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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 b  5  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 p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e for reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s o r her  g r a n t e d by  the head o f  representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain  s h a l l not be  a l l o w e d w i t h o u t my  permission.  Department o f  Biochemistry  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  April  30, 1 9 8 4  Columbia  written  ABSTRACT Cytochrome b  has a central role in many aspects of eukaryotic cellular metabolism.  s  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 physical 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 which  little attention  it  changes  has  been  directed  oxidation  state.  The  oxidation-reduction heme-substituted  equilibrium  chrome bi with  the  of  the  those  increases  functional  of  reduction  reports  kinetics  properties  of  a  of  dimethyl  the native protein reveals  of  the  reduction  support a role for one of the and  work  of  the  mechanism by  characterization bs  cytochrome  of  the  and  two  that  ester heme-substituted  esterification  cyto-  of the heme pro-  the reduction potential of the protein by 60 mV and significantly alters  electrostatics  protein  the  present  characterization  derivau'ves.  Comparison  pionates  and  toward  provide  direct  with  heme propionates  evidence  along the partially exposed  kinetics  for  electron  Fe(EDTA) 2 "  as  reductant  These  results  in stabilizing the oxidized form of the transfer  at  the  surface  of  the protein  heme edge.  The functional properties of deuteroheme-substituted  cytochrome b  5  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 kinetic  analysis  of  the  reaction  between  the  self-exchange  nadve  and  The second-order rate constant for this reaction after is 3.8 x 10:  M " 1 s_1  (u=  of the true self-exchange rate is attributed  deuteroheme-subsdtuted  proteins.  correcdon to a driving force of zero  0.1 M , p H 7 (phosphate),  25 ° C ) and represents  a prediction  rate of the native protein. The relatively low magnitude of this  to short-range  the site of electron transfer to be a generally useful  rate of cytochrome bi in  electrostadc  effects  arising  from the heme propionates  along the heme edge. This heme-subsdtudon  technique for studying self-exchange  ii  at  strategy appears  rates of other hemeproteins  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 b  s  Sequence Analysis  4  Crystallographic Analysis  4  Cleaved Forms of Cytochrome b  11  Detergent Solubilized Form  12  s  Biosynthesis of Cytochrome b  14  s  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 b  33  s  Reconstitution with Synthetic Porphyrins Preparation of Apo-cytochrome  fc5  34  Preparation of Deuteroheme- substituted Cytochrome b  36  s  Preparation of Protoheme IX Dimethyl Ester-substituted Cytochrome b  36  5  pH Titration of Cytochrome b  37  Spectroelectiochemical Measurements  37  Kinetics Measurements  40  5  Reaction of Cytochrome b with Deuteroheme-substituted Cytochrome b s  45  s  Nuclear Magnetic Resonance Spectroscopy  47  RESULTS Electronic Spectra of Native and Heme-substituted Cytochrome b  48  Proton Titration of Cytochrome b  48  s  s  Oxidation-reduction Equilibrium of Native and Heme-substituted Cytochrome b  s  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 b  s  62  Ionic Strength Dependence  63  pH Dependence  67  Temperature Dependence  69  Cytochrome ij - Deuteroheme-substituted  Cytochrome b Cross Reaction 5  71  Ionic Strength Dependence  72  Temperature Dependence  75  NMR Spectroscopy of Native and D M E - k  v  75  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 b  5  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 Cytochrome b  95  s  Rationale for Experimental Design  '.  96  Ionic Strength Dependence  96  Temperature Dependence  97  Theoretical Analysis of Reactions Rates  97  Significance of Results  99  Conclusions  •  BIBLIOGRAPHY  100  101  APPENDICES A. Cytochrome 65 Midpoint Reduction Potentials  vi  117  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  vii  137  LIST OF T A B L E S  I  Proteins Containing the "Cytochrome b fold"  10  II  Cytochrome £>5 Metabolic Functions  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  s  17,18  XIII Electron Tunneling Analysis XIV  94  Estimation of Protein-Protein Self-Exchange  viii  Rate  -  98  LIST OF FIGURES  1  Amino Acid Sequence of Cytochrome £>5  5,6  2  3-D Stereo Representation of the Tryptic Fragment of Cytochrome bt  3  Structure of Ferriprotphorphyrin IX  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  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  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  7 30  — 51,52  ix  .,  70  ABBREVIATIONS USED  ATP  Adenosine Triphoshate  CD  Circular Dichroism  DME-&5  Dimethyl Ester Heme-substituted  deuteio- i 5  Deuteroheme-substituted  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  Cytochrome b  5  Cytochrome bi  NADH-Cytochrome b Reductase (E.C. s  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 appreciated 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 invaluable 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 b transfers electrons from NADH to many physiot  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 particular, 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 methemoglobin (12). The sequence and three dimensional structure of cytochrome b have been deter5  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 cytochrome bs changes oxidation-reduction heme-substituted  oxidation state. For this equilibrium and  reason,  reduction kinetics  a  systematic  of  investigation  this protein and two  of the of its  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  membrane-bound form (17). The results provide reasonably  equivalent  to  that  of  the  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 b  f  and related proteins. In addition, some  theoretical possibilities for analysis of metalloprotein electron transfer reactions are considered.  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 postulated 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 compound 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. 1920!s when  Consequendy, the concept of Keilin, at  the  University of  additional  pigments  Cambridge,  was abandoned  "rediscovered"  until  the  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 distinctive 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 b  l  kidney (24),  isolated from liver (22) or bacteria (23), cytochrome fcj from pig  cytochrome b  7  which was inseparable  from the enzymatic activity associated  with yeast lactate dehydrogenase (25,26), cytochrome b  y  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 bi by Williams (31).  Soon afterwards, Strittrnatter  renamed cytochrome  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 b  5  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 preparations with pancreatic lipase released a soluble, heme-containing protein with the cytochrome bi spectrum (34). The relative ease with which this protein could be isolated from various mammalian liver sources prompted numerous investigations into its chemical properties (3). In the late 1960's, independent investigations by Sato in Japan (35) and Strittrnatter 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 b had 5  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 unequivocally  (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 intermembrane space (43). 3  soluble  A  form of cytochrome  6,  has  been  isolated  During maturation of the cell all subcellular organelles chrome b  s  erythrocytes,  are degraded.  is solubilized by an endogenase protease and which  maintain  a complete  subcellular  from mammalian  remains  organization  erythrocytes.  In this process cyto-  in the cytosol. In avian 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 cytochrome bi have been determined for several domains  of sulfite  oxidase (44),  been determined. A comparative (46),  between  these proteins,  and cytochrome  b  s  from  yeast cytochrome  b  2  (45)  and nitrate reductase (46)  have  sequence alignment, as discussed by Lederer and colleagues b from  cytochrome  bovine  species. In addition, the cytochrome 6<-like  (13),  rat  (47),  rat and  outer  mitochrondrial membrane  chicken (48)  is presented  (46)  in Fig  1.  The empirical molecular weight of cytochrome bi solubilized from bovine liver microsomes with Triton X-100  is 15,233 daltons  with trypsin releases a fragment  (including the heme). Solubilization of cytochrome  b  s  (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 b and proteins known to possess a similar heme crevice. s  1 1 BMC 2 RMC 3 ROM H2N 4 chicken  Ac  -  Ac -  5 b2  II  Asp H2N  Ala Gly  -  Asn  L Glx Glu  Gin  ii  Asp  Gin  Gly  II  Asp  Pro  Glx -  Lys  6 SOX 7 b557  1  Tyr  2  M  it  tt  II  3  It  Arg  it  II  II  Arg  tt  4  -  -  Thr -  5 6  He  7  Leu  Glu  1 2 3 4 5 6 7  Ser  =  Pro Arg  It  Tyr  -  tl  Asp Gin  -  II  = =  II  Cys Val  Arg  ?  