IHESIS é a”; 5' .- . : 31130 a"; ' RY a a A? {E (N: 5:3 {.91 gig"; @253} §tate : ivhlv‘v L University w‘ This is to certify that the dissertation entitled Structures of the Heme Chromophores in Cytochrome Oxidase presented by Patricia Mary Callahan has been accepted towards fulfillrn’ent of the requirements for Ph . D . degree in ChemiStrY Major professor Date June 10, 1983 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 vtr' v"- /‘ /~ 612.71? STRUCTURES OF THE HEME CHROMOPHORES IN CHROCHROME OXIDASE BY Patricia Mary Callahan A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1983 ABSTRACT STRUCTURES OF THE HEME CHROMOPHORES IN CYTOCHROME OXIDASE BY Patricia Mary Callahan Heme structures important for electron transfer and proton transfer in the enzyme cytochrome oxidase are dis— cussed. The application of resonance Raman spectroscopy, in conjunction with optical absorption, magnetic circular di- chroism and electron paramagnetic resonance spectrosc0pies, to cytochrome oxidase and appr0priate heme a model complexes, have enabled us to identify the individual heme electronic and vibrational properties. The pattern of vibrational fre- quencies, in particular the frequency of a polarized mode in the 1560-1600 cm”1 Soret region resonance Raman spectrum, is correlated to the heme iron spin and coordination geom- etry. After taking into account the porphyrin pyrrole ring B-carbon substituent dependence of this vibration, structural information can be obtained. It is found that cytochrome a3 is six coordinate and high-spin in the resting enzyme but five coordinate and high-spin in the reduced enzyme; Patricia Mary Callahan cytochrome a is six coordinate and low-spin in both redox states of the enzyme. Cytochrome a3 is observed to be in a hydrophobic environment based on the frequency position of its aldehyde substituent. The previously unassigned cyto- chrome a formyl stretching frequency is observed at 1650 cm"1 and 1610 cm-1 in the ferric and ferrous species, respec— tively. The formyl frequency down-shift and absorption red- shift of cytochrome a relative to low-spin heme a model com- pounds are interpreted to result from a hydrogen-bonding interaction between its position 8 formyl group and a nearby amino acid residue, possibly tyrosine. This hydrogen bond strength depends on the cytochrome 3 iron valence state and is estimated to differ by 2.0 - 2.5 kcal/mole between ferrous and ferric cytochrome a. This strengthening of the hydrogen bond upon reduction of the enzyme can be used to drive redox- linked events. Thus, the linkage between cytochrome g redox state and chromophore/protein interaction energy pro- vides a mechanism by which electron transfer events and protein structure are coupled. Two models that incorporate this linkage into a redox driven proton pump centered at cytochrome a in cytochrome oxidase are presented. To My Parents ii ACKNOWLEDGMENTS I gratefully acknowledge Professor Gerald T. Babcock for his contributions to all aspects of this research. I wish to thank Bob Ingle for the preparation of beef heart cytochrome oxidase, and Debra Thompson and Professor Shelagh Ferguson-Miller for the gift of rat liver cytochrome oxidase and Tatsuro Yoshida and Professor James A. Fee for the gift of HB 8 cytochrome oxidase from T. Thermophilus. I wish to acknowledge Brian Ward for his assistance in pre— paring the model compounds. Ron Haas is acknowledged for the predominant presence of a trouble-free laser system and the following peOple are acknowledged for the maintenance of my mental healthi Jerry, Charlie, Tony, Deb, Chris, Mark, Tom, Margy, Bob, Bob, Jimbo and Ron. iii Chapter LIST OF LIST OF CHAPTER I II III CHAPTER II TABLE OF TABLES. .-. . . . . FIGURES . . . . . . . l - INTRODUCTION. . Respiration. . . . . Cytochrome Oxidase . A. B. C. D. E. CONTENTS Components and Function. Biochemical Aspects. Chromophore Structure. Absorption Spectra Redox Titrations Catalytic Mechanisms A. B. Electron Transfer Intermediates. Energy Transduction. 2 - HEME ABSORPTION AND RESONANCE THEORY. . . . . . Introduction . . . . Absorption Spectra . A. B. C. Nomenclature . . Electronic Transitions Heme Absorption Spectra. Resonance Raman Spectrosc0py A. B. C. D. Classical Description. Quantum Mechanical Description Heme Resonance Raman Polarization and Symmetry Effects. iv Page vii ix 13 20 22 22 29 29 29 30 30 42 44 46 47 53 55 Chapter CHAPTER I II III CHAPTER I II III 3- MATERIALS AND METHODS . . . Spectroscopy . . . . . . . . . Protein Sample Preparation . A. B. Cytochrome Oxidase . . . Other Proteins . . . . . . Heme Model Compound Sample Preparation A. B. 4.. Heme a . . . . . . . . . Other Heme Model Compounds COORDINATION GEOMETRIES . . Reduced Cytochrome Oxidase . . A. B. Introduction . . . . . . . Soret Raman Scattering Frequency Dependence . . . . . . . . . . . . . Soret Excitation RRS of Ferric Heme Model Compounds. . . . . . . . . . . . . A. B. C. Previous Structural Correlations . . Peripheral Substituent Dependence of Vibrational Frequencies Vibrational Assignments. . Oxidized Cytochrome Oxidase. . A. B. C. Photoreduction . . . . . . . . . . Raman Spectra of Inhibitor Complexes of Cytochrome Oxidase. . . ....... .. Discussion . . . . . . . . . . . . . CHAPTER 5 - THE ORIGIN OF THE CYTOCHROME A II ABSORPTION RED-SHIFT. . . . . . . . . Introduction . . . . . . . . . . . . . . pH Dependent Spectral Shifts . . . . . . A. B. Oxidized Cytochrome Oxidase. . . . . Reduced Cytochrome Oxidase . . . . . Page 58 58 62 62 64 64 64 7O 71 71 71 73 81 81 84 93 101 101 109 113 123 123 128 128 135 Chapter BOND STRENGTH Change Model. III pH Dependent Structural Changes. A. Reduced Cytochrome Oxidase . B. Oxidized Cytochrome Oxidase. CHAPTER 6 - REDOX-LINKED HYDROGEN CHANGES IN CYTOCHROME 3 . I Introduction . . . . . . . II Hydrogen Bond Strength Correlations. . A. Manifestations in Heme 3 Species. . . . . . . . B. Quantification of Hydrogen Bond Energies. . . . . III Proton Pump Models . . . . A. Conformational B. Hydrogen Bond Chain Model. CHAPTER 7 - SUMMARY AND FUTURE WORK I Summary. . . . . . . . . . II Future Work. . . . . . . . REFERENCES. . . . . . . . . . . . . vi Page 154 154 164 168 168 169 169 180 184 185 186 193 193 195 202 Table LIST OF TABLES B-Carbon Substituents and Absorp- tion Maxima of Some Low-spin Ferrous Hemes. . . . . . . . . . . . . . . . . Spin-State, Coordination Geometry and Selected Raman Frequencies for Various Hemes and Hemeproteins . . . . Depolarization Ratios: Low-Spin Ferric Heme Compounds. . . . . . . . . Optical, Coordination and Vibra- tional Properties of Cytochrome a and 33 in Various Cytochrome Oxidase Species. . . . . . . . . . . . . . . . Absorption Maxima and Formyl Stretch- ing Frequencies of Cytochrome a and £3 with Corresponding Model Compounds. Absorption Maxima and Individual Chromophore Formyl Vibrational Assign- ments of Reduced Cytochrome Oxidase at Neutral and Alkaline pH . . . . . . Reduced Cytochrome Oxidase Soret Ex- citation Heme Chromophore Vibrational Assignments. . . . . . . . . . . . . . vii Page 45 85 96 112 124 137 153 Table Page 8 Spectroscopic Characteristics of 4. Hydrogen Bonded Heme a and Cu2 Porphyrin 3 Species. . . . . . . . . . . . . 177 viii Figure LIST OF FIGURES The structure of heme a. . . . . . . . . . . 6 Schematic of membraneous cytochrome oxidase. . . . . . . . . . . . . . . . . . . 8 Summary of the structure and ligation spheres of the cytochrome oxidase metal centers. . . . . . . . . . . . . . . . . . . 12 Optical absorption spectra of cyto- chrome oxidase: oxidized —————— and fully reduced ------ . Extinction coefficients are expressed per unit containing two hemes and two copper ions . . . . . . . . . . . . . . . . . . . . 15 Absorption spectral decomposition of cytochromes a, 33 and the CN complex of cytochrome a - oxidized 3. and reduced ------ . . . . . . . . . . . 19 Metalloporphyrin structure including atom labelling pattern . . . . . . . . . . . 31 Optical absorption spectra of the indicated heme a derivatives. The solvent is CH2C12, concentration ix Figure 10 11 12 13 Page is W14 uM, pathlength = 1 cm . . . . . . . . 32 Spatial and nodal characteristics of the lowest unfilled (eg) and highest filled (alu’ a2u) porphyrin orbitals . . . . 35 Stokes and Anti-Stokes Raman Scatter- ing Energy Levels: V0 is the laser excitation frequency, Vs refers to the scattered photon, v is the fre- k quency between v = 0 and v = l vibra- tional levels of the ground state, g and scattering occurs from an energy weighted sum of excited states, i. . . . . . 48 Schematic of the Raman spectrometer and flowing sample arrangement . . . . . . . 59 The Optical absorption spectra of several of the ferric heme a species used. The heme g concentra- tion was approximately 13 uM for all samples; the solvent was methylene chloride . . . . . . . . . . . . . . . . . . 67 Glassware used for hydrogen bonded heme 3 reduction . . . . . . . . . . . . . . 69 Optical absorption spectra of several of the cytochrome oxidase species used in the Raman experiments. The Figure 13 14 15 Page wavelengths of the laser lines avail- able are also shown in the figure. The cytochrome oxidase concentration was approximately 4 uM (in enzyme) for all four compounds . . . . . . . . . . . . . 74 Raman spectra of reduced beef heart cytochrome oxidase (a,b,c) and of reduced rat liver cytochrome oxidase (d) recorded with the laser lines indicated. Instrumental conditions: resolution, 6 cm-l; time constant, ls; scan rate, 50 cm-l/min. Raman lines which originate from the buffer systems used are indicated by aster- isks; the daggers indicate the Raman line of the sulfate internal standard. . . . 77 The Raman excitation profiles for the modes indicated have been calculated from Equation 6.1 in the text and are shown by the solid lines. The ex- perimental data obtained with the five available laser lines are shown by the closed circles and estimated error bars. The experimental and theoreti- cal points are arbitrarily normalized xi Figure 15 16 l7 18 19 Page at 441.6 nm. The three low frequency points and the two high frequency points have been connected by dashed lines which have no theoretical basis. . . . 79 Resonance Raman spectra of several heme a model compounds dissolved in methylene chloride. Solvent and non- resonance enhanced ligand vibrations are indicated by asterisks. Instrum- ental conditions as in Figure 14 . . . . . . 92 (Ct-N) A distance plotted versus vibrational frequency for several Soret excitation structure sensitive modes. . . . . . . . . . . . . . . . . . . . 99 Resonance Raman spectra of several cytochrome oxidase species recorded with 406.7 nm excitation. Instrumental conditions: resolution, 6 cm-l; time constant, 1 s; scan rate, 50 cm-l/min. The instrument gain was constant in recording spectra a,c,d and e; for spectrum b this was increased by a factor of three. . . . . . . . . . . . . . . 104 The incident laser power dependence of the resonance Raman spectrum of xii Figure 19 20 21 22 23 Page resting cytochrome oxidase. The ex- citation frequency and power incident on the sample are indicated. Instru- mental conditions: resolution, 6 cm-l; time constant 1 s; scan rate, 50 cm-l/ min. . . . . . . . . . . . . . . . . . . . . 108 Resonance Raman spectra of the oxi- dized (a,d) and partially reduced (b,e) complexes of cytochrome oxidase with formate and cyanide. In c, the spectrum of oxygenated cytochrome oxi- dase, formed by air oxidation of the dithionite reduced enzyme, is shown. Instrumental conditions: resolution, 6 cm-l; time constant, 1 s; scan rate, so cm‘l/min. . . . . . . . . . . . . . . . . 111 Possible structures for the cyto- chrome 33 dioxygen reducing site . . . . . . 117 Schematic of the cytochrome oxidase oxygen reducing site . . . . . . . . . . . . 121 Resonance Raman spectra of oxidized cytochrome oxidase at several pH levels obtained with 413.1 nm excita- tion. Enzyme concentration was ap- proximately 30-50 uM (heme a basis). xiii Figure 23 24 25 26 27 Page Instrumental conditions: resolution, 6 cm-l; time constant 1 s, scan rate 50 cm-l/min. . . . . . . . . . . . . . . . . 130 Optical absorption spectra of oxidized cytochrome oxidase at several alkaline pH levels. Enzyme concentration was approximately 10-15 uM (heme a basis) for all samples. . . . . . . . . . . . . . . 132 Optical absorption spectra of reduced cytochrome oxidase at several pH levels. Enzyme concentrations are approximately 6-8 uM . . . . . . . . . . . . 136 MCD spectra of reduced cytochrome oxidase at pH 7.4 and 11.5. Enzyme concentration of enzyme is 10 uM . . . . . . 140 Titration curves as a function of pH for several spectral parameters of reduced cytochrome oxidase. Open circles are normalized a band half- widths at half height. The solid line is that calculated for pK = 10.5. Filled circles represent data points of MCD trough/peak (436/446.7 nm) ratio. Open triangles represent the normalized intensity ratio of xiv Figure 27 28 29 Page v(C=O)/v4; the solid line is that calculated for pK = 9.3. . . . . . . . . . . 