k , 2 ) or (Ref 268) A G 1 2 * = 0.592 (29.46 - In k 1 2 ) k c a l / m o l or k 1 2 = 6.21 x 10 exp( G 1 2 ± / 0.592) [5] The f ree energy for the r e a c t i o n i s a composi te of f ree energy terms a s s o c i a t e d w i t h a t t a i n i n g the a c t i v a t e d s t a te ($) and that from the p o t e n t i a l d i f f e r e n c e between the r e a c t a n t s 1 35 ( d r i v i n g fo rce = df ) AGobsd = AG* + Gdf or AG* =AGobsd - Gdf [6] S u b s t i t u t i n g 2 and 5 i n t o 6 0 . 592 (29 .45 - l n k , 2 * ) = 0 . 592 (29 .45 - I n k , 2 obsd) -(-23.06 (AE)) [7] Rearrange and s o l v e for k1 2+- ( c a l l e d k 1 2 a d ; ] i n Eq 19) k 1 2 * = 6 . 2 1 X 1 0 1 2 e x p ( l n k , 2 o b s d - 29.45 38 .95(AE)) [8] 1 36 APPENDIX E F i r s t - o r d e r ra te c o n s t a n t s for the r e d u c t i o n of n a t i v e c y t o -chrome b5 by D M E - f c 5 . Na phosphate b u f f e r pH 7 in a l l c a s e s . C o n t a i n i n g the p h o t o - a c t i v e r e d u c i n g system d e s c r i b e d in the t e x t . I Temp [DME-6 5 ] k obsd 0.05 25 5.0 M .0283 .001 .0289 .001 10.0 .0383 .001 15.0 .0464 .001 15.0 .0417 .001 0.10 5.0 .0231 .001 5.0 .0233 .001 11.4 .0233 .001 11.4 .0228 .001 11.4 .0387 .001 11.4 .0398 .001 15.0 .0376 .001 15.0 .0374 .001 0.20 5.0 .0207 .001 5.0 .02014 .001 0.30 5.0 .027 .001 5.0 .0257 .001 10.0 .0405 .001 10.0 .0404 .001 1 37 0.4 5.0 .0194 .001 5.0 .037 .001 10.0 .0518 .001 10.0 .0524 .001 15.0 .0662 .001 15.0 .066 .001 0.5 5.0 .0395 .001 5.0 .0385 .001 10.0 .0691 .001 15.0 .0753 .001 15.0 .0728 .001 1 38 


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