Leu  5 6 7  tt  -  Thr -  ." " " "  Val II  -  it  It  II  tl  II  He  Lys ti  Arg it  Arg -COOH  50 Leu -  II  II  Glu -  Gin Ala tt ti  Trp -  " " " "  ti  He He  Val  Thr II  Glu  1 2 3 4  1 2 3 4 5 7  Ser  Leu -  II  Gin Glu  II  Leu H2N  Ser -  Asp -  Glu II  Ala  Lys ti  T Lys - A l a it  Pro Met Pro  His II  II  Asl H2N  It  Asn  Gly Lys  Ser  Tyr  Lys  Val  Ala  Glu Glu Glu  Val  II  II  it  Val Val  Glx Ala Gly  it  II  it  Arg  II  He  Lys  Gin  Tyr  II  Leu -  30 His -  Leu Val Met He Val Val  40 Leu -  He II  Val II  Val Thr M  tt  He Val He His  II tl II  Asn Gly  Ii  Tyr His Gly His Gly Thr tl  -  ' Thr Arg  Asp  it  Thr II  Arg 7  Lys II  Arg Arg Tyr Asp ti  Lys Lys  Val -  Glu -  His -  Pro -  Gly -  it  Gly -  II  ti  II  It  it  II  II  II  II  II  II  it  Ser Asp Pro  II  II  tl  II  II  II  7  II  11  II  Leu ti  Lys  II  II  11  ii  II  II  11  ii  tt  II  11  ii  II  ii  Phe  Asn  -COOH  5  II  Ala it  II  Lys  Glu -  "  it it  11  tt  Gin 7  Thr -  60 Glu -  II  II  ii  II  II  II  ti  II  II  II  II  Glu  11  II  Val  "  II  •t  it  Asp  *  tt  Ala -  it  *  tt  Asp -  11  Gin Asp Glu  "  II  Gly -  Tyr -  Glu  Phe  II  Gly -  Pro Pro 7  7  II  Ala -  II  11  II  Gin -  Ala  M  It  II  Glu -  Lys  It  tl  *  Arg -  Ser -  11  tt  7  -  11  He  li  Lys Arg Asp  Pro H2N  11  11  Asn  Tyr li  Gin  tl  B Glu -  Lys -  II  20 Asn '- Asn -  Glu  Arg  10 Val -  Asp  II  tt  Phe -  He -  Ser -  Ala  Asp tt  Asp Asn Ser Asn He  1 9  Phe  -  Glu -  *  Asp -  B  Val -  II  ii  H  it  3 4  II  ti  II  II  II  II  II  tl  5  tl  II  1  Ser  2  n  -  Lys II  II  •t  II  it  II  ii  "  Pro  Leu  =  Thr -  Phe Tyr  =  Tyr  = =  = = = = Lys  II  -  -  Tyr  He  Ala  Pro  Glu  1  Asp  Asp -  T Arg -  Ser -  Lys -  1 2 5  He  II  M  n  II  Ser  Met  II  Pro  He II  Tyr II  80 He -  70 Asp -  II  It  tl  II  II  II  tl  II  Asn  Val  He  Ala Asp  Gly -  Glu -  Leu -  His  II  II  II  II  Thr -  Lys -  11  ii  11  Val  Asp n  II II  Pro  H e II  Leu Lys  Lys Pro  Arg  Pro  Pro  Glu  Thr -  H e - Asp Val Glu L y s -COOH  Ser -  Pro -  H e -  Ala  II  Pro 11  Ser -  II  II  Trp  1 2  He Met  -  11  _  II  Leu •Val  He -  Tyr -  His Arg  II  M  Lys  - Pro 11  II  n  II  it  Glu  Gly  Ser  - He  tf  Thr  Leu  Pro  Tyr  Ala  Pro  Trp -  Trp -  Thr  it  L y s -COOH -COOH Leu  Val  Cys  Asn -  Pro Ser  II  110  Glu  Gly  1 2  Thr -  Met  LE Glu -  100 -  II  II  E"  II  Pro  G l u - Leu  it  II  Leu  Arg -  II  "  Lys  Ala -  It  90  II  II  Asn  II  -  Thr - Pro  Ala  II  Glu  3 4 5  Gin  Ser II  it  -  His It  3 4 5  2  Leu  Gly -  II  II  Leu -  Ala II  Tyr II  II  II  130 Thr Met  II  Ser 11  Ala II  Ser Ala  Glu If  Ser II  120 Leu ti  tl  Phe - V a l II Val  A s n -COOH 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 , s p a c e 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 * residue involved i n charge-pair interactions B l i g a n d t o t h e heme i r o n E b o n d c l e a v e d i n e r y t h r o c y t e b5 L b o n d 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 preparations T b o n d c l e a v e d by t r y p s i n BMC b o v i n e m i c r o s o m e s RMC r a t m i c r o s o m e s 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  11  If  Ala It  - Asn II  - Leu II  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^, G l u 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 b is non-covalendy bound, the propionates are oriented towards the surface of s  the protein, and two histidine ligands are coordinated to the iron (His^ and His^. These characteristics  are  generally  c-type cytochromes (54).  more  reminiscent  of  The three-dimensional  myoglobin and  hemoglobin (17)  homology of these structures  than  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 publication 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 b can exist in either one of two orientations that differ by a 180° rotation 5  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 homogeneous preparations forms (t^2  0 3  of either  form.  The relatively  rapid  interconversion between these  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  8  surface  area  of  the  protein (53). One  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 accessibility 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 other b cytochromes, which have the "bi fold", were placed on the backbone of the cytochrome 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 hydrophobic 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 b t  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 b ) 2  Multiple Heme-Binding  blood  63  Plasma Protein  Nitrate Reductase  Neurospora crassa  46  (cytochrome b ) i51  Sulfite Oxidase  liver  44  structure for the carboxyl terminal sequence of the protein binding segment as; residues  91-97 98-102 103-112  flexible link, random coil e sheet £ 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  b  s  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 related 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 cytochrome b . The electronic spectrum of cytochrome b s  s  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 observation 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. Overlap between the iron d orbitals and the heme T T 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 ferricytochrome bs has a net charge of -6.5 at pH 7 while the charge on the tryptic fragment 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 membrane 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 structure, 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 generally 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 phobic tail (84). Each molecule of cytochrome b  5  lipid surrounds the hydro-  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 < A G ° r g 25° C=  25±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  $  profile.  Some  exhibits a broader, more complex denaturation  evidence suggests that the hydrophilic and hydrophobic domains undergo  thermal denaturation at different rates during the unfolding of the protein (80). The instability of the detergent-solubilized form relative to the trypsin-cleaved form was attributed to reduced protein-water interactions between domains (72). Recent scanning calorimetry results 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 relative importance of inter-domain interactions or of phospholipid headgroup effects on this complex process remains to be determined.  13  Biosynthesis of cytochrome b  5  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 b ranges from a high of 0.88 nmole/mg protein in the liver to only 0.10 s  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 b was not ins  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 b  5  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 b is translated on free rather than membrane bound polysomes. It s  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 b will 5  aggregate to form octamers until the lipid content reaches the critical micellular concentration (37,83,99,100). Cytochrome b then partitions into the lipid phase, but in contrast to s  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 distinguished 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). However, migration of the hydrophilic C-terminus (...Glu^ ^ s n ] j 3 COOH, bovine liver microsome, 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 interactions. 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 b  s  will remain loosely associated with the membrane. The oc-  curence of an exchange of newly synthesized cytochrome b between subcellular organelles s  is uncertain (38,94,98). 15  Physiological Roles of Cytochrome 6< Initial studies of cytochrome b, revealed that this cellular pigment could be selectively reduced upon addition of NADH  to microsomal membranes isolated from the he-  patocyte. However, no information was available regarding the significance of this observation. Three  metabolic  roles  for  cytochrome  b  s  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 b may provide the second electron to cytochrome P-450 in its catalytic cycle s  (113,114). This synergistic function has now implicated cytochrome b in a wide variety of s  metabolic processes (Table II). NADH cytochrome b reductase (EC from calf contains an FAD prosthetic b  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 incorporated  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 b , s  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 hydrolyzed cytochrome b as the electron acceptor, the rate limiting step is a stereospecific pros  ton transfer from NADH  to FAD. The reductase catalyzes the transfer of two electrons  from a molecule of NADH to two molecules of cytochrome b , in sequential s  steps.  