143 Visible excitation resonance Raman spectra of reduced cytochrome oxidase a), and partially reduced inhibitor complexes b) and c). The spectrum in c) was obtained with a flowing sample arrangement. Enzyme concentra- tion was approximately 200 uM. The sample conditions in d) are m200 uM heme a, 0.5 M 2-methy1imidazole, in 0.07 M CTAB, 0.1 M sodium phosphate, 0.001 M EDTA, pH 7.4, with sodium dithionite as reductant. Instrumental conditions: resolution 5 cm-l; a)—c) time constant ls, scan rate 50 cm-l/ min; conditions in d) time constant 2.5 s, scan rate 20 cm-l/min . . . . . . . . 146 Visible excitation resonance Raman spectra of reduced cytochrome oxi- dase at several pH levels, with exci- tation wavelength as noted in the figure. Enzyme concentration was 200-300 uM (heme 3 basis). Instru- mental conditions: resolution 5 cm-l, XV Figure 29 30 31 32 33 Page time constant 2.5 s, scan rate 20 cm-l/min . . . . . . . . . . . . . . . . . . 149 Soret excitation resonance Raman spectra of reduced cytochrome oxidase at neutral and alkaline pH (a-e). Enzyme concentration was approximately 40 uM. Sample conditions in f) are m50 uM heme g, 0.7 M_N—methyl imi- dazole, 0.07 M CTAB, 0.001 M EDTA, 0.1 M sodium phosphate, pH 7.4. Instrumental conditions: resolution 6 cm-l; a)-e) time constant 2.5 s, scan rate 20 cm-l/min; f) time constant ls, scan rate 50 cm-l/min . . . . . 152 Schematic of proposed structural changes of the oxidized and reduced heme 3 chromophores at several pH levels. . . . . . 162 Visible region absorption Spectra of reduced bis-imidazole heme a in CH2C12 and reduced cytochrome oxi- dase . . . . . . . . . . . . . . . . . . . . 170 Postulated active site structure for cytochrome a in cytochrome oxidase. The heme 3 iron is six coordinate with histidines occupying the two xvi Figure 33 34 35 Page axial ligation sites. The peripheral formyl group is involved in a hydrogen bond with a proton donor designated as X-H, which is associated with the polypeptide backbone . . . . . . . . . . . . 171 High frequency Soret excitation Raman spectra of cytochrome oxidase and low- spin heme 2 model compounds. In (a) and (c), the beef heart enzyme, dis— solved in 0.05 M Hepes, 0.5% lauryl maltoside, pH 7.4, was used. In (d) cytochrome oxidase (91333) from Thermus thermophilus in the Hepes/maltoside buffer was used. In (b) and (e) the bis-(N-methyl imidazole) heme a com- plex was dissolved in methylene chloride. The carbonyl stretching frequency for cytochrome 33 is indicated by i in (a), (c) and (d) . . . . . . . . . . . . . . 173 Absorption red-shift for cytochrome g, heme g or Cu porphyrin a species as a function of hydrogen bond en- thalpy as calculated from Equation (6.1). The points are numbered according to the compounds listed in Table 8. . . . . . . 179 xvii Figure 36 Page A possible mechanism for the redox- driven proton pump in cytochrome oxidase. The cytochrome a heme a moiety is indicated by Fe C=O; the iron valence is indicated. In (a), the stable oxidized form of the cyt a site is indicated. A hydrogen bonded chain in its higher energy configura- tion occurs to the right of the formyl group and is designated as Hd-Rr....; a hydrogen bonded chain in its lower energy configuration occurs to the left of the formyl and is designated as '°'Hb-R£' Reduction is shown as a two step process which results in (c), the stable reduced form of the site. The arrows in (a) and (b) indicate the changes in structure which occur during steps 1 and 2, respectively. Reoxida- tion occurs by a two step process, with (d) as an intermediate, to regenerate the stable, oxidized cyt a configuration. The hydrogens involved in the pumping action are subscripted with letters a-d to identify their motions during the various steps. . . . . . . . . . . . . . . . 189 xviii CHAPTER 1 INTRODUCTION I. Respiration Cellular respiration is the process whereby reducing equivalents generated from foodstuffs pass through a series of membrane bound electron carriers to a terminal electron acceptor such as oxygen. The synthesis of adenosine tri- phosphate (ATP) which provides the major energy source for the cell, is coupled to this electron transfer process. A transmembrane pH gradient generated at several locations in the respiratory chain is essential for ATP synthesis but the detailed mechanism of oxidative phosphorylation is not known. In eukaryotes the respiratory components are found in the inner mitochondrial membrane. These components, labelled complexes I-IV, contain flav0proteins (FP), iron-sulfur cen- ters (Fe-S), quinones (Q) and heme and copper containing proteins. (For a historical perspective of the heme pro- teins (cytochromes) and comprehensive reviews see (Keilin, 1966; Lemberg and Barrett, 1973 and Tzagoloff, 1982)). The sequence of electron transfer reactions was determined largely after the introduction of dual wavelength spectro- photometry by Chance and Williams (1955, 1956). This technique enabled the observation of the absorption spectra of individual components of mitochondria while in turbid suspensions. This new spectroscopic method along with specific inhibitors of the electron transport chain and artificial electron acceptors identified the sequence of carriers shown in Scheme 1. Succinate dehydrogenase Complex II FP2 NADH—>FP1(4Fe-S)+é§+2(Fe-S)-+cytb_(Fe-S)cyt_c_l+cytg+Cu,cyt§g3—>O2 NADH dehydrogenase Cyt 221 complex Cytochrome oxidase Complex I Complex III Complex IV Site 1 Site 2 Site 3 Scheme 1 Complex I, called NADH dehydrogenase, accepts electrons from reduced nicotinamide adenine dinucleotide (NADH) and reduces ubiquinone (Q). The active redox components of this complex are flavin and iron-sulfur centers. Complex II or succinate dehydrogenase comprises flavin and iron-sulfur redox components also, and feeds reducing equivalents into the mitochondrial electron transport chain at ubiquinone. Ubiquinone, an electron and proton carrier, is present in approximately a six fold excess relative to the 1:1 stoichiometry of complexes I-IV. Complex III is composed of two b cytochromes, the Rieske iron—sulfur center, as— sociated quinone molecules and cytochrome 31' After oxidiz- ing Q, this complex transfers electrons to the water soluble redox protein cytochrome 3. As the penultimate carrier, cytochrome 3 reduces the copper and heme 3 containing pro- tein, cytochrome oxidase, which then carries out the reduc— tion of dioxygen to water. The redox potential spanned from NADH to O is 1.14 2 volts. The large decrease in free energy from the passage of two electrons through this series (53 kcal/mole) is liberated as heat in hibernating and hairless newborn ani- mals and some flowering plants (Nicholls and Locke, 1981). In coupled mitochondria, however, this free energy is utilized to translocate protons against a pH gradient. Sites 1, 2, and 3 in Scheme 1 contribute to the transmembrane pH gradient and therefore ATP synthesis with a stoichiometry of approxi- mately three ATP molecules formed per two electrons passed through the respiratory chain. The soluble and membrane bound components have been isolated and studied in great detail. This thesis is con- cerned with a spectroscopic study of Complex IV. II. Cytochrome Oxidase A. Components and Function Complex IV or cytochrome oxidase (ferrocytochrome g: O2 oxidoreductase; E C 1.9.3.1) enjoys a central role in the field of membrane bioenergetics because it is responsible for more than 90% of the oxygen consumed by living organisms. Cytochrome oxidase catalyzes the oxidation of ferrocyto- chrome g and the four electron reduction of dioxygen to water. In addition, it contributes to the transmembrane pH gradient by the translocation of two protons per elec- tron (Wikstrom and Krab, 1979). The overall reaction can be written as: 2+ 3+ + + 4cyt g + 02 + 8Hin+4cytg + ZHZO + 4Hou t where Hin refers to protons in the internal mitochondrial matrix phase and ng refers to protons in the cytoplasmic t phase. This multisubunit enzyme is composed of four metal cen- ters; two heme a chromophores, designated cytochrome grand cytochrome g3 and two protein bound c0pper ions, designated Cu grand Cua3. These four metal centers seem to function in pairs. Cytochrome a and Cua are responsible for the oxida— tion of ferrocytochrome g and further intramolecular elec- tron transfer. Cytochrome a has recently been implicated in the proton pumping action of the enzyme (Wikstrom, 1977). The second pair of metal centers make up the oxygen reduc— ing site, and cytochrome a3 is the site of ligand binding such as substrate 02 or inhibitors (HCN,HN3,CO,NO). The structure of the heme a_macrocycle is shown in Figure l. The distinguishing features of this structure relative to the more common protoheme containing proteins (hemo- globin, myoglobin, cyt b) are the hydrophobic hydroxyfarnes- ylethyl tail at position 2 and the formyl substituent at position 8. The pyrrole nitrogens of the porphyrin ligand fulfill four of the central metal iron coordination posi- tions and fifth and sixth axial ligands can bind to the iron from above and belowtflmaplane of the ring. The heme a chromophores in this protein are amenable to a variety of spectroscopic probes. These chromOphores and the two copper centers have been studied by absorption, electron paramag- netic resonance (EPR), electron-nuclear double resonance (ENDOR), circular dichroism (CD), magnetic circular di- chroism (MCD), infrared (IR), resonance Raman (RR), mag- netic susceptibility, Méssbauer and extended x-ray absorp- tion fine structure (EXAFS) spectroscopies. Because of its complexity, cytochrome oxidase has also been the subject of many biochemical investigations. The following discussion focusses on information obtained by biochemical methods and Chromophore structure and reactivity as determined by spec— troscopic investigations. (For reviews see Malmstrom, 1979; Wikstrom‘EE 31" 1981 and Wikstrom et 31., 1983). Figure 1. The structure of heme a. B. Biochemical Aspects Cytochrome oxidase from eukaryotic organisms has a molecular weight of approximately 160 kD and is composed of at least seven subunits. Of these, the three largest are coded for by mitochondrial DNA (Subunits I, II and III) and the remainder are of cytoplasmic origin. In contrast, bac- terial cytochrome oxidases consist of only two or three poly- peptides. The properties of these subunits resemble those of the largest eukaryotic subunits (Ludwig, 1980). With the use of labelling techniques, it is found that several subunits (I, II and III) are accessible from both sides of the mem- brane (Azzi, 1980; Wikstrom gt gt., 1981; Capaldi, 1982). Electron microscopy and image reconstruction data show that the monomeric enzyme spans the membrane in the shape of an inverted Y and it is found to protrude m50-60 A into the cytoplasmic (C) phase and about 10-15 A into the M or matrix phase (Henderson gt gt., 1977: Frey gt gl., 1978) (See Figure 2). The aggregation state of solubilized cytochrome oxidase is dependent on detergent. In most detergents it is oligo- meric, however, in lauryl maltoside, the enzyme seems to be dispersed as a monomer (Ferguson-Miller, 1983). The effect of aggregation on catalytic function or the aggregation state of cytochrome oxidase $2.21X9 is not known. Subunit III has been implicated in the enzyme's ability to form dimers (Georgevich gt gt., 1983) and removal of this subunit .ommoflxo .v.1...¢ ouar.... ‘10-; 9...: $035 mEoEooSo oEonnoouao msomcmuneoa mo oaumamcom N onsmflm has little or no effect on the spectral or electron trans- fer properties although some evidence suggests that it does eliminate the proton pumping function of the enzyme (Wik- strom EE.§$" 1981, Penttila, 1983). Wikstrom gt gt. (1981) have also implicated the dimeric state in proton pumping by the oxidase. By monitoring a controlled denaturation of cytochrome oxidase Winter gt gt. (1980) concluded that the four metal centers are located in subunits I and II. This is supported by the fact that the two subunit bacterial oxidases have the same spectral and catalytic properties as the mammalian enzyme (Ludwig, 1980; Fee gt gt.,l980; Yamanaka gt gt., 1981; Powers gt gt., 1981; Gennis gt gt., 1982; Sone and Yanagita, 1982). C. Chromophore Structure Cytochrome g, the physiological electron donor to cyto- chrome oxidase, binds in its high affinity site to subunit II of the enzyme (Bisson gt gt., 1982). Resonance energy transfer measurements of the distance between fluorescence cytochrome g derivatives and the nearest heme g Chromophore result in approximately 25-35 A heme g - heme 3 distance (Vanderkooi gt_gt,, 1977, Dockter 23.21-1 1978). Since cytochrome g is the first electron acceptor from cyto- chrome g (Halaka, gt gt., 1982, Antalis and Palmer, 1982), it is postulated that cytochrome g and its functionally 10 associated Cu, (Cua) are located on subunit II (Wikstrom gt 31., 1983). Cytochrome 3 has been shown to be ligated by two histidine residues by comparison of the EPR, MCD and resonance Raman spectra of the 13 3139 Chromophore with heme 3 model compounds (Blumberg and Peisach, 1979; Bab- cock gt 31., 1979). Examination of the invariant histidine residues of subunit II, and the possible folding pattern of this polypeptide locates the cytochrome 3 binding site close to the cytoplasmic membrane (Wikstrom gt 31., 1983). This location is consistent with the EPR defined location of cytochrome a and Cua. With the use of water soluble para- magnetic probes it was concluded that these metal centers are close to the cytoplasmic membrane (Ohnishi, gt 31., 1979). The EPR signal of Cua occurs about g2 but it is atypi- cal of known cupric copper signals; its hyperfine splitting constant can be understood if the unpaired electron spin density is delocalized onto the copper ligand(s). One and possibly two cysteines have been suggested as ligands of Cua on the basis of EPR data (Peisach, 1978; Blumberg and Peisach, 1979; Chan gt 31., 1978, 1979). Two invariant cysteines found in the primary structure of subunit II lie in an area of the polypeptide that has sequence homology with blue c0pper proteins (Steffens and Buse, 1979). The in- variant cysteines and histidine found in this region agree 15 with the recent EPR and ENDOR data of N-histidine and 2H-cysteine in which Stevens gt 31. (1982) conclude that 11 at least one histidine and one cysteine are ligands to Cua. See Figure 3 for a summary of the ligation properties of cyt 3 and Cua. The other two metal centers are most likely located in subunit I. Cytochrome 33+ and Cu:+ are not detectable by EPR in the resting enzyme. Howevei, EPR signals arising from cyt 33 and Cu23 can be induced by addition of ligands and/or reduction of the other metal center (Stevens gt 31., 1979; Brudvig gt 31., 1980 and Reinhammar gt_31., 1980). Magnetic susceptibility measurements suggest that this pair of metals is antiferromagnetically coupled in an S=2 ground state with |2J|>2oo cm”l (Tweedle gt 31., 1978). This in- dicates that the metals are in close proximity to each other. This distance has been measured by EXAFS to be 3.75 i 0.05 A (Powers gt 31., 1981). In the resting enzyme, Powers gt 31. (1981) suggest the presence of a bridging sulfur ligand be- tween the iron of cyt 3§+ and Cu:+. However, the amount of enzyme with this bridging sulfur—ligand varies with the type of isolation procedure used. Heterogeneity in the spectra and properties of cyt 33 in the resting enzyme has been observed previously (Kumar gt 31., 1983, Brudvig gt 31., 1981). MCD spectra of ferrous cytochrome 33 indicate a five coordinate high-spin iron geometry (Babcock gt 31., 1976). The identity of the fifth, axial ligand as histidine has been obtained from EPR studies of nitrosyl ferrous cyt 3 (a2+ —3 3 - NO) (Blokzijl-Homan and Van Gelder, 1971). Since 12 OH CISHZ'I 'OOC 'OOC H0 'Nhis [ \ x \ \ N 79 N / o\ \ i/ / N . C O 0' ins COO’ cyt o 0 Figure 3. Summary of the structure and ligation spheres of the cytochrome oxidase metal centers. 13 no EPR signal from Cu:+ —3 the nature of its ligands remains obscure. is observed under most conditions, The currently accepted model for the arrangement of cytochrome 33 and Cu33 is described as the "front-side" model and is shown in Figure 3. The close proximity (m4 A) of these metal ions as determined by magnetic susceptibility and EXAFS data is satisfied by this model. Further evi- dence for this structure is supplied by carbonmonoxide flash photolysis studies. After photolyzing the inhibitor from cyt 3§+, the carbon monoxide is observed to rebind to the CuE3 site (Alben gt 31., 1981); therefore a model in which the two metal centers are capable of binuclear ligand binding is suggested. D. Absorption Spectra The focus of this thesis is the determination of struc— ture by analysis of the spectral properties of the heme 3 chromophores of cytochrome oxidase. Although optical absorp- tion spectroscopy is the most commonly used spectroscopic probe, a complete understanding of the enzyme's absorption spectrum has not been reached. The visible absorption spec- tra of oxidized and reduced cytochrome oxidase are given in Figure 4. The heme 3 chromophores dominate the spectrum in the near UV and visible regions because of the intense n-w* transitions of the porphyrin macrocycle. Typically, e oxi- ytochro: C p O - spectra 0 ‘ reduced - - fullv and .3 e .g‘ .3 .l C e S a .C 10118. - per -d two cop Y“ O. a a... :u : C I t. ’I‘ E. mM" cm" 15 200 ISO IZO Oiriiliiiiliiiilinr 500 550 600 650 700 owemm ‘ ilriiiliiiilriiilriiiJir 650 700 750 800 850 Wavelength, nm Figure 4 16 three absorption maxima are observed for metalloporphyrin species, namely, an intense Soret band (also y band) at 400-450 nm, a weaker a band at 550-600 nm and a 8 band at approximately 1100 cm-1 to higher energy than the a band. A more detailed discussion of heme electronic properties will be given in Chapter 2. The reduced enzyme's absorption maxima are 443, 555, 565, and 604 nm. The Soret maximum of oxidized cytochrome oxidase is broad and centered at 418-424 nm. Its visible spectrum is structureless except for the a maximum at 598 nm and a weak shoulder at 655 nm. The 655 nm band is present only if the enzyme is oxidized in the presence of oxygen rather than other chemical oxidants. In the case of ferricyanide oxidation a transient high-spin heme g6 signal which accounts for 100% of one heme is ob- served (Beinert and Shaw, 1977). This signal has been as- 3+ 3 . sults is that Cua is reduced and the exchange coupling —3 between cyt 33 and Cua is broken, therefore the 655 nm —3 band is present only when the exchange coupling between signed to cytochrome 3 The explanation for these re- these two metals in their higher oxidation states, is present. Aside from the 655 nm shoulder and 830 nm band (which has been assigned to Cu:+), the major spectral features arise from cytochrome 3 and cytochrome 33 electronic tran- sitions. Because of their similar chemical nature the heme 3 Chromophore absorption spectra strongly overlap. 17 In 1966, Vanneste took advantage of the photodissociability of the electron transfer inhibitor, carbon monoxide, from ferrous cytochrome 33. From the photochemical action spec- trum he was able to identify the individual contributions of cytochrome 3 and 33 to the total absorption spectra. These deconvoluted spectra are shown in Figure 5. In the 3+ are 427, 550 bottom panel the absorption maxima of cyt 3 and 598 nm and of cyt 32+ are 443 nm (420 nm shoulder), 520, 555 and 603 nm. Comparison of the extinction coeffic- ients of cyt 3 and cytochrome 33 (middle panel) in the visible region show that cyt 3 dominates the spectrum in both oxidation states. In the Soret region, on the other hand, cyt 3 is the major absorber in the oxidized enzyme but the intensities of cyt 3 and cyt 33 are comparable in this region for the reduced enzyme. Vanneste's deconvoluted absorption maxima of cyt 3§+ are observed at 414, 560 and 2+ 3 y/o intensity ratio for reduced cytochrome 33 is indicative 600 nm and of cyt 3 at 442, 565 and 600 nm. The large of a high-spin species (Lemberg, 1969), which is in agree- ment with other spectroscopic data (Babcock et a1., 1976, 1979). The low-spin character of cyt 3 is indicated by its smaller y/a intensity ratio. An anomalous aspect of these spectral assignments is the Soret wavelength maxima of the reduced components. In general, the Soret maxima of high and low-spin species are not the same, as is observed for the Soret maxima of cyt 32+ and cyt 3§+ at approximately 443 nm. 18 Figure 5. Absorption spectral decomposition of cyto- chromes 3, 33 and the CN complex of cyto- chrome 33; oxidized —————— and reduced ------ . (From Vanneste, 1966.) E, mM" cm" 19 11111111151111. —a"3 - CN ---o’§ - CN IOO l20 - . --- 0‘3 11111111111111 400 480 560 640 Wavelength, nm Figure 5 20 E. Redox Titrations This spectral decomposition method is valid only if the heme chromophores have independent spectral properties and the redox state of one Chromophore does not effect the elec- tronic transitions of another. A controversy on this point arose in the mid-seventies. By monitoring the absorption intensity at 604 nm as a function of redox potential, Wil- son gt 31. (1972) concluded that the heme chromophores have fixed midpoint potentials of 285 mV for the cyt 33+/2+ couple and 350 mV for cyt 333.”2+ and that their spectral properties are strongly interacting. Nicholls and coworkers (1974) and Wikstrém gt 31., (1976) presented arguments favor- ing an alternate description; that is, fixed spectral prop— erties but varying redox potentials. Evidence for the alternate interpretation was given by Babcock gt 31. (1976, 1978) who established the individual Soret region MCD spec- tra of the heme 3 components and monitored both the ab- sorption and MCD spectra of the enzyme during reductive titrations. Their data show that the spectral deconvolu- tion of Vanneste is valid (142;! non-interacting electronic properties). This conclusion is reached because under all experimental conditions the reduction levels of cytochromes 3 and 33 are comparable. This result cannot be reconciled with a model in which the two heme centers have well re- solved potentials. Addition of one electron to the enzyme lowers the redox 21 potential of the second heme electron transfer reaction. This negative cooperativity has been estimated to involve an approximate 2 kcal/mole coupling between the heme cen- ters (Carithers and Palmer, 1981). Under the equilibrium conditions present in redox titrations, the two hemes 3 behave essentially as a two electron system in analogy with classical two electron carriers such as ubiquinone (Wikstrbm gt 31., 1983). A recent different absorption spectral study involving several different inhibitor complexes of cytochrome oxi- dase (Blair gt 31., 1982) also supports the independent Chromophore spectral model and Vanneste's deconvolution. This study does, however, point out small spectral interac- 3+ + + + 3 , Cu2 and cyt 33 , Cual a —3 —3 species. These results can be understood in terms of an tions especially for the cyt 3 electrostatic effect, whereby the charge on CuE3 effects the absorption properties of nearby cyt 33. Although Van- neste's results are generally accepted, simple heme 3 model complexes of the spin and ligation states described above for cytochromes 3 and 33 do not reproduce the de- convoluted absorption spectra (Lemberg, 1962). This re- sult will be discussed in Chapters 4 and 5. 22 III. Catalytic Mechanisms A. Electron Transfer Intermediates Up to this point only the structural and spectroscopic properties of the static forms of the enzyme have been dis- cussed. When reduced cytochrome oxidase is reoxidized with 02 at room temperature, at least three different conforma- tions are sequentially formed. The first which is formed within 2 ms at room temperature and decays in a few seconds is known as the 95 conformation. This form of the enzyme has a Soret maximum at 427 nm, broad absorption at 580 nm when compared to the resting enzyme, a 655 nm absorption band and EPR signals at g=5, 1.78 and 1.69. These EPR transitions were interpreted in terms of an S=5/2 ferric cyt 33 interacting with a nearby paramagnetic center (Cui3) (Shaw et a1., 1979). Another possible interpretation of the g=5 EPR spectrum involves an S=3/2 system that is composed of ferryl cyt 33 and cupric copper (Wikstrdm gt 31., 1981). Following formation of the 95 species, a second conforma— tion of the enzyme called oxygenated cytochrome oxidase is formed. This is a misnomer in the sense that 02 is not bound to cyt 3§+ as in oxyhemoglobin but the oxygenated des- cription of the form of the enzyme with the following charac- teristics has become common usage; the absorption maxima occur at 427 nm and 600 nm, the 655 nm band is present, the enzyme requires four electrons for complete reduction, and 23 the EPR spectrum is identical to that of the oxidized en- 3+ and Cu:+ are EPR undetectable). The —3 oxygenated enzyme's reactivity to exogenous ligands is fast zyme (i.e., cyt 3 and monophasic in comparison to the resting enzyme which binds ligands sluggishly and in a multiphasic manner (Kumar gt 31., 1983). This suggests that the oxygenated enzyme may be the catalytic starting point for oxygen reduction. On the time scale of hours, the oxygenated enzyme reverts to the rest- ing form. Since no intermediate oxygen reduction products such as superoxide, peroxide or hydroxyl radical are released from the 33 site and because of the unfavorable thermo- dynamic barrier to transfer of the first or third electron to oxygen, it has been proposed that a concerted two elec- tron transfer process takes place (Malmstrdm, 1974; Bab- cock gt 31., 1978; Reed and Landrum, 1979). The initial reaction sequence would then reduce dioxygen to the level of peroxide. The low temperature triple-trapping technique of cytochrome oxidase reaction intermediates developed by Chance and coworkers (Chance gt 31., 1975) has permitted a preliminary identification of the catalytic mechanism of oxygen reduction. In this technique the enzyme, or mito- chondrial suspension, is saturated with carbon monoxide 3+ a2 2+ —3 +°CO compound, and then cooled in the presence of ethylene under reducing conditions to form the 3 +-CO or 3 2 33 glycol to about -30°C. Oxygen is then added