This  transfer  is  eliminated  when  16  a  free  sulfhydryl  is  one-electron  blocked  with  Table II. Metabolic Reactions Which Utilize Reducing Equivalents from Cytochrome b$  JLe^cjion  Sceciss  Cholesterol biosynthesis  Rat Liver  Eel  (4 demythylation) (A 5 desaturation) Ergosterol biosynthesis  5. cerevisae  (4 demethylation)  117  (A 5 desaturation)  118  Fatty Acid Chain Elongation  Rat Liver  4,119  A 5 desaturation  Rat Liver  120  A 6 desaturation  Rat Liver  121  A 9 desaturation  Rat Liver  5,122  Hydroperoxide reduction  Pig Liver  123  Hydroxylamine reduction  Rat Liver  124  Methemoglobin reduction  Erythrocyte  78  Plasmalogen biosynthesis  Pig Spleen  125  Prostaglandin synthesis  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 b  s  by Fp^ is dramatically increased  when the two  proteins are restricted to lateral diffusion in the plane of a membrane bilayer (133). Presumably 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 modification 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 G l u ^ , Glu^g, G l u ^ . Asp^  a n  d  the single exposed heme propionate  on cytochrome b . However, the interaction domain may extend beyond this region. Cytos  chrome b , which is thought to have a similar heme edge environment, reacts thirty-fold 2  slower with Fp^. Cytochrome c, interacting only at the exposed heme edge of cytochrome bs and cytochrome b , exhibits identical rates of electron transfer with each protein (136). 2  Lack of an hydrophobic patch on the surface of cytochrome b  2  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 b (133). s  As with Fp^, this reductase (Fp^.) also requires the same complementary charge pair interactions at the exposed heme edge for activity (137). Since the primary acceptor of electrons 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 cytochrome b , as this was the only microsomal component known to be reducible by all s  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 nonheme 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 b ) and is solubilized at concentrations of Triton s  X-100 which do not solubilize the 53,000 dalton A 9 desaturase still buried in the membrane (121). Microsomal fatty acid elongation is dependent on cytochrome b (4, 119). The res  quirements for this reaction are a fatty acid-CoA thioester, two carbon equivalents from malonyl CoA, ATP and M g 2 \ Cytochrome b supplies electrons in the reductive steps; kes  toacyl CoA to hydroxy CoA and trans a,p-unsaturated acyl CoA to elongated acyl CoA. Reducing  equivalents  are  supplied  primarily from  NADPH  NADH can drive the reaction at 30% efficiency (119).  19  for  de novo synthesis,  but  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 b involvement during the 1970rs (8,114,115,127.130.141-150). Results from reconstitution and s  immunological studies have ranged from obligatory, to insignificant, to even inhibitory  ef-  fects of cytochrome b on the reaction rate. A recent report by Bosterling et al cautions, s  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 inhibited 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 reductants in many processes (Table II). However, the list of reactions in which cytochrome b  t  is known to participate continues to expand. Although most efforts in elucidating the metabolic roles of cytochrome b have fos  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 b could greatly stimulate this s  activity (152), it was not until twelve years later that Hultquist and Passon established that a soluble form of cytochrome b occurs in erythrocytes and has a similar stimulatory efs  fect (153). Subsequent immunological studies showed that hepatic cytochrome b  s  and  reductase  erythrocytic methemoglobin reductase have similar antigenic properties and indicate that  methemoglobin reductase is in fact a cytochrome b reductase (154,155). As previously diss  cussed,  sequence  analysis  has  established  the 20  identity of  the  hepatic  and erythrocytic  cytochromes (10). A direct correlation between cytochrome b  s  content and methemoglobin  level has been observed in the aging red blood cell (156,157). Interactive computer graphics studies have now been used to generate a specific, hypothetical mode! for the interaction 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 complementary 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 activity arises from residual cytochrome i 5 and reductase which are not cleaved during maturation 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 b  f  is still controversial (162-165). Recent work has  suggested a potential role for calmodulin in the regulation of cathepsin D proteolytic activity 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 metal complexes  have  made  concerning electron transfer  such processes one  of  chemical reactions (169). In general, electron transfer  21  the  reactions between simple  best understood  categories of  between two coordination centers is  discussed  in  terms  of two mechanisms:  an inner-sphere  bridging ligand or molecule and an outer-sphere  mechanism  which  involves a  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 reasonable 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 coordination complexes and metalloproteins are considered (at least initially) rather than reactions 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 intensely colored chrbmophores is eliminated. Two general approaches electron  transfer  reactions.  have been developed in attempts to predict the rates of  Marcus  theory  employs  what  might  semi-empirical approach in which two known rate constants,  be  considered  the self-exchange  as  a  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 b kinetics presented here.  22  5  Marcus Theory Electron transfer reactions can be distinguished according to their degree of electronic 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 complex, 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:  tA-  +  A <= = = >  f A + A-  According to Marcus theory, the rate of reaction between two reactants predicted from the self-exchange  can be  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 =  kj,=  1 x 10n M '  1  s"1 (or 1 x 10")  self-exchange rate constant for reactant 1  k 2 2 = self-exchange rate constant for reactant 2  Kn=  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 reactions. 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 together 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):  AG*  In Eq 2 A G =  = £ J * ( 0 ) [l + (AG/4 AG+(0))]3  A G  0  +  Wp -  [2]  Wr, in which A G °  24  is the standard free energy change  (-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 barrier 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 reaction  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  self-exchange  approach  was  required  for  analysis  rates are not generally available  Wherland and Gray (174)  was to use  of  such  reactions  because  the  for proteins. The approach developed by  the measured  protein/reagent  cross-reaction  rate  constant, the reagent self-exchange rate constant, and the driving force of the reaction between 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 self-exchange  theory  may  be  used  to  produce  an  electrostatics-corrected  protein  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 nacorr 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 25  hydrophilic ligand  structure  (e.g.,  Fe(EDTA)2") TT-conducting  give  rise  ligands  to (e.g.,  low  ku  values  Co(phen)3?*)  produce  while  complexes  relatively  large  with values  hydrophobic, for  k„corr.  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 hydrophobic 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 reactivity, 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) interactions 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 exponentially 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 quantitative 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 experimental 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:  kab=  6.023 x 10-4 (2 T T / h) |Tab (r)|2 (4 TT ^  x (2  TT  x  3  T )->''  r/R ) exp[-(Ea - E^ - A )2]/(4 k^ T A )]  [3]  with the activation parameters AH*=  (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 become. Finally X, is the characteristic decay constant defined as:  1/2(0.72)-' = 0.7 %  Within the  Hopfield  [7]  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 cytochrome 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 b reductase (135) is thought to occur at s  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 b reveals that one of the heme t  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 environment 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  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 negative 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 b in light of the s  structural information that is now available, the present investigation reports the characterization of the electrochemical properties of cytochrome b and two heme-substituted derivas  tives. The structure of ferriprotoporphyrin IX and the deutero- and dimethyl ester derivatives 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 b  s  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 = - C H = CH2. R2= R l = H, R2= H; Dimethylester heme Rl= - C H = CH2, R2= -CH3.  