to the suspension 24 by stirring or mixing with O2 saturated buffer. The oxygen will not react with this form of the enzyme owing to the slow dissociation of CO at this temperature in the dark. The oxygenated suspension is then brought to -196°C. The enzymic reaction with oxygen can be initiated by an intense flash of light which photodissociates the CO and allows the oxygen to bind. The reaction proceeds in the temperature range -60 to -130°C and can be stopped at any point by rapid freezing in liquid nitrogen. EPR and absorption spec- tra of the reaction intermediates prepared in this way have been monitored (Clore and Chance, 1978, Clore gt 31., 1980). The stable intermediates that have been formed and charac- terized by this method are called Compounds A, B and C. Compound A has been identified as an oxy cyt 32+ 3 (cyt 3§+°02). This conclusion is reached on the basis of species its Optical absorption Spectrum which is similar to the CO complex but non-photolyzable. Oxy heme 3 model studies (Babcock and Chang, 1979) are consistent with the spectrum of Compound A. A later intermediate of the mixed-valence (33+ 3§+:CO) compound called Compound C is characterized by a difference spectrum absorption peak at 607 nm (e = 12 mM”1 cm-l) and weak Soret absorption intensity. This species has been suggested to be the u-peroxy form of the 33 site (33+ - O - O - Cu:+) or ferryl iron and cuprous c0pper (Fe:+ = O - O - Cu;+) (Wikstrom gt 31., 1981). If the re- —3 -3 action intermediate sequence is begun with fully reduced cytochrome oxidase (32+ 3§+-CO) a different structural 25 intermediate is formed (Compound B) following the formation of the oxy intermediate (Compound A). The exact nature of Compound B is difficult to determine because there is par- tial oxidation of cyt 3 and Cua, also (Wikstrom gt 31., 1981). — Similar spectral intermediates have been observed by ATP dependent partial reversed electron flow through cyto- chrome oxidase (Wikstrdm, 1981). In this work, Wikstrom suggested that the previously observed energy dependent shift of ferric cyt 33 (Erecifiska, et a1., 1972) could be attribut- ed to reversed electron transfer. Under highly oxidizing conditions, the addition of ATP to a mitochondrial suspension causes a red-shift in the Soret region of cytochrome oxidase and a broad absorption increase at 580 nm, similar to Com- pound B described by Chance and coworkers. At a higher phosphorylation potential a moderately intense band in the a region at 607 nm is observed. This spectral form re- sembles Compound C. These results are good evidence for the physiological nature of the low temperature inter- mediates. Much research is now being conducted on the intermed- iates of the cytochrome oxidase oxygen reduction reaction. The structure of cyt 33 in the oxygenated enzyme as de- termined by RRS and a possible reaction mechanism that is consistent with the data obtained to date will be presented in Chapter 4. 26 B. Energy Transduction It has been known for thirty years that electron trans- fer from cyt g to O is linked to ATP formation (Maley and 2 Lardy, 1954, Lehninger gt 31., 1954). The approximately 500 mV spanned by this reaction contains enough free energy for the energetically uphill proton translocation against a pH gradient. In recent years, isolated cytochrome oxi- dase reconstituted into liposomes has been shown to gen- erate a chemical potential across the membrane (Hinkle gt 31., 1972) in agreement with Mitchell's chemiosmotic coupling hypothesis (Mitchell, 1961). Mitchell described the uptake of protons by the 33 site in the formation of water as the only source of proton motive force in Complex IV (Mitchell and Moyle, 1983). This does not agree with the stoichiometry of protons and charges translocated across the membrane, however (Wikstrém and Krab, 1979, Reynafarje gt 31., 1982). This is problematic because cytochrome oxidase con- tains redox components that are only formal electron car- riers (two hemes 3 and two copper ions) and therefore the result of H+ translocation by cytochrome oxidase is not completely accepted (Mitchell and Moyle, 1983). Evidence is accumulating, however, that the proton pumping of cyto- chrome oxidase is a physiological function (Wikstrom gt 31., 1983, Casey and Azzi, 1983) and that specific treatments can inhibit this function (Casey gt 31,, 1980; Wikstrom gt 31., 1983; Penttila, 1983 and Maroney and Hinkle, 1983). 27 Subunit III seems to have a specific role in the trans- location of protons. Casey gt 31 (1980) found that di- cyclohexylcarbodiimide (DCCD) appeared to block H+ trans- location in cytochrome oxidase reconstituted into lipo- somes. Under these conditions, DCCD was predominantly bound to subunit III. Treatment of the enzyme to remove subunit III results in the inhibition of proton translocation, (Sarraste gt 21" 1981; Penttila, 1983) but the electro— chemical proton gradient is formed with only 50% efficiency. This result can be interpreted in the following way: 1) an inhibition of the proton translocating segment of the pro- tein and 2) a continuation of electron transfer and the 33 site proton uptake necessary for water formation (Wikstrdm, 1983). It is surprising that proton translocation can be decoupled from electron transfer with no effect on the rate of the latter but the possibility of long range interactions between the redox element and the proton source cannot be excluded. The identification of the redox element of the proton pump as cytochrome 3 is based on 1) its pH dependent mid— point potential (Artzatbanov gt 31., 1978) and 2) its kinetic heterogeneity which may reflect two conformational states (Wikstrom gt 31., 1981). A necessary element of a redox-linked proton pump is that there must be a connection between the redox component and the polypeptide backbone or proton source. Such a structural connection will be 28 discussed in Chapter 5 and its possible role in the proton pumping function of the enzyme will be presented in Chapter 6. CHAPTER 2 HEME ABSORPTION AND RESONANCE RAMAN THEORY Introduction A great deal of structural information can be obtained from the absorption spectra of metalloporphyrins. When the absorption spectrum alone fails to identify or characterize a particular sample, the selective vibrational technique, resonance Raman spectroscopy, can supply additional in- formation. Because of the importance of absorption and resonance Raman spectroscopy (RRS) as applied to the heme 3 chromophores of cytochrome oxidase in this thesis, an understanding of the theoretical considerations is neces- sary. This chapter will present a description of metallo- porphyrin electronic transitions and of resonance Raman spectroscopy as applied to these chromophores. I. Absorption Spectra Metalloporphyrins and related compounds are responsible for the red, orange, brown and green coloring of a variety of biological materials; including red blood, green plants and the brilliant coloring of some species of birds. It 29 30 has long been recognized that this class of compounds derive their light absorption properties from n-w* transitions (Platt, 1956). A. Nomenclature The basic structure of the porphyrin macrocycle is given in Figure 6. The four pyrrole rings are joined at the methine bridge or meso positions, labelled a, B, y and 6, and various peripheral substituents can be linked to the B-carbon posi- tions labelled 1-8. The remaining carbon atoms adjacent to the pyrrole nitrogens are called o-carbons. For this specific example of an iron-substituted metalloporphyrin, the mole- cule is called a heme. In this form the porphyrin ligand exists as a dianionic species which upon removal of the central metal atom is protonated at opposite pyrrole nitro- gens to form the free base compound. B. Electronic Transitions 1. Descrtption of Porphyrin Spectra The aim of several spectroscopic theories has been to explain the Optical absorption spectra of porphyrin and metalloporphyrin species. Typical spectra of these two compounds are shown in Figure 7. An intense band in the 1 near UV (5 z 100 mM- cm-l) called the Soret (or B) band 31 .CMOpumm mcflaaonma Eoum OCHUOHOGH ousuosuum cfinwnmnomOHHmuoz .m musmflm 32 .50 H u camcochmm .25 vae we :OHumuucwocoo .NHONmU ma pco> iHOm one .mm>flum>finmp m mew: pODMOflch on» NO muuommm coflumuomnw HMOHMQO .n ousmflm «ES £86663 one cow 0mm 9.8 9.8 8+ 0mm _ mm_ .0 ONO T aouoqmsqv man d 5:298 a m Sizeod III mmflv — — _ _ :0 II 33 and a weaker (e 2 20 mM-1 cm-l), a (or Q) band are the two main transitions in metalloporphyrin spectra. The B (or 001) band at approximately 1100 cm"1 higher energy than the a band is a vibronic overtone of the a band. In D4h symmetry these two electronic transitions are of Eu sym- metry and therefore doubly degenerate and x, y polarized in the plane of the ring. For the free base porphyrin, the protonation of Opposite pyrrole nitrogens lowers the sym- metry of the ring to D2h and the x, y degeneracy is lifted causing a doubling of transitions in the visible region (ttgt, Q30, 031. QUO’ le, from low to high energy, respec- tively). 2. Historical Development In the free electron model of porphyrin spectra, Simpson (1949) was able to predict the relative energies and relative intensities of the Q and B transitions. By analogy with benzene, the 18 n electrons of the porphyrin ring are placed in orbitals of increasing angular momentum. The top-filled levels have angular momentum, L=4 and the lowest empty orbitals correspond to L=5, therefore these two levels can give rise to transitions of AL = :1 or AL = :9. By Hund's rule, the forbidden transition, AL = :9 lies lower in energy. This development qualitatively predicts the intense B transition (AL = 11) and a weak or forbidden transition to lower energy (AL = 19). A similar description 34 of the porphyrin n states, the cyclic polyene model used by Moffitt (1954), reached the same results as the free elec- tron model and also correctly accounted for the splitting of the QX and Qy energy levels in the free base absorption spectra. In an effort to relate the porphyrin electronic transi- tions to structure, Longuet-Higgins gt 31. (1950) employed molecular orbital (MO) calculations. They obtained two top-filled orbitals, a2u and alu (with alu of lower energy) and lowest empty degenerate orbitals, eg (Figure 8). This identifies two electronic transitions, (a + e ) and 2n 9 (a lu + eg), corresponding to the Q and B bands, respec- tively. However, this develOpment incorrectly predicts equal intensities for the two transitions. The shortcoming of this method lies in the neglect of the fact that since the two calculated transitions are both of Eu symmetry their Coulomb repulsion overlap matrix is finite. This causes the states to mix and drives them apart in energy. 3. The Four-Orbital Model Gouterman (1959) recognized the necessity of configura— tion interaction (CI) in the description of porphyrin elec- tronic states. Configuration interaction arises because of the neglect of electron correlation from the restric- tion to one-center integrals in Hfickel MO theory. The solutions then are not solutions of the complete Hamiltonian, 35 Figure 8. Spatial and nodal characteristics of the lowest unfilled (eg) and highest filled (alu' a2u) porphyrin orbitals. (From Gouterman, 1961.) 36 but rather solutions of an effective Hamiltonian, A Heffw = Ew. The complete Hamiltonian can be represented as A = A ' H Heff + H , where H' is predominantly the electron repulsion term, e2/ri This Hamiltonian can cause states Of the same j. symmetry to mix, specifically the singly excited configura- ). tions (alueg) and (aZueg Since the singly excited configurations are not an ac- curate representation of the system, a better starting point for describing the excited states of a metallopor- phyrin are linear combinations of the two excited con- figurations. The components of the zeroth order Q and B electronic states are: 0 _ 1. IBy> — 7% [(aZuegy) + (aluegx)] o ._ 1 _ le> - 2 [ (aluegx)1 IB°>=-l—[(a e )-(a e )1 X 5 2ng lugy lc2">=——l [(a e )+ (a e )3 x /§ 2u gX lu gy 37 It can be seen that B and B; give rise to the allowed Soret transitions while Q and Q are the forbidden visible I‘ where wo is the ground state wavefunction and integration is over all space. As stated earlier R 2 R 1y 2y’ intensities are approximately equal. The measure of ab— i.e., the sorption intensity is the dipole strength which is the square of the transition moment, 2. For IB;>, the transi- tion moment squared equals 2 _ 1 2 q — §[<(a2ueg )IYIwO> + <J y x _1 2 - EERZY + Rly] and for |QO> Y 2 _ 1 _ 2 q - 2[<(a2ueg )lylwo> <(alueg |y|¢0>1 y X _ 1 _ 2 — 2ER2y R1yJ ° 38 The corresponding dipole strengths of the x-polarized transi- tion can be determined as follows: let R 1x and R2X = then 2 o _ 1 2 q (IQX>)- 2[ + (alueg |x|w0>1 Y _ 2 _ [RZX + R1xJ and 2 O _ 1 _ 2 q (IBX>) — 2[ 1 _ 1 _ 2 - 2[R2x R1xJ ' At first glance the x-polarized transition moments seem to contradict the statement that the B transition is strongly allowed and the Q transition forbidden, but we still need to relate the x and y components of the transition moments to each other. This is done by defining an axis system in Figure 8 which leads to the following relations: 39 C4x + y C4egx + eg C4alu + alu Y C4y + -x C4eg + -eg C4a2u + a2u y x Therefore, C4R1x I - _ ’R1y and C4R2 I = R2y Using these transformations 2 o _ 1 _ 2 and 2 o _ 1 2 similar to the y-polarized transitions. The final result is that R = 1/2 (R1 + R2)2 and r = 1/2 (R2 - R1)2 for the B and Q transitions, respectively. For both the x and y- polarized transitions the intensities add in the Soret and subtract and cancel in the visible region. These 50/50 mixed configurations still do not adequately 40 represent experimental data. By allowing these zero order wavefunctions to "unmix" depending on the magnitude of a configuration interaction parameter, 0, the intensity of the Q band can be varied continuously relative to the B tran- sition. The coordinate transformation which allows coupling only between states of the same polarization can be defined by: le> = COSGIQ:> - sin0|B§> le> = coselQ3> - sine|33> le> = coselB:> + sine|Q:> and o . o B > = 8 B > + > | cos I y Sinele The restriction to coupling between states of the same polarization can be relaxed by considering a more complete vibronic description (Shelnutt, 1981). Previously we defined O z o and O _ o rx : (Qxlxlwo> 41 Similarly we define the unmixed transition moments as 7:! III . o o < : Bxlxlwo> s1n6rX + coseRX and H Ill 0 . o x coserX s1n0RX , with analogous equations for the y-polarized transitions. It is usually assumed that r: vanishes as shown in the pre- ceeding section, which results in the approximate transi- tion dipoles SO 22 o coseRX and '1 22 . o SineRX . This predicts that the Q transition gains intensity at the expense of the B or Soret band. Furthermore, it can be shown that for the case where 0 is small, the energy sepa- ration between the Q and B transitions is at a minimum; this separation then increases as the configuration inter- action parameter increases (Gouterman, 1959). The four- orbital model gives a good qualitative and quantitive 42 description of metalloporphyrin absorption spectra. The above description of metalloporphyrin electronic transitions has ignored the effect of changing internuclear coordinates on excited state interactions. Briefly, vibronic coupling effects lead to additional intensity in the a band arising from inter- and intra-manifold coupling of vibrational states of the appropriate symmetry. This will be discussed in Section II of this chapter. C. Heme Absorption Spectra Calculation of heme Optical absorption spectra are complicated by the presence of the iron d orbitals which are of the apprOpriate symmetry and energies to couple with the porphyrin n orbitals. Furthermore the range of iron ligation states (four to six ligands are possible) results in several possible crystal field symmetries. In an octa- hedral field, exemplified by an iron ion ligated by the porphyrin nitrogens and two axial nitrogenous ligands, the five degenerate orbitals are split into two groups. The lower energy group (dx d d 2) comprises orbitals with Y'YZX axes oriented at 45° to the pyrrole nitrogen axes and those at higher energy (dx2_ 2 and dZZ) are oriented along the pyr- Y role nitrogen axes and along the axial ligand directions, respectively. From this axis system it can be seen that the high energy dzz orbital will be most sensitive to axial ligation. Zerner gt 31., (1966) performed extended 43 Hfickel calculations on ferric and ferrous porphyrins and were able to predict the spin-state of each species. As expected the spin states vary according to the position of the axial ligands in the spectrochemical series. In general this series can be listed from strong field to weak field ligands as: CO, CN-, nitrogenous ligands, sulfur ligands, oxygen ligands and halogens. In addition to spin-state variation, several oxidation states of iron are possible in heme species. The two most common are ferric and ferrous iron. Ferric (or oxi- dized) heme spectra are usually broader and less intense than the ferrous (or reduced) cases. Addition of an elec- tron to the heme n system in the ferrous compound results in population of the w* antibonding orbitals. This explains the red-shift observed in the Soret maximum upon reduction. In general, however, the wavelength maximum of the a band of low-spin hemes changes very little upon reduction. Charge transfer (CT) transitions have been shown to be important in the spectra of hemes and heme proteins (Smith and Williams, 1970). Several charge transfer bands exist in the visible and near IR region of the spectrum and are most prominent for high-spin hemes. These bands have been interpreted as porphyrin n to metal dTr (Smith and Williams, 1970) and metal to axial ligand (Asher, 1981) charge trans- fer transitions. Three important hemes of physiological significance 44 significance (heme 3 found in cytochrome g, protoheme or heme 3 found in hemoglobin and myoglobin and heme 3 found in cytochrome oxidase) vary in their pattern of peripheral sub- stituents and absorption maxima. They all have propionic acid residues at position 6 and 7 (Figure 6), presumably to anchor the Chromophore to the polypeptide backbone 313 hydrogen bonding. These acid functionalities are insulated from the porphyrin ring by two carbon atoms and therefore the ring system "sees" these substituents as alkyl groups. Their presence or absence does not affect the spectra. Table 1 summarizes the substituents and absorption maxima of these three species. The optical properties of this series of hemes supports two aspects of Gouterman's formula- tion of metalloporphyrin spectra. First, the more closely the heme approximates D4h symmetry, the weaker is the in- tensity of the a band relative to the Soret, and second, the red-shifted a maxima in the lower symmetry cases is an ex— ample of the larger splitting between the Soret and 0 bands predicted by the four orbital model. These symmetry con- siderations will be important in Chapter 5 for the inter- pretation of the absorption and Raman spectra of cyto- chrome 3 in cytochrome oxidase. II. Resonance Raman Spectroscopy Resonance Raman spectroscopy has been widely used in the study of hemes and heme proteins (for reviews see nmmwau I OI Heep Hwnuoammmcumm mo mmo mxoutsnum n mam .AmmIOIV mmmxcfla nonsmoflsu u as m .H>Eu0m u m .Oflom OflGOHmOHm n m .Hmcfl> u > .Hxnuoa n o: "mCOADMH>muQnm cflmno opflm 45 ”Hugo omovmv Aauso «mamas a: mae a: 0mm 62 a a m: as m: as m: m mama “Hugo emmmmv AH-eo omaeav at ome 2: emm m: a a m: > m: > m: m mam: “Hugo Gmmmmv Aauso sommav an omv a: mam m m m m: > 6: mum m: m mew; umuom a m n m m e m m H Kw: .mmEom msouuwm .CHQmIBOA oEom mo mafixmz coflumHOmnfi pom mucmsuflwmnsm connmolm .H OHQMB 46 Felton and Yu, 1978; Rousseau gt 31., 1978; Clark and Stewart, 1979 and Asher, 1981). The selective enhance- ment of only the vibrations of the resonant chromophore allows protein-associated pigments to be studied owing to the "invisibility" of the protein matrix to the laser ex- citation. The types of information available from RRS studies of heme proteins are the identification of 1) oxidation and ligation state of the iron, 2) solvent en- vironment of the heme chromophore as indicated by the fre- quency position of peripheral substituents, 3) covalent or electrostatic interactions at the ring periphery, 4) local protein environment effects arising from symmetry perturbations, 5) the nature of axial ligands, and 6) transient effects (ps, ns) on the heme spectra. A. Classical Description Classically, Raman scattering is caused by the inter- action of electromagnetic radiation and the molecular polarizability. Light from a laser source at frequency Vo induces oscillations in the electron cloud of a mole- cule with an amplitude proportional to the polarizability. The resulting oscillating dipole moment radiates at fre- quency v0; this is called Rayleigh scattering. This oscil- lating dipole can be modulated by the oscillating field created by harmonic vibrational motion of the nuclei. The resulting beat frequencies occur at v0 1 vk where Vk is 47 a normal mode of the molecule. Thus vibrational Raman scattering yields light shifted in energy relative to the incoming laser frequency at lower energy (00 - vk) called Stokes scattering and at higher energy (v0 + vk) called anti-Stokes scattering (Figure 9). Stokes scattering is more intense than anti-Stokes because it arises from the zeroth vibrational level of the ground electronic state, therefore it is the usual experimentally observed process. The intensity of the Raman scattering process is given by GP 4 scattered = c(vO-vk) IO 2 I(a ) GFIZ DIG CO (2.1) where G and F are ground and final vibronic levels, respec- th tively, I is the laser intensity and a is the po 0 OO element of the molecular polarizability tensor (o,p = x, y and 2). To describe further the polarizability tensor in terms of wavefunctions of the ground and excited states a quantum mechanical approach must be used. B. Quantum Mechanical Description of Raman Theory The Kramers-Heisenberg-Dirac dispersion formula relates the polarizability tensor and quantum theory (Heitler, 1954). The polarizability tensor elements can be des- cribed as: 48 .H .mwumum pmufioxm mo Esm pmusmflos hmumco so Eouw musooo mcfluouumom paw m .memum pcsoum on» NO mao>oa Hmcoflumunfl> Hn> cam ou> cmm3uon hocooo Iouw mnu ma V_> .couonm pouwuumom may 0» mummmu m> O .mocosooum coflumufioxm Honda can we 9 "mHm>OA amumcm mCAHmuumom :mem mmxoumufluc< pom moxoum > n__>\~ 03% —O nu >> 9320 "mm oxN a? .01 m llnll/leuulnlo moxoemuzcd. octmtoom coEom .m musmflm £3.05 on Ou> O _u> lllu + 90 I (EI-EG)-hv -1r 0 I I F . (E -E )+th-1FI (2.2) where G and F are the initial and final vibronic levels as stated before and the summation is over all excited vibronic levels, I. The dipole moment Operator, u = Zeiri weights i the overlap of the initial and final state wavefunctions with all intermediate excited states. EG, EF and EI are the en- ergies of the vibronic levels, PI is the lifetime or band- width of state I, and hvo is the laser excitation frequency. The first term in Equation (2.2) is important for resonance Raman and both terms are used in the formulation of normal Raman scattering. The wavefunctions of the vibronic levels, G, F and I are usually not known so the Born-Oppenheimer approximation is made which separates the nuclear and elec- tronic coordinates IG(r,Rk)> = Ig(r;Rk)>In(Rk)> where In(Rk)> are product harmonic oscillator wavefunctions and the electronic wavefunction Ig(r;Rk)> is parametrically dependent on R This approximation enables the integra- k. tion in Equation (2.2) to be performed over the nuclear coordinates. For example, the first matrix element is 50 = _ O , — Mgi(r.Rk) where we have defined 0 _ , . Mgi(r,Rk) — This is the usual description of an electronic transition where Mgi specifies the intensity and the band shape is described by the Franck-Condon overlap integrals . The Mgi are usually weakly varying functions of nuclear coordinates so they can be expanded in a Taylor series about the equilibrium nuclear position, where R0 = 0, 3M . _ 0 91 Mgi(R) - Mgi(RO) + [aRk]R Rk + . . . (2.3) o where . [3M .] M , = i gi aRk R0 I and Mgi can be written as 8H . . O RO M . = X M (2.4) gl s¢i gs Ei ' Es 51 This is the key point in the connection between electronic scattering and changing nuclear coordinates. The Raman process is an electron scattering phenomenon yet the fre- quency of light Observed is a direct measure Of nuclear vibrational frequencies. This formulation is strictly analogous to vibronic coupling between electronic states in absorption spectroscopy whereby the changing nuclear configuration scrambles the fixed-nuclei electronic wave- functions. Transitions that originally were weak or for- bidden increase in intensity from coupling to nearby allowed transitions. The modes that are group theoretically capable of coupling electronic states may be determined by examin- ing the matrix elements in Equation (2.4). The two excited states 5 and i in D4h symmetry of hemes are of Eu symmetry. The vibrations that will result in non—zero matrix elements are those contained in the direct product Eu x Eu = A 19 +A +B 29 19 29’ Substituting Equations (2.2) and (2.3) into (2.4), +3 for the resonant case and considering only one excited electronic state leads to the following expression for the polarizability tensor A+B+... Q ll )-hv6-if y ll 2 [Mgi(RO)I z (E. (2.5) k ik-Egn k 52 B = M 1mm.) 5: + 91 O 91 o k (Eik-Eev)-h\)O-11‘k (2.6) These equations indicate that Raman intensity can be produced by two mechanisms, termed A-term (or Franck-Condon) scattering and B-term (or Herzberg-Teller) scattering. The A-term scattering intensity results from three factors; the Franck-Condon overlap integrals, the transition moments Mgi and the frequency dependence of the resonance denomin— ator. The Franck-Condon factors are non-vanishing only if there is a displacement of the potential in the excited state along a nuclear coordinate or if there is a change in frequency of a normal mode between ground and excited states. The first mechanism is the predominant cause of A-term in- tensity and therefore only totally symmetric modes give rise to A-term scattering (Clark and Stewart, 1979). B-term enhancement results from vibronic borrowing of intensity between the resonant electronic transition and a nearby electronic transition through Méi in addition to the resonant denominator. Non—totally symmetric modes are enhanced by this scattering mechanism as seen by the har- monic oscillator matrix elements = 6 i.e., n,kil’ modes along which there is no coordinate diSplacement in the excited state will have non-vanishing integrals (Clark and Stewart, 1979). 