30  H; Deuteroheme  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 combination electrode was used for all pH measurements. Conductivity readings were taken with a Markson Model 10 conductivity meter. Reagent grade chemicals were used throughout except as noted. Concentration of protein solutions was (Amicon-12 and -52)  fitted  achieved by ultrafiltration in stirred cells  with Amicon YM-5 (5,000 molecular weight cutoff)  mbranes. The concentration of ferricytochrome b  s  me-  was determined from its electronic ab-  sorption spectrum based on the published extinction coefficient (E 4 1 2 . 5 =  117,000 cnr 1 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 accept 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 operations 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 (measured 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 debris. 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. M i crosomes were stored at -70 ° C .  Trypsin Solubilization In a typical preparation, about 3 liters of the frozen microsomes were used. Subjectively, 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,  L-l-(tosylamido)-2-phenylethyl chloromethyl ketone (TPCK, Sigma #  and  0.07  g of  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 b The  protein solution  was  s  loaded  (Whatman DE-52®) that was equilibrated  onto a 7.5x5.0 cm column of DEAE-cellulose 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  ^° ^  valu  e ) greater than 2.5 were pooled for further purification. This  protein was dialyzed against cold, distilled water until the conductivity of the protein solution was 2.1 mmho or less. The protein solution was then rechromatographed over a second DE-52 column employing the same conditions. Fractions with an R value greater than 3.5 were concentrated to a volume of 2-3 70x2.5 cm column of Sephadex® NaPi, pH 7.2 pooled,  G-75  at 4 ° C . Fractions  concentrated  (as  above) and  ml. The concentrate was eluted over a  (superfine) that was  equilibrated  with an  5:A280  stored  in liquid nitrogen.  33  r a t  *°  °^  0 r  Fractions  with 20 mM 8reater with a  w e r e  ratio  between 4.0 and 5.8 were pooled, concentrated and rechromatographed over the gel filtration column as described above. Approximately 60-70 mg of cytochrome b, with a R value >  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 b  5  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 2-butanone  (Burdick and Jackson) and gende  by addition of 1 volume of  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 organic layer. The apo-protein was then dialyzed (Spectra-Por® tubing) against a liter of 0.6 mM  NaHC0 3  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 NaHC0 3 . The recovered protein was centrifuged for 3 min in a Silencer model H-25F1 microcentrifuge (12,000 rpm) to remove  a  small  amount  apo-cytochrome b was > s  apo-cytochrome b  s  of  material  which  0.5 mM (E 2 ! 0 =  did  not  redissolve.  The concentration  of  10,500 mM" 1 cm"1 (186)). All manipulations of  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 b . 5  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 b  s  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 (E 3 8 3 =  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, reconstitution was complete in 1 hour. The protein solution was adjusted to pH 7.2 and concentrated 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 centrated. The deuteroheme-substituted (E, 03 =  *°  ~  ^  P°°'e^  w e r e  *  ~  c o n  122,000 M " 1 cm"1 (186)) and stored in liquid nitrogen.  Dimethyl  ester protoporphyrin IX  was  Cytochrome b  obtained  s  commercially (Porphyrin Products  90181) and was checked for purity by thin layer chromatography  TLC®  a n c  derivative was recovered with a yield of 80-85%  Preparation of Protoheme IX Dimethyl Ester-substituted  Lot  r a t  (Gelman Instant  system) (194). A single species was observed both in solvent I (2,6-lutidine:H20  20:1) with R f = A  1.1  0.92 and in solvent II (hexane:CHC13:methanol 1:1:0.2) with R f = molar equivalent of DME-heme  to apo-cytochrome  b  t  0.72.  (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 b completed,  the solution was placed  s  maintained on ice. When the addition was  in a refrigerator  for  24 hours. An additional  0.15  equivalents of DME-heme was then added, and the solution was returned to the refrigerator 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 (superfine)  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 reconstituted protein was >  r a u 0  °^  u n e  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 described 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 b  5  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 b  s  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 (Dilut-it®,  Baker) was added  spin bar.  The pH was monitored as  0.01  N NaOH  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 spacers 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 electrode 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 together 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  water bath (Lauda electrical  RM-3). This  salt bridge connected to a refrigerated, circulating  bridge was  Tilled with a saturated  KCI solution, and  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  above. Temperatures could be reliably controlled over the range of 5-40  described  ° 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 triply recrystallized (196) Ru(NH3 ) 6 Cl 3  (12 u M) (Alfa) to facilitate equilibration of the protein  with the working electrode was introduced into the OTTLE cell. After temperature equilibration, 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 oxidized. Potentials measured against the SCE were converted to the hydrogen scale by adding the factor 244.4 mV (25 ° C ) (197).  Kinetics Measurements The reduction of cytochrome b and its heme-substituted derivatives by Fe(EDTA)2" s  was studied with a stopped flow spectrophotometer. Buffered reductant solution was prepared anaerobically at controlled pH and ionic strength (198). Humidified, oxygen-free nitrogen gas was prepared from commercial bottled stock by passing the gas through a train of  two vanadous bubblers (199)  The  and one photo-reduced methyl viologen bubbler (200).  latter bubbler contained 50 y M 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 vigorous 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 temperature 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 stopped-flow  motor to permit acquistion of spectra of the contents  of the  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 maximum  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 equipped with two 8"  2/10  16 bit minicomputer (Data  General Corp, Westboro, MA)  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 spectra were written in collaboration with OLIS.  42  Fig 5. Schematic drawings of modifications to the stopped-flow spectrophotometer. All dimensions 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  1.70  ".006 O ring  2.25  166  .385-ir  _J  .07 5 ^ri— -; -j.325 •1.06  3 / 8 ' PTE  .330 .2 85  r  — 1.20  .128  i  2/32' PTE  I  .575 3.35 -  h  . 1 90  .325 .315  f-.078  .262  .3 1 7 .2 12 .137  H  .063  .240 •  3.76  .150  B  \  I  .325  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 D M E - i j  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 constants were obtained from weighted linear least-squares analyses of concentration dependences. Activation parameters were obtained from weighted linear least-squares analyses of Eyring plots.  