53 C. Heme Resonance Raman Scattering The use of the equations Obtained in the previous de- velOpment of resonance Raman theory are not strictly appli- cable to the electronic transitions of heme species because of their degenerate nature. Use of a more general develop- ment yields essentially the same results with bulkier nota- tion (Shelnutt, 1981), so the approximate expressions of the previous section will be used for purposes of discussion. Franck-Condon scattering is the dominant mechanism for strongly allowed electronic transitions, so this mechanism holds for excitation frequencies in the region of the in- tense Soret band. Writing out the intensity of the A- term for this electronic state leads to: I W IAI2 % i<9°JB°>I _ l<9llBl> (2.7) EEO-th-iFBO EBl-th-iPBl where the summation over k in Equation (2.5) is carried to the first vibrational overtone and Bo and B1 refer to the excited state lowest and first vibronic levels, respectively, with energies E and E B0 B1‘ As stated above, vibrations which display an excited state displacement are active in this scattering mechanism. In D4h symmetry, Alg modes are allowed. Since the Soret transition is of Eu symmetry, those vibrations contained in 54 the symmetric direct product of {Eu x Eu} = Alg + Blg + B29 may cause a Jahn-Teller distortion in the excited state. Blg and B29 modes can cause shifts of opposite sign in the potential minima of the Ex and By components, therefore re- sulting in enhancement of these modes with Soret excitation (Shelnutt gt 31., 1977). Examination of the frequency de- pendence of Equation (2.7), an excitation profile, would reveal two peaks in the intensity, one at the 0-0 vibra- tional transition and the other at the energy of the ac- tive normal mode, hvk to higher energy. Constructive inter- ference between the 0-0 and 0-1 intensity peaks and des- tructive interference in the wings is observed. B-term scattering is important for electronic transitions that gain their absorption intensity by vibronic coupling. Excitation into the a band of a heme sample results in the following approximate equation: (2.8) I+ EQO-hVO-IFQO EQl-th-il‘Q1 The vibrations active in the Herzberg-Teller scattering mechanism for D symmetry are those active in vibronic 4h coupling. As stated earlier, these vibrations are those . . . = + contained 1n the direct product of Eu x Eu Alg + A29 B modes are expected to be weak or + B , although A lg 29 lg absent (Perrin gt 31.,1969). The frequency dependence of 55 the B-term intensity also results in two maxima, and the interference effects are constructive (minus sign in Equa— lg’ Blg and B29 modes and destructive (plus sign) for modes of A2g symmetry. tion (2.8)) for A D. Polarization and Symmetty Effects The four different symmetry vibrations (Alg' A2g' Blg and B29) allowed in heme resonance Raman spectra can be identified by their depolarization ratio. Because of the linearly polarized nature of the laser excitation, Raman vibrations can be characterized by the extent to which they retain this polarization. The depolarization ratio is de- fined as the ratio of the Raman intensity scattered per- pendicular to the incoming radiation relative to the in- tensity of the parallel scattered component, pi = Ii/II Depolarization ratios can be estimated for molecules of a given symmetry by use of the three polarizability tensor invariants: the isotropy G0, the symmetric anisotropy S and the antisymmetric anisotropy Ga. To calculate these G tensor invariants it is convenient to define the symmetric and antisymmetric tensors: S = %(o + o ) and A = %(o - o ). The relative elements of apoanxaavailable (McClain, 1977). Then 56 G = %|Tr{s}|2 G = Tr{S}{S+}-GO Ga = Tr{A}{A+} . For 90° scattering geometry I p = -L = 3G5 + 5Ga g I|| lOGO + 4Gs In D4h symmetry, Alg modes are polarized (p) with p2 = 1/8, B1g and B29 3/4. and A2g modes are anonamously polarized (ap), p2 = m. Anomalous polarization reflects asymmetry in the scat- modes are depolarized (dp), p2 tering tensor and is only Observed at resonance with the a band (Rousseau gt 31., 1978). These depolarization ratios are strictly valid only for molecules of D4h symmetry. For molecules of lower symmetry, there is dispersion in pi and the values become 33/4 for polarized modes, =3/4 for depolarized modes and >3/4 for inversely polarized vibra- tions. The variation in pl with excitation frequency provides information about the symmetry and environmental perturbations of the heme macrocycle (Shelnutt 1980, 1981). However, the use of the depolarization ratio Obtained at a single excitation frequency to assign mode symmetries can 57 be misleading because ap modes can be polarized in the Soret, and polarized modes may be depolarized in the vis- ible region (Shelnutt, 1981). None of the physiological relevant hemes is strictly of D4h symmetry. In fact, all of them are Cs’ yet the predictions made for D molecules 4b are observed in most cases. When deviations occur, a slight reduction in the molecular symmetry classification is usually sufficient to interpret the data. In the worst possible case of Cs symmetry, all mode symmetries are A and their depolari- zation ratios range from 1/3 to 2. This description of the factors that influence heme ab- sorption and resonance Raman spectra provides a background for the discussions in the following sections. The experi- mental aspects of RRS will be covered in the next chapter. CHAPTER 3 EXPERIMENTAL PROCEDURES I. Spectroscopy The setup of a Raman experiment is given in Figure 10. The incident laser frequency impinges upon the sample from the bottom of a clear cuvette and the scattered light is collected at 90° to the incident beam. The scattered light is then focused, passed through a polarization analyzer (optional), a polarization scrambler and a double mono- chromator to the cathode of a photomultiplier tube. The polarization scrambler is used because of the differential detection of perpendicular and parallel polarized light. The scattered intensity versus frequency data is then dis- played on a strip chart recorder. The Raman spectrometer used in the experiments reported here is a Spex 1401 double monochromator in conjunction with the associated Ramalog electronics. Scan speeds of 50, 25 and 10 cm-l/minute were used along with the respective time constants of 1, 2.5 and 5 sec. The delta frequency position was calibrated before each experiment by using benzene as a standard, and the position of the polaroid analyzer was calibrated by reproducing the literature depolarization ratios of 58 59 . HGOE Imocmuum oamfimm mcflsoam cam HouoEOuuooom :mEmm osu mo oaumfionom .oa ousmflm muted mmpzaoo I1 024 zepora mmo< «0520:1820: fi .52.. 358 E «.sz «3.68 . tut/3 o JQQPRO QZDQ thpflmwu awszomhuwdm Zqzdml U_._.HOm oxy “monEom Ham MOM 2: ma maouoEonuomo mos cofluouucooaoo m oEo: one .mpsum ucomonm on» SH poms moeoomm m oEon Ofluuow onu mo Houo>om mo onuoodm COHumuomno Hooeumo one .HH mucosa Ha ousowm “ES Iewzw4w><>> 67 co» one cow one cow one ooe on» _ _ _ A _ _ 00.0 . N.O O..O WC mzv so cud mo mNAYI. n!— ..¢O_U tum oEoc ......... 0nd? .6 .nm oEoc in ~._ .166 Naomzeau mew; !! and]. LUNAEEZ ano oEoc ll LT. EDNVQHOSBV 68 bonded heme 3 (NMeIm)2 or copper porphyrin 3 model complexes were formed by addition of a hydrogen donor (gtgt, phenols, acids and alcohols) until no further Spectral shifts were observed. Heme 3(NMeIm)2 could not be used with acids with pKa m5 owing to the preferential protonation of the axial imidazole nitrogens. Reduction of heme 3 (NMeIm)2 in aprotic solvents was carried out by the method of VanSteelandt-Frentrup gt 31. (1981). For the hydrogen bonded heme 3 model compounds the reduction procedure was Slightly different. The heme 33+ (NMeIm)2 in solution and hydrogen donor were kept in separate arms of the glassware shown in Figure 12. Follow- ing several freeze-pump-thaw cycles and addition of the methanolic solution of 2-2-2 cryptand solubilized sodium dithionite to reduce the heme 3, the ferrous heme 3 (NMeIm)2 could be mixed with the hydrogen donor. Absorption and Raman Spectra were recorded in the attached quartz cuvette. In cases where the hydrogen donor was a liquid, rigorous degassing was necessary beforetjmareduction step. An al- ternative reduction method involves the use of tetrabutyl- ammonium borohydride. A small amount is placed in the cuvette sidearm of the glassware, the freeze-pump-thaw cycles are performed as before, the heme 3 solution is poured over to mix with the tetrabutylammonium borohydride and then the ferrous heme 3(NMeIm)2 complex can be mixed with the hydrogen donor present in the third arm. This 69 /' ‘ M—d Cr 4,11 . 493 1 b L Figure 12. Glassware used for hydrogen bonded heme 3 re- duction. . 70 method eliminates the necessity of any additions once the glassware has been closed off from the atmosphere. Care must be exercised in the amount of tetrabutylammonium borohydride used. Just as with excess sodium dithionite, facile reduction of the heme 3 peripheral aldehyde is Ob- served. Solvents were of spectral grade and dried over molecular seives prior to use; other reagents were obtained commercially and, when necessary, were further purified by distillation. B. Other Heme Model Comppunds Hemin chloride was obtained from Sigma and the low-Spin ferric bis(NMeIm) complex prepared by addition of 0.7 M N-methylimidazole to a solution of the heme in 0.1 M sodium phosphate, 2% SDS, pH 7.4. Protoporphyrin IX dimethyl ester was obtained from Sigma and the iron insertion was carried out as described by Lemberg gt 31.(1955) to form the Fe3+ protoheme dimethylester chloride compound. Ferric iron etioporphyrin I chloride and ferric iron octaethyl- porphyrin chloride were the kind gifts of Professor C. K. Chang. Preparation of the six coordinate high- and low- spin species was achieved as described above for the heme 33+ complexes. During the preparation of the various derivatives, the optical absorption spectra were monitored to insure complete formation of the Species of interest. CHAPTER 4 COORDINATION GEOMETRIES I. Reduced Cytochrome Oxidase A. Introduction Raman spectroscopy offers the potential to resolve several of the outstanding questions regarding cytochrome oxidase. The technique provides information on both the immediate coordination sphere of the heme-bound iron (Spaulding gt 31., 1975; Spiro gt 31., 1979; Callahan and Babcock, 1981) and on the conformation of porphyrin ring peripheral substituents (Salmeen gt 31., 1973, 1978). Given the unusual formyl substituent of heme 3, this latter in- sight is expected to be extremely useful (Babcock and Sal- meen, 1978). The application of Raman spectrOSCOpy to the enzyme, however, is complicated by facile photoreduction of the metal centers in the laser beam (Adar and Yonetani, 1978; Adar and Erecifiska, 1979) and by ambiguities in the optical properties of the two heme chromophores. The photoreduction difficulty has been avoided recently by using frozen samples and laser excitation in the visible region (Bocian gt 31., 1979) or by using flowing oxidase 71 72 samples (Babcock and Salmeen, 1979). By using the flow technique and several Soret region laser lines to obtain oxidase Raman spectra, the optical properties of 3 and 33 have been clarified (Babcock and Salmeen, 1979; Ondrias and Babcock, 1980). Recent oxidase Raman studies carried out by Woodruff gt 31., (1981) have confirmed these observa- tions. The absorption properties of 3 and 3 determined 3 by Raman Spectroscopy are in close agreement with those originally postulated by Vanneste (1966) but which had been clouded somewhat during the past decade by the uncertain manifestations of heme—heme interaction (MalmstrOm, 1979). The majority of the oxidase Raman work we have done has used Soret excitation. The scattered intensity under these conditions is controlled by Franck-Condon overlap factors and is distinct from the Herzberg—Teller nature of heme Raman spectra obtained with excitation in resonance with the a and 8 bands (Felton and Yu, 1978; Rousseau gt 31., 1979; Clark and Stewart, 1979). In Section II Soret excita- tion Raman spectroscopy is used to extract heme structural information through the use of model compound data (Calla- han and Babcock, 1981). In Section III vibrational band assignments for cytochromes 3 and 33 are made and in con- junction with heme 3 model compound data the spin and co- ordination states for the two oxidase iron chromophores are determined. The Soret excitation frequency dependence of the reduced oxidase Raman spectrum has been explored and 73 the data can be interpreted qualitatively by assuming that the polarizability is dominated by a single electronic state and its Franck-Condon overlap with the ground state. Finally, the mechanism of cytochrome oxidase photoreduc- tion has been studied in some detail in order to clarify the nature of this process. B. Soret Raman Scattering Frequency_Dependence Studies concerned with the frequency dependence of Raman scattering in the a, 8 region of heme protein electronic spectra have been carried out, for example on cytochrome g (Friedman et a1., 1977), and have been interpreted to first order in terms of a Herzberg-Teller scattering mechanism, and more quantitatively by incorporating both non-adia- batic and Jahn-Teller effects (Shelnutt, 1980). No system- atic investigations of this type have been carried out for Soret excitation, with the exception of a study carried out by Champion and Albrecht (1979) of the oxidation state marker band of cytochrome 3. Because the Soret absorption spectrum of cytochrome oxidase is considerably red-shifted compared to other hemes and heme proteins, fixed frequency ion laser lines can be used to construct a crude excitation profile in order to explore in more detail the frequency dependence of Soret scattering. Figure 13 Shows the Optical spectra of several oxidase derivatives and the relationship of the five available 74 I T T i I 2T Ox Cyt ox r: T -_._. Ox Cyt ox +HCOOH .._.- Ox Cyt ox * KCN .‘ ...... Red Cytox ' \ 0.8 0-6 Absorbonce O b 0.2 360 400 Figure 13. Optical absorption spectra of several of the cytochrome oxidase species used in the Raman experiments. The wavelengths of the laser lines available are also Shown in the figure. The cytochrome oxidase concentration was approxi- mately 4 uM (in enzyme) for all four compounds. 75 laser lines to these Spectra. In reduced cytochrome oxi- 2+ 2+ dase, both 3 and 33 have maxima at 443 nm (Vanneste, 1966). Two of the laser lines are thus on the long wave- 1 length Side of the absorption peak (by 571 and 735 cm- ) and the remaining three are on the Short wavelength side of the peak (by 71, 1634, and 2015 cm-l). Figure 14 shows Raman spectra of the reduced oxidase obtained with the three highest frequency lines as well as the Raman spectrum of reduced rat liver cytochrome oxidase with 441.6 nm excita- tion. The spectra obtained with the 457.9 and 454.5 nm lines were similar to those recorded by Nafie gt 31. (1973). Equation (2.7) predicts two peaks in the Soret excita- tion profile for a given mode: one at the absorption peak (l;2;' at the 0-0 fundamental) and a second at a frequency given by the sum of the 0-0 fundamental and the vibrational quantum. Constructive interference is predicted in the frequency region between the two peaks and destructive interference is predicted in the wings. In Figure 15 excitation profiles of six of the modes in Figure 14 are plotted according to a form of Equation (2.7), where the Franck-Condon factors in the numerator are set equal and approximated by the extinction coefficient at the Soret maximum. The bandwidth chosen in Equation (2.7) was 475 cm-1, estimated from the Optical data of Vanneste (1966). Two peaks are Observed in the calculated excita- tion profiles unless the bandwidth is of the same magnitude Figure 14. 76 Raman spectra of reduced beef heart cyto- chrome oxidase (a,b,c) and of reduced rat liver cytochrome oxidase (d) recorded with the laser lines indicated. Instrumental conditions: resolution, 6 cm-l; time con- stant, ls; scan rate, 50 cm-l/min. Raman lines which originate from the buffer systems used are indicated by asterisks; the daggers indicate the Raman line of the sulfate in- ternal standard. k Romon Intensity o)44l.6nm n. m ( b)4l3.l nmm c) 406.7 nm d)Rot Liver 4416 nm 77 Reduced Cytochrome Oxidase I358 3 342 $4 l (000 500 Av(crn" Figure 14 Figure 15. 78 Raman excitation profiles for the modes in- dicated have been calculated from Equation 2.7 in the text and are shown by the solid lines. The experimental data Obtained with the five available laser lines are shown by the closed circles and estimated error bars. The experimental and theoretical points are arbitrarily normalized at 441.6 nm. The three low frequency points and the two high frequency points have been connected by dashed lines which have no theoretical basis. 79 me wusmflm ATEunbECcoaootu 5.6390 com 98 gem 93 com Q- emu 03 0.8 0.8 _ _ _ _ _ 1m 7.506. 8 7638. m. 4 u. A m/ a C m. I m l m 1 u. . .A m - L L . _ . m _ _ _ e _ _ _ _ _ cow 03. 69 09. owe ox. owe 00.. .558» 362.6 oEoEuoSO pooapom Co oEotn. cozozoxw coEom oococomom 80 as the vibrational quantum, in which case the peaks are not resolved. Included in these theoretical plots are data points obtained from the experimental data of Figure 14. There is rough agreement between the calculated and experimental points in that the decreased intensity ob- served for the low frequency modes with 406.7 and 413.1 nm excitation is predicted. However the experimental data also Show deviations from Equation (2.7): a) the scattered intensity on the low frequency Side of the 0,0 peak and on the high frequency side of the 0,1 peak is greater than that calculated from Equation (2.7) which predicts des- tructive interference in these regions, b) the amplitude of the 0,1 scattering is greater than that of the 0,0 and c) the effects in (a) and (b) become more pronounced as the frequency of the vibrations increases. These phenomena have been observed in a band excitation profiles of heme proteins and have been accounted for by including a non- adiabatic term to allow for the effects of Soret excited states (Shelnutt gt 31., 1976; Rousseau gt 31., 1979). The dramatic increase in intensity observed for the peripheral aldehyde stretching vibrations of cytochromes 3 and 33 at 1610 cm‘1 1 (see Chapter 5) and 1665 cm- can be interpreted as arising from nonadiabatic coupling to a transition at higher energy. Carbonyl substitution has been shown to induce additional absorption bands in the spectra of chlorin species (Weiss, 1972). Raman excitation in the 81 near UV region of these Species results in an additional enhancement of the peripheral substituent vibrations (Lutz gt 31., 1982). A similar mechanism may be Operative here for the heme 3 chromophores in cytochrome oxidase. With the Franck-Condon nature of the Soret RR Spectra established, structural information can be extracted from the Spectra by comparison with appropriate model compounds. II. Soret Excitation RRS of Ferric Heme Model Compgunds A. Previous Structural Correlations The geometry of the porphyrin macrocycle in hemes and heme proteins is reflected in the frequency positions of several vibrational bands observed by resonance Raman Spectroscopy (Spiro, 1974; Felton and Yu, 1978; Kitagawa gt 31., 1978; Rousseau gt 31., 1979). One of the most useful empirical correlations between heme structure and Raman frequency has been develOped by Spaulding et a1., (1975) who showed that an inverse relationship exists be- tween the frequency of an anomalously polarized mode in the 1550-1610 cm"1 region and Ct-N, the distance from the center of the porphyrin to the pyrrole nitrogens. This was later confirmed and extended (Huong and Pommier, 1977; Scholler and Hoffman, 1979; Spiro gt 31., 1979); plots of Raman frequency vs. porphyrin core size (C -N) for the ap t mode (denoted Band IV) as well as for a polarized mode in 82 in the 1600-1650 cm"1 region (Band IV) could be fit by an empirical equation of the form (4.1) where 3i is the frequency of the vibration under considera- tion, d the Ct-N distance and Ki and Ai are adjustable th vibration. Equation (4.1) parameters Specific to the i has thus far been established for the three vibrational bands indicated above, all of which have appreciable methine bridge bond stretching character (Spiro gt 31., 1979). As a consequence, the correlations indicated by Equation (4.1) are relatively insensitive to the nature of the pyrrole B-carbon substituents, provided that these are linked by C-CS bonds. However replacement of the hydrogen at the methine bridge carbon by, for example, phenyl groups or deuterium causes shifts in these bands unrelated to core size change and Equation (4.1) is no longer applicable. In addition, Bands II and V are also somewhat sensitive to the nature of the axial ligands and shift to higher fre- quency as the n acceptor character of the ligands is in- creased. With the establishment of sound correlations between porphyrin core size and Raman frequency, it has become possible to interpret scattering data to determine both spin state and iron coordination number for several heme 83 proteins (Spiro gt 31., 1979; Seivers gt 31., 1979). How- ever research which has been done thus far to link por- phyrin core size and Raman vibrational frequencies has been carried out with d, B excitation. The vibrations Ob- served upon Soret excitation of hemes and heme proteins are, in general, distinct from those enhanced by a, B excitation and the empirical correlation between core Size and Raman frequency described above for Herzberg-Teller active modes may not necessarily apply to the Franck-Con- don vibrations. Sporadic reports have appeared in the literature which relate the frequency of a polarized mode in the 1560-1600 cm.1 region, observed with Soret excitation, to heme iron spin state. For example, Yamamoto gt 31. (1973) reported such a correlation for hemoglobin and myoglobin compounds, Remba gt 31. (1979) reported analogous data for chloro- peroxidase and Babcock and Salmeen (1979) tabulated the frequency of the band as a function of heme 3 iron spin state. However, a systematic study of this phenomenon has not yet appeared. In this section, the Raman Spectra of a number of heme model compounds and heme proteins are in- vestigated and an empirical correlation between the fre- quencies of the observed modes, the porphyrin core size and the pattern of B-carbon peripheral substituents is establish- ed. In the next section this correlation is applied to the interpretation of Soret excitation Raman spectra of cyto- chrome oxidase and a number of its inhibitor complexes. 84 B. Per1pheral Substituent Dependence of Vibrational Fre- guencies 1. Protoheme §pecies Soret excitation Raman spectra of several protoheme con- taining Species are presented in Table 2A. These have been selected to represent the commonly encountered combinations of spin state and iron coordination: a) Six coordinate low- Spin, b) six coordinate high-Spin, and c) five coordinate high-spin. The Raman spectra of the cyanide and fluoride complexes of ferric HRP have also been investigated and it is found that they resemble the azide and fluoride metmyo- globin complexes, respectively (Babcock gt 31., 1981). AS expected for Soret excitation, the principal vibrational modes Observed are polarized (pi i .75) (see Table 3). However the depolarization ratios rarely achieve the 1/8 value ex- pected for D4h symmetry, indicating that the effective sym- metry is reduced. This effect, plus the conjugation of the vinyl substituents with the porphyrin n system (Adar, 1975; Choi gt 31., 1982) contributes additional bands to the high frequency Raman Spectra of protoheme derivatives when compared to the symmetric heme species described below. The Soret excitation Spectra are distinct from those Obtained for the same compounds with a, 8 band excitation, particularly in the 1550-1600 cm"1 region, where the 85 mmma mmma mmma omma mmma moma m m\a +mm use oema mmma moma oema amma eoma m «\a maSaozzv+mommmo u--- nun- u--- I--- omma moma m m\a mauzov+momemo aema mmma moma oema mmma moma m ~\a mxeaozzm+mmm0aum omma oema mama omma mmma amea m mxm cmaemo+mmmv mmma mmma mmma mmma mmma mmma m «\m nao+mmmoaum mama mmma amea mama mama mmma m m\m mlomzom+mmm0aum Oouousuom mcoauamom momm Ham nuaz ooEom .m mmma m nun In- nu: .mmma mmma moma «\a ASHozzv+mommzaumm oema mmma mmma moma .emma mmma moma m m\a NASaozzC+mom osonoaoum omma nu- In- nu- .amma mmma moma m m\a mznzuez mmma mmma mmma amma mmma mmma m m\m sao+mommzoumm omma mmma ooma omma mmma mmma m ..m\m mmm mouamaxo oama omma mmma mmma mmma mmma m m\m Naomzov+mommzoumm aama mmma amea mmma mmma mmma m «\m cmmnzumz moma emma mmma mmma mmma mmma m ~\m ensues moma mmma mmma mmma mmma mmma m m\m mnmuoz moaoomw oEonouoam .¢ do do a .UHOOO ououm USSOQEOO xo pouom n xoK camm oanmma> u 4 moaocosooam GoEom «momocosooum coEom .mcaouoam oEom poo moEom msoaao> How moaocosooam :oEom pouooaom one .mauoEooU GOHDMSHOHOOU .ououm swam .m oanoe 86 .nommav .AM.MM nooonom na opoE oao munoanmammo mm one m oEOHnOOu>O one .Amhmav ++ .NM NM muo>oam Omam one Amemav ooEouaoz oom .moosmmnfio umnBoEOw ma nammlnman ousm mo mmm mo unoEnmammm one xx .lmemav .mm_mw ouaem an mam Aeneas amm_mw mueeeam me mmaaaeoo mmahmu seem ¥ ehoa -- -- -- .aema omma moma m m\a -zo.+mm use omma I -- -- -- .aema omma moma m m\a m emu oema N Am -- -- -- .mema omma moma m m\a ASaozzv+mm meow mmma l -- -- -- .mmma amma mmma m m\m -ao+mm osmm mmma -- -- -- .mama mmma mmva m ~\m mooom.