Reaction of Cytochrome fc5 with Deuteroheme-substituted Cytochrome fc5 The stopped-flow  oxidation system  of  deutero-fc5  by  the  native  cytochrome was  studied  with  the  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. Consequently, the choice of this wavelength was based  on computer-assisted  spectroscopy. The electronic spectra of native and deutero-£ 5  optical difference  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 b (1 uM) was mixed with res  duced deutero-fcj (10-30 uM) to assure psuedo-first-order dizes readily in the presence  of dioxygen, so  45  precautions  conditions. Deutero-65 autoxiwere required to assure its  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 desiccator and dried for 48 hours before use. Activated platinum black was stored in vacuo. A 5PM  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 efficiently retained by a single, thick glass-fiber filter (Gelman Type 66075). To ensure anaerobic conditions, all transfer Na2Sj04  lines and stopped-flow  surfaces were flushed with 3 mM  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 syringe barrel of the stopped  to the upper  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 concentrated 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 determined 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 previously by Strittmatter DME-fcj  and Ozols (186). The absorption spectra  of reduced and oxidized  are virtually identical to those of the native protein. The principal changes in  the DME- b  s  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 derivatives 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 b  s  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 approaching 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 monitored 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. Cytochrome 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 y M , 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- b (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. 5  51  ABSORBANCE ABSORBANCE  ABSORBANCE  Table IV. Extinction Coefficients from Electron Absorption Spectra (Fig 6)t Soret  £band  a band  oxidized  reduced  reduced  reduced  Cytochrome b  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)  Protein 5  t  values are reported as wavelength {nm}(extinction coefficient) {M_1 cm-1}.  ± values normalized to be equivalent to the native cytochrome b oxidized Soret peak. s  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-6 5 . All derivatives were well behaved and could be cycled repeatedly between oxidized and reduced states with no detectable 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 experimental 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 associated slope measured at each solution condition for each cytochrome derivative is presented in Appendix A. As cytochrome b has a net negative electrostatic charge and the mediator is posis  tively charged, the effect  of mediator concentration on midpoint reduction potential was  evaluated to determine whether the use of Ru(NH3)6 protein. Variation of the [b ] :[Ru(NH3)6 s  '*  24 3  perturbed the behavior of the  ' *] ratio over several values in the range from  2+ 3  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 expression (210):  E ° , .= obsd  where  E ° - RTA/F (q  2  ox  n  - qM  2 ) f( yp ) red  [8]  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 (Q ox = clrec}  +  Various  forms  of  (210-212). The difference "extended  ion  size",  non-linear regression  R ,  the  function  f( y )  were  evaluated  as  previously  suggested  between these functions is equivalent to an evaluation of the on  the  calculated  protein  charge.  In  each  case, weighted  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 conductivity 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 Potential. Different "Extended Ion Size" Radii Incorporated into the Function y ' ' V (1 + (Rp .329)y''2).t  R =6.08 P  R =3.04 P  R =0 P  -36.8  -22.1  -17.7  -10.6  20.8  24.0  27.1  34.3  Protein  Parameter  R =17.0$ P  R  Cyt-fcj-  E  -39.2  y  = 0, mV  E y = inf, mV 20.5  Deutero- 6S  DME- ^  =15.5  q M ox  -5.95  -5.24  -1.76  -0.97  -.24  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 M ox  -6.6  -6.5  -2.1  -1.2  -.032  E y = 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  -8.6  -7.5  -2.2  -1.0  -0.19  q  t  P  ox  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 protein 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 following equation: E  =  E +  R T 2.3nF  m  The  In K red + [ H * ] K + [Hi ox  [9]  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 manner of Schejter et al (216). As shown above, cytochrome b and  s  is a highly charged molecule,  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 between the oxidized and reduced states of the protein can be divided into two terms at zero ionic strength: an electrostatic term, A G ° j , corresponding to the free energy change caused  by adding  one  electron to the oxidized protein, and a  non-electrostatic term  AG° ,, that involves formation of metal-liaand bonds, lieand-field stabilization for calcunonel lation of the electrostatic components are:  K r  e  ta x 0  -  1  }  1  (  R  P  AG°,= el  e2 N v(1-q )/(R M oxy v p  ASel=  e2 N £  AHel =  e2 N / R  (q Q x -  D  )  [  D) = A H ° - T '  1) / (R  D2)  (1 - qox)  (a-  58  A S°  1  1  0  ]  [11] J  [12]  2f> T)/D 2  [13]  •FlEp  3in  =rt <7-3Wa (0) :ro = " 5  -OI/Q  01 UJ  S3IBbS-lSB3[ 'IB3UIJ . 'pSiqgtSM  8 3IB S3UI] piJOS JA{ TO -  ^-ojsansa ( ) -Vi ?'0 = (nj -Vi VO = (•) '<? auioiqo :(3ieqdsoqd) QV Hd IB [Bnusiod uoryonpsi sqi jo souspusdsp siniEisdiusj. 'II 3 i J v  rt  rt  s  Table VI. Parameters Calculated from an Analysis of the pH Dependence of the Reduction Potential (25 ° C ) . Protein  Ionic Strength c  pK ox  y  y  pK , red  E  (A/)  (mV vs NHE)  Cytochrome b,  0.1  5.8  6.2  11  Cytochrome Z>,  0.5  5.7  5.9  25  Deutero- b  0.1  5.9  6.2  -13  .0.1  5.6  5.8  81  s  DUE-b,  The variables used in these equations are: N= R =  P  Avogadro's number, e=  electronic charge,  distance of closest approach, D = dielectric constant of water, p= DebYe-Huckel con-  "  stant (0.4), and a = lectrostatic  the constant 196.9. The contributions of these electrostatic and none-  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. Electrochemical 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 reduction potentials at lower ionic strengths by this technique were prevented by extremely slow  electrochemical  equilibration.  A  listing  60  of  the  midpoint  reduction  potential  and  Fig 12. Dependence of Fe(EDTA) 2- reduction potential cn pH [u= 0.25 M Nal, pH 7] .The solid line has no theoretical significance.  0.25 M phosphate, +  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 =0.1  y  M  Cytochrome b Obsd  DME- b  s  =0 M  y  =0.1  M  y=0.1  AH° M  u=0.1 M  -37 mV  5.1 mV  -37 e.u.  -11 kcal/mol  Elec  -80  -80  -5.1  -2.5  Nonelec  43  85  -32  -8.5  Obsd  -99  -44  -45  -12  Elec  -82  -82  -5.2  -2.6  Nonelec  -17  38  -40  -9.4  Obsd  8.3  70  -31  -11  Elec  -116  -116  -7.3  -3.6  Nonelec  124  184  -24  -7.4  s  Deutero-65  y  AS°  E° m  Em  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 program 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-6 5 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 regression fit to a pseudo-first-order reversible rate equation (219,220):  f(t) =  Ajjbj {(Keq + 1) ffKeq Keq  where Q =  [1 +  + n + expfk, O tfl -1)} (Keq + 1) exp(k2 Q t)]  1 + 2/Keq  and, A obs is the observed change in absorbance at time t and Keq = The  [14]  k 2 /k. 2 (vide infra).  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 N a 2 S 2 0 «  to the stock protein solution and record-  ing (Cary 219 spectrophotometer) the absorbance change at the wavelength used in monitoring 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  •  exp(- K RQ1 1 + k R ^  - Z,Z, e; {exp(- K R3) + 4eeo:tfkTR2--1+IcRl  [15]  where the pre-exponential constant evaluates to 3.576 at 25 ° C , y = protein radius, R2 is the radius of Fe(EDTA)3on Fe(EDTA)1-  (-2),  Values estimated for k  R = R) + R2, and Z and Z  t  x  0.329 P !'-\ R i is the  (4 A.), Z 2 is the net electrostatic charge  is the net electrostatic  charge on the protein.  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 _ 4eeo 2kTu  {111 + Z2i - (Zl 1 + R^ 1+R2K  where the constant has a value of 1.174  + Z2)?  [16]  ;  1 + RK  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.  4 e eo7T2kT where ?i cos 0 angle 8  -  P,ros9  K Jk)  ( I + K R,)  eR  [17]  describes the effect of a protein dipole Pi on molecule 2 (P2 = 0) at an  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).  to  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 ) . Wherland-Grav (Eq 15)  DebyeHuckel (Eq 16)  van Leuween (Eq 17)  Z,  k„  Z,  kn  kn  dipole (Debye)  R = P  15.5 A  Cyt-fc  -7.3  0.41  -13.5(8) 0.25  420(20) -12.8  0.11  Deutero- bs  -7.2  0.13  -12.2(2) 0.17  139.5(2) -9.6  0.19  DME- bs  -4.3  45  -6.9(2) 97  4310(66) -3.9  200  R = P  17.0 A  f  266  212  110  102  inf  -10.2  •  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  138(2)  0.14  111  112  DME-b5  -5.1  35  -8.0(3) 73  180  inf  -25.2  t  0.09  -10.8  4270(70) -4.3  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 ---->  Fe(EDTA)2" + cyt-6,  products  >  products 67  Fis 15. pH dependence of cytochrome reduction rate [25 ° C , phosphate]: (n) cytochrome 6,W(V= 0.5 M); ft) Deutero-b (y= 0.1 M); (0) DME-fcj (u= 0.1 M) (rate * 10'5). s  CL  68  cyt-fc-H*  < = = = = > cyt-fc5 + H*  In this case, the following relationship can be derived (81): k13 =  k. [Hj  +  K  K  [18]  " "lrT]—+"^ir*~ a  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,1 J = M  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 derivation 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) 0.5 M ) , 1.1 x 10° ±  tive cytochrome b (u= s  102 ±  0.49 for DME-b  5  (u=  1.69 x 101 ±  0.4 for deutero- b  5  (u=  0.26 for na-  0.1 M ) and 7.98 x  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 temperatures (pH 7.0). In the case of native cytochrome b , the temperature dependence of the s  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  (M)  k  b  (protonated  (unprotonated  form)  form)  (M" 1  s-1)  (M-1 s"1  Cytochrome b  0.5  5.85  720  250  Deutero- b  0.1  5.84  390  87  DME-6 5  0.1  5.74  4400  1800  s  s  Table X. Activation Parameters for the Reduction of the Cytochrome Derivatives in Phosphate Buffer. Protein  Ionic Strength  (kcal/mol)  (e.u.)  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)  (M) Cytochrome b  s  0.5  AS*  AH*  pH  Deutero- b  0.5  7.0  16.8 (1)  -26.5 (4)  DME- bi  0.1  7.0  4.1 (1)  -29.9 (2)  s  Cytochrome b /Deuteroheme- substituted Cytochrome bi Cross Reaction s  The kinetics of cytochrome b pseudo-first-order  s  reduction by deutero- b (red) were studied under s  conditions with the reductant in excess. The electronic spectra of oxi-  dized and reduced deutero- b  s  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 cytochrome b  s  and deutero- b with ionic strength is shown in Fig 18. The data were fitted s  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,Z  2  x  q 2 /(4TTe eo  k  T  R)  /  + (ZP)(1 + K R ) + (PP)(1+KR)2) q2 f(k)  [20]  (u)  where  ZP = (Zi P cos 6 + ZP,cos 6 ,) 2  PP = P, P  2  f(k) =  2  2  cos 6 ,cos 6 : /(q R)2  (1- exp(-2KR2))/(2 < R 2 (l+< R0)  and, q is the elementary which interacts at the angle  charge,  P (in Debye) is the dipole moment of the protein  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 (reduced - oxidized). Cytochrome b , 0.1 M (- - -);Deutero-i5 at 10, 20 and 30 uM ( ). The extinction coefficient for the changes in absorbance at the deutero- b isosbestic points are 5.0 M" 1 cm"1 (405 nm) and 70 M " 1 cm-1 (429nm). $  s  5  A  404.5  405.5  427  429  W A V E L E N G T H (nm)  73  431  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, Z 2 = 5015 M ' 1 s"1.  With this analysis, P,cose,=  -6.5, R i = R2 =  15.5 %. and k =  109(14) Debye and P 2 cose 2 =  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  thermodynamic parameters calculated ± AH -  is shown in Figure  19. The  from a weighted least-squares analysis indicates that ±  16+1  kcal mol"1 and AS — 11 +  4 e.u. Autoxidation of cytochrome b  s  lowing its reduction was not found to be a problem as the observed  fol-  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- b  s  technique. With the platinum black technique, a substantial  reduced by the photochemical proportion of ferrodeutero- b  s  underwent autoxidation during the 25 min thermal equilibration period despite the precautions described deutero- b  s  previously. The photoreduction method was successful  in maintaining the  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 b  illustrated in Fig 20A. A similar analysis of DME-fc5  is  is illustrated in Fig 10B. The de-  pendence of heme-ring hyperfine shifts on pH is illustrated graphically in Fig 21.  75  s  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)  z,t  z.  van Leeuwen (Eq 20)  Wherland-Grav (Eq 15)  z,t  z2  k12±  z,t  z,t  (M-« s- ')  Dipole 1 Dipole (Debye)  (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)  -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)  R =17.0 A P  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) Ul(t) B  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)  E  Q a  CO  S a a.  78  Fig 21. Hyperfine shifts, of selected peaks in the low-field NMR (A) and DME-fc, (B). Conditions as in Fig 20.  79  spectra of cytocrome bs  DISCUSSION Oxidation-Reduction  Equilibria  of  Native and Heme-Substituted Cytochrome b  t  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 investigation  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 (Stellwagen (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 parameters, vary each independentiy and determine the relative effect of each factor. Spectroscopic and crystallographic analyses of cytochrome b permit a qualitas  tive examination of the factors which control the reduction potential in this hemeprotein. The two histidine ligands are fixed in a nearly coplanar orientation by hydrogen bonds to the polypeptide backbone. This alignment of ligand planes is consistent with a preferential  derealization of unpaired electron spin from the oxidized  iron into the TT orbital system of the porphyrin ring (227). This stabilization contributes to a lower reduction potential. The coordinated histidine also interacts electronically with the' iron atom. As the strength of the hydrogen bond between the histidine N 6 1 nitrogen atom and the polypeptide backbone increases (equivalent to deprotonation of the imidazole ring) the electron density at the coordinating N e 1 nitrogen 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 b is low spin in both the oxidized s  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 crevice of cytochrome bi was found to be nearly identical to that of other c-type cytochromes, 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 b to the relative great exposure of its heme s  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 b . s  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 influence the electronic distribution in the heme ring. In cytochrome b , many aromatic s  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 support of the Argos-Mathews model; as a consequence of esterificatipn of the heme propionate, the proposed coulombic stabilization of the ferric protein would be abolished and the reduction potential would increase. A similar shift"-in reduction potential ( + 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 aqueous 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^= is incorporated into myoglobin (237)  -240 mV) (237). However, when deutero-heme or cytochrome b  s  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- b relative to the native pros  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 Ru(NH 3 y'' J *  presumably  results  a dependence of the reduction potential on  from, the 82  inability of  the  redox-linked site to  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 mediator was negligible in comparison to that of the supporting electrolyte. The  net charges estimated  for cytochrome b  s  and for deutero- b  s  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 cytochrome 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. Uncertainty in this approach arises from simplifying assumptions required when estimating the net charge from the amino acid sequence. In both Euglena cytochrome c and D M E - 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 dependence of the reduction potential. Studies on horseradish peroxidase and c-type cytochromes suggest a mechanism  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 interaction between the histidine and the iron is sufficient to account for this observation. An alteration in the axial  ligand field strength may also be involved. The  change in reduction potential observed with cytochrome b and its derivatives is s  +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  84  for this effect An  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 discussion is limited to a few measurements  of c-type cytochromes (215). The most  striking feature of these data is that A H ° for cytochrome b is 4-5 kcal/mol more t  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 (calculated at q ^ox  =  5.1, pH 7.0) y  are A S °  '  .=  -33.5  e.u. and A H °  nonel  .=  -14.3  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 b and its derivatives would argue that the observed s  difference with cytochrome c is due to redox linked conformational changes in the latter protein, and not to a coulombic stabilization of cytochrome b  s  by the heme  propionate. Significandy, the non-electrostatic entropy component is different for each derivative. 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 b  s  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): -  A +  ki k, D- < = = >. A D - < = = >  k3 -AD <==> - A + D  k-s  k.3  With cytochromes, this reaction is studied by following changes in the electronic absorption  spectrum  (k2/k.j).  Under  of the protein, which pseudo-first  order  occurs  conditions  during the electron transfer step  ([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 residues on the surface of the protein is spherical. With increased electrolyte concentration, 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 reduced, 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 surrounding cytochrome c has been reported (249). From this analysis, electrostatic forces appear 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 effect  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 significantly reduced. In the DME-Z^ derivative, the charge at the heme edge is decreased by two, and the discrepancy between the calculated charge and the sequence charge is decreased presumably because of reduced electrostatic interactions. These results provide reasonably direct evidence that electron transfer occurs through the exposed 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 previouslyestimated (570 debye) (223). In addition, the dipole moments of the derivatives are reduced. The accuracy of these estimations will be clarified ultimately when the dipole 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 relationship to estimate the net electrostatic charge on a protein from small reagent redox 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 regard.  88  in this  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 b reduction reaction over the s  pH range 5.5-8.0. This relationship is linear in either the A H i  +  - AS  T  plane or in  ±  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 difference between the reduction potentials of the reactants. Both Fe(EDTA)2" (Fig 12) and cytochrome b (Fig 10) exhibit a dependence in their reduction potentials with s  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 reduction 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 protein 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 distance 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  LQ  O  LQ  O  K  K  <S  <5  1  LO  ( |OUJ/|D3>l)jHV  90  I  O  I  determined at this time.  Temperature Dependence The reduction  activation parameters measured reaction  non-electrostatic  may  also  be  from the temperature  regarded  as  composites  dependence of the  of  electrostatic  and  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 reaction, 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 b heme propionate. This explanation appears to be incorrect for s  three reasons. First, DME-b  s  Second,  has the same activation entropy as the native protein.  the cytochrome c parameters were  not measured  under identical solution  conditions, so the electrostatic component may account for some of the observed difference. 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 of  the  apparent protein self-exchange rate, k n c o r r ,  Marcus  relationship  (Eq  1)  to  the  reduction  estimated rate data  by an application 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 91  and its derivatives is  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"') chrome  1.2 x 10' for cytochrome c and 2.3 x 10! for cyto-  CJJI.  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 c 3 j l . In the current work the increase in reaction rate with DME-6 be correlated with a decrease in the distance  5  can  (by 0.7 A.) across which electron  transfer occurs.  Electron Tunneling Theory Analysis of the Fe(EDTA)3" reduction reaction data by the multiphonon electron 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 cytochrome c the vibronic coupling parameter was fixed at 0.5 eV (258). As a first approximation, 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  the theory.  92  distance is accomodated within  Table XII. Marcus Analysis of Fe(EDTA);"reduction rate data Units  Cytochrome bi  Deutero- b  113  36.4  5.1  -44  -13.5  -12.3  3.7  3.85  INPUT k„ mV vs NHE  El Z,  OUTPUT . corr kn  M" 1  \vl2  kcal/mol  1.15  1.05  w21  kcal/mol  0.618  0.567  wll  kcal/mol  2.04  1.70  A'Gi|COrr  kcal/mol  16.7  16.6  AG0  kcal/mol  2.02  3.15  f factor  0.018  0.028  K  0.0033  0.0049  M"  116  67.5  M"  790  213  kcal/mol  14.6  14.9  mV vi- NHE  -64.3  -115.6  A  2.7  2.9  s"1  eq oo  K„ 00  oo  AGn E  m  rpt t  (*») '  v  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  (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  P  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- b /Fe(EDTA) 2 - ; s  (D)cyt- c/cyt- b ;(E) cyt- b /deutero- b . s  b  }  94  Metalloprotein Electron Transfer Self-exchange Reactions General Considerations Direct measurement of metalloprotein electron transfer possible with optical techniques because the spectra idendcal. Consequentiy, the only have involved  self-exchange  rates is not  of the reactants  and products are  meaurements of this type available  for metalloproteins  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 investigated 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 studies. The present work develops an alternative approach in which the cross-reaction between 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 protein. 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  ku0rT  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 electronic absorption spectrum of deutero- b relative to that of the native protein allows the 5  reaction between the two proteins to be monitored optically.  Ionic Strength Dependence The rate of electron transfer between native cytochrome b and deutero- b increases 5  significandy  with ionic strength as  expected  s  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 interactions, hydrogen bond formation and conformational rearrangements are not expected to stabilize 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 ± S' . 1  From the reaction with Fe(EDTA)2",  kn  =  1.4 x 102 M " 1 s'1.  0.4 M " 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 cytochrome bi has been estimated at 6.0 x 102 M " 1 s"1 (u = 0.1M, pH 7, 25 ° C ) by Marcus 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 (k n = k22). The consistent self-exchange rate constants estimated for other hemeproteins under similar conditions are (189); k n 107 for cytochrome c  5SU  ( M - 1 s~') =  2.8 x 10s for cytochrome da,  4.6 x  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=  Values from  0.1 M).  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  -7.5  -13.2  z  -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  4.31  4.31  k22  4.31  4.31  600  600  600  600  600  3  k22  2  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  kI3  3515  6566  k22  10.2  74.3  10.3  10.6  16.6  16.6  13.1  13.1  13.1  13.1  13.1  48.8  46.2  G „  10.7  11.1  f  -.01  -.008  -.008  -.008'  -.01  -.01  -.01  -.01  -.01  AG°° AE A  OO  AG  1 2  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 b  s  (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- b /cytochrome b electron transfer is taken as evis  t  dence that the presence of the photo-activated reducing system did not influence the reaction mechanism.  Significance of Results Calculation of the degree of heme exposure in cytochrome b by Stellwagen indis  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 inconsistent with such a prediction. Presumably the slowness of the cytochrome b  s  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 rearrangements 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 dependent upon chemical and structural variables. As clearly shown by this work the heme propionates can funcdon to control electron transfer, not been fully recognized before  the significance of this finding has  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 cytochrome participates is regulated by the presence of this propionate. 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S t a n d a r d d e v i a t i o n s a r e i n d i c a t e d  CYTOCHROME  b  5  PH  Temp  I  Med  5.50  25.0  0.1  0. 1  Potent i a l 13.3  ±  0. 1  slope 59.0 +  1 .0  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  5.67  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  0. 1  5. 1  0.4  58.3  0. 6  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  -11.8  0.2  63. 1  0.2  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  6.51  7.00  7 .00  10.0  10.0  0.5  11 7  15.5  0.4  60.6  0.5  16.6  0.5  58.3  0.6  30.0  13.6  0.4  60.7  0.5  35.0  5.4  0.4  62.6  0.5  0.03  -4.5  0.4  59.6  0.6  0.05  0.0  0.8  60.0  0.1  -2.3  0.6  59.6  0.7  0.2  9.2  0.3  58.2  0.4  0.3  14.0  0.2  59.5  0.3  0.4  17.8  0,3  59.2  0.4  4.4  0.5  62.6  0.6  5.9  0.7  59.3  0.8  0.5  6.1  0.3  60.1  0.3  1 .0  5.7  0.4  59.6  0.5  2.0  4.2  0.3  59.7  0.4  2.5  4.7  0.3  59.9  0.4  3.5  4.7  0.3  58.9  0.4  3.5  5.3  0.2  59.9  0.5  15.0  6.7  0.4  60.3  0.5  0.1  4.0  0.1  60.0  0.2  15.1  0.2  59.9  0.3  0.5  63.1  0.7  0.9  57.0  1.0  25.0  7.00  25.0  0.1  7.00  7.50  25.0  25.0  0.1 0.5  8.00  25.0  0. 1 0.5  0.1  3.414.1  DEUTEROHEME-SUBSTITUTED CYTOCHROME b  s  ' pH  Temp  I  Med  5.56  25.0  0.5  0.1  Potent i a l  slope  -18.2  0 .9  60.0  2.0  5.67  -18.7  0.2  58 . 0  0.3  5.76  -19.5  0. 2  57.7  0.3  5.87  -21.9  0. 1  57 . 1  0.2  6.13  -24.8  0.4  58 . 1  0.5  6.47  -27.6  0.4  59'-. 1  0.5  6.80  -30.2  0.2  59.3  0.3  -27 .9  0.2  56.7  0.3  -26.5  0.9  56.0  1 .0  -26.6  0.3  54.1  0.4  -30.3  0.5  56.9  0.7  -35.3  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  0.05  -52.3  0.2  59.0  0.3  0. 1  -48.8  0. 1  58.9  0.2  -39.5  0. 1  58.0  0. 1  1.0  -49.4  0.2  58.9  0.3  0. 1  -39.5  0. 1  58 .4  0.3  0 .2  -41.2  0.2  59.6  0.2  0.3  -38 .2  0 .3  60.2  0.6  -25 . 1  0.2  59. 3  0.2  -30.3  0.6  59.6  0.9  -31.8  0.3  60.5  0.4  7.00  10.0  0.1  15.0  .20.0  7.00  25.0  0.01  0.4  11 9  0.5  7.00  30.0  0.1  35.0  7.55 8.09  25.0  0.1  -29.0  0.6  60.4  0.9  -33.1  0.3  60.6  0.5  -50.9  0.2  60.5  0.2  -56.1  0.2  60.0  0.3  -34.2  0.4  62.7  0.6  -33.4  0.2  59.6  0.3  1 20  DIMETHYLESTERHEME-SUBSTITUTED pH  Temp  I  Med  5.46  25.0  0.1  0.1  CYTOCHROME  b  5  Potent i a l  Slope  77 . 4  0. 1  58 . 3  0.2  5.66  75. 1  0.3  58 . 2  0.2  5.76  75. 1  0.6  58.8  0.2  5.97  73.0  0.1  59.5  0.5  6.27  71.8  0.3  58 . 5  0.9  6.50  69. 7  0.7  59.0  1 .0  84.9  0.6  58.4  1.6  10.0  79.3  0.3  54 . 1  0.7  11.0  74.2  0.6  57.8  1.2  15.0  76.8  0.5  54.0  1 .0  20 . 0  74.9  0.4  58 .6  0.4  20.8  75.0  0.6  59.6  0.7  68.0  1 .0  61 .0  1 .0  69.6  0.4  58.8 '  0.8  0.2  73.4  1 .3  61.7  0.7  0.3  77.8  0.9  58.0  1 .0  0.4  82.5  1 .2  57.6  0.3  0.5  84.6  0.3  59.2  0.6  0.1  69. 3  0. 1  60.0  0.2  69. 1  0.3  61.3  0. 1  33 . 4  64 . 3  0.9  63.0  1.0  35.0  61 .7  0.9  62.. 3  0.7  68.4  1 .0  58.0  2.0  68 . 2  0.6  59.4  0.5  0.3  54.6  0. 1  7 .0.0  7.00  7.00  7.52 7.70 7.97  8.4  25.0  30.0  25.0  0.1  0.1  0.1  0 .1  65.8 12 1  APPENDIX  B  Midpoint  reduction potentials  ( v s N.H.E.) f o r F e ( E D T A ) " 2  sodium p h o s p h a t e  buffer.  contributed  from  N a l . A l l measurements  m e t e r s were  obtained  t o the N e r n s t  Ionic  from  s t r e n g t h i s 0.5 M w i t h  a weighted  equation. Standard  a r e a t 25.0  linear  deviations  0.25  Reduction Potential (mV)  5.50  115.7  6.00  ±  least-squares  f i t  are i n d i c a t e d  (n >  Slope (mV)  0.7  60.8  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  M  °C. P a r a -  6) . pH  in  ±  0.8  APPENDIX C F i r s t - o r d e r r a t e c o n s t a n t s f o r t h e r e d u c t i o n of t h ecy t o chromes l i s t e d by F e ( E D T A ) " i n p h o s p h a t e b u f f e r . 2  CYTOCHROME pH 5.50  5.50  6.00  6.00  b  s  Temp  +  Fe  kobs  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  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  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  0.0125  6.65  0. 08  0.01  5.06  0.08  0.0075  3.457  0.004  0.005  2.280  0.003  0.0025  1 . 1 67  0. 003  34.4  25.0  34.5  25.0  I 0.5  0.5  0.5  0.5  1 23  6.5  6.5  7.0  7.0  0.00125  0.501  0.005  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  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  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  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  40.0  25.0  40.2  25.0  0.5  0.5  0.5  0.5  124  7.0  7.0  7.0  7.0  7.0  25.0  25.0  25.0  25.0  25.0  0.4  0.3  0.2  0.1  0.05  0.01  2.70  0.06  0. 008  1 . 97  0.04  0 . 006  1 .49  0.03  0.004  0.969  0.007  0.002  0.491  0. 003  0.001  0.221  0.003  0.01  2.081  0.004  0.008  1 .686  0.004  0.006  1.142  0.004  0.004  0.841  0.002  0.002  0.456  0.005  0 . 001  0.251  0.005  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  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  0.0012  0.053  0.001  125  8.0  8.0  0.0009  0.040  0.001  0.0006  0.025  0.001  0.0003  0.0145  0.0001  0.00015  0.0078  0.0001  0.0125  5.54  0.04  34.4  4.56  0.02  29.8  3.92  0.02  25.0  3.37-  0.04  20.1  2.985  0.008  15.5  2.53  0.01  0.0125  3.28  0.02  0.01  2.548  0.005  0.0075  1.89  0.01  0.005  1.24  0.01  0.0025  0,638  0.004  0.00125  0.291  0.005  40.1  25.0  0.5  126  DEUTEROHEME-SUBSTITUTED CYTOCHROME b  s  pH  Temp  I  Fe  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  0.008  1.72  0.03  0.012  2.10  0.04  0.016  3.22  0.04  0.020  4.33  0.04  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  0.002  0.348  0.005  0.004  0.510  0.007  0.008  1 .270  0.021  0.012  1 .75  0.04  0.016  2 . 32  0.04  0 . 020  2.86  0.04  0.000125  0.0048  0.003  5.90  6.14  6.5  7.0  25.0  25.0  25.0  25.0  0.5  0.5  0.5  0.05  1 27  kobs  ±  7.0  7.0  7.0  7.0  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  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  0.00025  0.0139  0.0005  0.0005  0.0272  0.0005  0.001  0.0468  0.0005  0.0015  0.0600  0.005  0.002  0.081  0.001  0.0025  0.093  0.002  0.0005  0.035  0.003  0.001  0.060  0.006  0.002  0. 126  0.005  0 . 003  0. 189  0.005  0.004  0.2526  0.0001  0.005  0.327  0.001  0.001  0.074  0.005  0 . 002  0.155  0.005  9.8  25.0  25.0  25.0  0.5  0.1  0.2  0.3  1 28  7.0  7.0  7.5  8.0  25.0  25.0  25.0  25.0  0.4  0.5  0.5  0.5  0.004  0.315  0.005  0.006  0.479  0.003  0.008  0.609  0.007  0.01  0.804  0.013  0.001  0.095  0.005  0. 002  0. 19  0.01  0.004  0.36  0.01  0.006  0.566  0.018  0.008  0.719  0.005  0.01  0.980  0.024  0. 002  0.276  0.003  0.004  0.518  0. 003  0.008  0.914  0.007  0.012  1 .30  0.03  0.016  1 .66  0.03  0.02  1 .96  0.02  0.002  0.21  0.002  0.004  0.391  0.003  0.008  0.804  0.006  0.012  1.18  0.012  0.016  1 .49  0.025  0.02  1 .92  0.035  0. 002  0 . 1 55  0.003  0.004  0.314  0.003  0.008  0.692  0.007  1 29  0.01  0.012  1 .04  0.016  1.41  0.02  0.02  1.81  0.04  1 30  DIMETHYLESTERHEME-SUBSTITUED CYTOCHROME b  s  pH  Temp  I  5.45'  25.0  0.1  5.65  5.74  5.94  Fe  kobs  ±  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  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  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  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  13 1  0.0025  6.930  0.02  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  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  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  0.0025  2.84  0.355  15.5  3.837  0.35  19.9  4.287  0.344  24.8  4.941  0.342  28  54  00  00  0.05  5.95  0.1  1 32  25.0  7.00  7.00  7.00  7.0  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  0.0025  5.626  0.338  34.4  6.31'6  0.041  39.1  7.003  0.041  0.0005  1.319  0.016  0.001  2.540  0.015  0.002  5.304  0.014  0.003  7.986  0.022  29.5  25.0  25.0  25.0  0.1  0.2  0.3  0.4  0.004  10.42  0.04  0.005  12.94  0.02  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  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  7.0  7.54  8.03  25.0  25.0  25.0  0.5  0.1  0.1  0.008  26.70  0.02  0.010  33.01  0.02  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  0.00025  0.466  0.002  0.0005  0.900  0.011  0.001  1 .800  0.0015  2.730  0.019  0.002  3.700  0.021  0.0025  4.650  0.023  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  0.017  APPENDIX D Derivation From t h e  of  Eq  19.  standard  relationship  for  free  energy  AG = - n F ( A E )  [1]  which a f t e r s u b s t i t u t i o n v o l t s ) (Ref 267) AG=-23.06 From t r a n s i t i o n k,2 where  =  for  the  constants  (AE)  state  t h e o r y at  (kB T ) / h  h = P l a n c k ' s constant= =  T = Temperature  (K)  k12 rearranging  = rate  in  kcal/mole  [2]  ,  [3]  G12/RT)  constant^  R = Gas c o n s t a n t  (potential  25 ° C  exp(-  kB = B o l t z m a n ' s  is  3.298  1.584  x  x  10"2U  1 0"3"  cal/deg cal/sec  1.9872 c a l / d e g mol =  constant  298  (M~ 1  s"1)  3 to  -In  (h k , 2 / k B  T)  =  AG12*/RT  or ln(kB/h) and  substituting AG12*/RT  -  ln(k,2/T)  the  (Ref AG12*=  [4]  constants  = 23.76  -  = 29.46 or  = AG12*/RT  (-5.697) -  ln(k,  2  -  ln(k12)  )  268) 0.592  (29.46  -  In  k12)  kcal/mol  or k12= The  free  terms that  6.21  energy  for  associated from  the  x  10 exp(  the  G  1  2  reaction  with  potential  ± / 0.592) is  attaining  a composite the  difference  1 35  activated between  [5] of  free  state the  energy ($)  and  reactants  (driving  force  =  df)  AGobsd = A G *  +  Gdf  or AG* Substituting (-23.06  =AGobsd 2 and  and s o l v e k  1 2  *  [6]  5 into 6  0.592(29.45 (AE))  Rearrange  Gdf  -  lnk,2*)  for  = 6.21X10  k 1 2 +1 2  = 0.592(29.45 -  (called  k  exp(lnk,2obsd  1 36  1 2  a d ; ]  -  Ink,2  i n Eq  29.45  obsd)  [7]  19) 38.95(AE)) [8]  APPENDIX  E  First-order chrome  b  5  Containing  rate  constants  by D M E - f c 5 .  for  the  reduction  Na p h o s p h a t e b u f f e r  the p h o t o - a c t i v e r e d u c i n g  of  native  pH 7 i n a l l  cases.  system d e s c r i b e d in the  text. I  Temp  [DME-6 5 ]  k obsd  0.05  0.10  0.20  0.30  25  5.0  M  .0283  .001  .0289  .001  10.0  .0383  .001  15.0  .0464  .001  15.0  .0417  .001  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  5.0  .0207  .001  5.0  .02014  .001  5.0  .027  .001  5.0  .0257  .001  10.0  .0405  .001  10.0  .0404  .001  1 37  cyto-  0.4  0.5  5.0  .0194  .001  5.0  .037  .001  10.0  .0518  .001  10.0  .0524  .001  15.0  .0662  .001  15.0  .066  .001  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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