+mm umo m mmma ml -- -- -- .mama mmma mesa m mxm +me emu mmma -- -- -- .mama mema mmma m m\m mnemzov+mm oEom ++moaoomm m oeom .U do do a nouom n xoa .puoou wwmww UGSOQEOU oanama> u xoa moaonosooum noEom . «moaonooooum noEom .posnaunoo .m manta 87 structure sensitive anomalously polarized mode (Spaulding gt 31., 1975) is replaced by polarized modes. Yamamoto gt 31. (1973) were the first to point out that these polarized modes are sensitive to spin state. For the Six coordinate myoglobin derivatives they investigated with 441.6 nm ex- citation; the ratio of intensities for the bands at 1566 and 1584 increased as the high-spin content increased. The data confirm this correlation and demonstrate that it also applies to six coordinate high-spin and low-spin protoheme compounds. Moreover, five coordinate high-spin protoheme compounds Show a strong polarized mode at 1576 cml, inter- mediate between the 1584 cm-1 low-spin and 1566 cm1 Six coordinate high-spin marker bands. Thus the high frequency polarized mode Showsthe same general dependence on heme structure as reported for Band IV, the high frequency ap mode: as the Ct-N distance increases from six coordinate low-spin through five coordinate high-spin to six coordinate high—spin, the frequency of the band decreases. However, in the case of Band IV the overall decrease amounts to 30 cm.1 for the heme complexes examined whereas for the polarized mode the corresponding decrease is only 18 cm-l. This observation indicates a less dramatic Ct-N distance dependence for the polarized mode. Table 2 summarizes the positions of the prominent bands of protoheme, FeOEP and heme 3 Species as well as vibrational frequencies Observed for the same compounds with excitation in the visible region. 88 With a, 8 band excitation a polarized mode in the 1500 cm—1 region is observed (Band II) which is sensitive to heme iron spin and coordination. A band in the same region is also prominent in the Soret excitation Raman Spectra we report and exhibits the same dependence; we conclude that Band II is enhanced by both a, B and Soret laser lines. For six coordinate high-spin derivatives Band II occurs at 1 1482 cm—1 and Shifts to 1496 cm- for five coordinate high- spin species. In Six coordinate low-spin protoheme com- pounds the observed frequency is 1507 cm-1. Because other bands are also apparent in the 1470-1510 cm-1 region, cor- relations based only on Band II positions are somewhat tenuous. 2. Hemes with Saturated Ring Substituents Attempts to extrapolate the correlations develOped in the previous section between protoheme structure and Raman frequencies to g—type cytochromes and to heme 3 containing species were only partially successful. For Band II the same pattern emerges. However for the polarized mode in the high frequency region inconsistencies are apparent. This behavior is demonstrated for species with saturated ring substituents in Table 2B where we have collected data of representative six coordinate high—spin, five coordinate high-Spin, and Six coordinate low-spin compounds. The 3+ Raman spectrum of bis(cyano)Fe OEP has also been recorded 89 and it is found that its vibrational properties are similar to those of the bis(NMeIm) complex; its principal high fre- l 1 quency vibrations occur at 1590 cm- , 1505 cm- , and 1376 cm—1. In the 1500 cm.1 region, the low-spin species show Band II at 1507 cm-1; for the high-spin compounds Band II occurs at 1496 cm-1 for five coordinate complexes and at 1 1482 cm- when the iron is Six coordinate. In the high frequency region, there is a positive Shift of about 10 cm-1 in frequency for the structure sensitive polarized mode for the hemes with saturated substituents compared to the corresponding protoheme derivatives. Thus the six co- ordinate 1ow-spin indicator occurs near 1590 cm-1, the five 1 coordinate high-spin band is at 1584 cm- and the Six co- ordinate high-spin mode Shows at 1575 cm-1. AS with the protoheme Species, however, the total decrease in the fre— quency of the high frequency polarized mode as the Ct-N distance increases from its six coordinate low-spin value (1.989 A, (Collins t 1., 1972)) to its six coordinate high- spin value (2.045 A, Mishiko gt 31., 1978)) is approximately 18 cm’l. Protoheme is distinct from the species in Table 2B in that it has unsaturated substituents at two of the B-carbon positions, 1;E;r vinyl groups at carbons 2 and 4. The Raman data above thus indicate that upon Soret excitation of iron porphyrin compounds the observed frequency of the mode in the 1560-1600 cm.1 region reflects three different 90 structural aspects of the chromOphore: (a) the spin state of the iron, (b) the coordination number of the iron, and (c) the nature of the peripheral substituents. For Band IV Observed with d, 8 band excitation, it is apparent that the frequency position reflects only C -N distance, i.e., t structural features (a) and (b) above, since similar values of A and K in Equation (4.1) can be obtained from Raman data for metalloporphyrins with either saturated or un- saturated B-carbon peripheral substituents. An explanation for the dichotomy in behavior of these two modes can be constructed based on the normal coordinate analysis of Abe gt 31, (1978) and is considered below. 3. Heme 3 Species The data above demonstrate the usefulness of Soret ex- citation Raman data to the assignment of heme geometries provided that proper account of the pattern of peripheral substituents is taken. In heme 3 these substituents are distinct from those of protoheme and of the compounds in Table 2B in that a hydroxyfarnesylethyl group occurs at position 2, a vinyl at ring position 4 and a formyl at ring position 8. Thus we have carried out a classification of the structure sensitive heme 3 modes observed upon Soret excitation and show representative spectra of the various derivatives in Figure 16 and Table 2C. Six coordinate low- 3+ 1 spin heme 3 (Figure 16d) shows bands at 1506 cm- and Figure 16. 91 Resonance Raman Spectra of several heme 3 model compounds dissolved in methylene chloride. Solvent and non-resonance enhanced ligand vibrations are indicated by asterisks. In- strumental conditions as in Figure 14. 92 :2 g :- xex=4067nm .«2 :2 «.2 l I l 0] O) Y. ' — o) Heme 93*CI -0 a #- 9§ ? g l I ¢ T 9(- * b) Heme 93‘00; >- N N I- i; I~ _ _ n * U) | ‘T E- .- m -— E: I; c) Heme g3*om no“ .mOUOE ®>HUHmmem QHDUUSHUW COHkuuHUXO umuom >Ocosvouw amnOauounfl> msmuo> woquHQ oonoumap < AZIuUV .ma onsmam 99 ea oasmam .15va 89 0mm. 08. 08. q u 4 q u 8.. a too e... a sateen :5 o oEozostd . o o oEoI .m._ “:08 m com. o m We .2 228 m Now emo 98-1 com .m: 2095 no.5? $.39. no.9 or 882 2,528 9.28.5 - mmm 8:266 stem 100 bis(N-methylimidazole) and bis (cyano) compounds are com— pared with the histidine methionine combination in cyto- chrome g. This may reflect a real axial ligand dependence or the fact that the substituents in the 2 and 4 positions on the ring periphery in cytochrome g are involved in thioether linkages. The observations and differences described above regard- ing the behavior of the four structure sensitive modes summarized in Table 2 can be rationalized by reference to the recent normal coordinate analysis of Abe EE.3£° (1978). Bands II, IV, and V correspond to vibrations v4(A1g), v19 (A29) and v10 (Blg), respectively, in their normal co- ordinate analysis and the polarized mode we observe in the 1560-1600 cm.1 region with Soret excitation can be assigned as the analog of their v2 (A Abe gt El- (1978) have 19" shown that v3, v19 and v10 have considerable a-carbon/ methine-carbon stretching character whereas v2 involves primarily B-carbon/B-carbon stretching. Consequently, the more dramatic Ct-N distance dependence of Bands II, IV and V relative to v is a manifestation of the fact that the 2 pyrrole ring maintains a fairly rigid structure and shifts in porphyrin geometry as a result of core expansion are accommodated largely by distortions in the a-carbon/ methine-carbon/a-carbon bond angle. Substituents to the pyrrole B-carbons, on the other hand, are predicted and observed to alter the frequency of v2 without perturbing 101 v3, v19 or v10 to a Significant extent. In this regard, Kitagawa and coworkers (Abe et al., 1978) have shown that 11' the Blg mode which represents the out of phase analog to v2, is also sensitive to the nature of the 8 carbon sub- V stituents. The data presented above demonstratethat Soret excita- tion Raman spectroscopy of hemes and heme proteins can yield structural information analogous to that obtained with visible excitation. This information is, however, somewhat more difficult to extract because the frequency of the structure sensitive modes is also a function of the nature of the peripheral substituents and because the changes in frequency upon change in structure is less dramatic than for Band IV. Nonetheless, there are a number of advantages associated with Soret Raman, particularly in the less severe demands upon protein concentration and laser power. In the following section Soret excitation Raman results of oxidized cytochrome oxidase are presented which apply the structural correlations developed here. III. Oxidized Cytochrome Oxidase A. Photoreduction Photoreduction of resting cytochrome oxidase by the incident laser light at the powers required for Raman spec- trosc0py is a major obstacle to the application of this 102 technique to the oxidized enzyme. Moreover, uncertainty exists in the literature regarding both the mechanism (Seiter and Angelos, 1980) and manifestations (Woodruff 33 31., 1981) of cytochrome oxidase photoreduction. Figure 18 presents results obtained with the flowing sample tech- nique developed to avoid the photoreduction problem (Bab- cock and Salmeen, 1979). Under flow conditions (Figure 18a) and with 406.7 excitation the oxidation state marker band 1 with no shoulder to lower fre- l is observed at 1373 cm- quency, the oxidized cytochrome a3 modes at 1572 cm- and l 1676 cm_ are present (Ondrias and Babcock, 1980) and the l 1 cytochrome §_modes at 1506 cm- , 1590 cm- and near 1650 cm 1 are observed. (See Table 4 for a summary of the vibrational properties of cytochromes a and 33.) When the flow is turned off and the Spectrum of the static sample recorded (Figure 18b), a number of changes occur. In particular new bands are apparent at 1622, 1612, 1520 cm"1 and a shoulder appears on the low frequency side of the 1 3+ 3 are still present. The spectrum of the 1372 cm' band. The cytochrome 3 bands at 1676, 1572, and 1478 cm-1 partially photoreduced enzyme in Figure 18b bears a strong resemblance to that of the enzyme in the presence of for- mate and a mild reductant (Figure 18d), a treatment which 2+ a§+-HCOOH (Nicholls is known to generate the Species a and 976). When the flow is restored the sample again exhibits the spectrum of the oxidized enzyme (Figure 18c). Figure 18. 103 Resonance Raman spectra of several cytochrome oxidase species recorded with 406.7 nm excitation. Instrumental conditions: resolution, 6 cm-l; time constant, 1 S; scan rate, 50 cm-l/min. The instrument gain was constant in recording spectra a, c, d and e; for spectrum b this was increased by a factor of three. 104 mhnT. a) Resting - flow on. b) Resting '- flow Off. 1:13 flow on. d) Partially red c) Resting +HCO0H e) Reduced. I600 l500 I400 l300 A; (cm") I700 >._._mzm._.z_ 24248 Figure 18 105 The results in Figure 18, particularly the comparison between 18b and 18d indicate that in aerobic samples of cytochrome oxidase, the photoreduction process leads first to reduction of cytochrome 3 followed by reduction of cyto- chrome 33. This redox situation, iLEL, reduced cytochrome a and oxidized cytochrome 33, corresponds to that found in the aerobic steady state (Nicholls and Petersen, 1974) and suggests the following mechanism for the photoreduc- tion process. Illumination produces reducing equivalents external to the oxidase (most likely via a flavin contamina- tion (Adar and Yonetani, 1978)) which subsequently reduce the enzyme. In the presence of oxygen the predominate species is that corresponding to the aerobic steady state (a2+ 3+ _ 33 2' (Adar and Yonetani, 1978). This proposal is compatible with ). Upon depletion of 0 full reduction is achieved previous results with 441.6 nm excitation which showed minimal formation of reduced cytochrome 3 upon illumina- 3 tion of static oxidase samples (Salmeen gt_31., 1978), with the observations of Adar and Erecinska (1979) who saw a delay in the appearance of the 1665 cm.1 band, characteris- tic of reduced cytochrome 33 in the initial stages of photoreduction at -10°C, and with the results of Bocian et 31. (1979) who Showed that Soret excitation of frozen oxidase samples produced photoreduction but that visible excitation did not. This latter result is expected if the photoactive Species is a flavin, which has a strong ab- sorption in the blue but not in the red region (>550 nm) 106 of the spectrum. The photoreduction process has been studied in more detail by carrying out laser power studies on flowing oxidase samples (Babcock gt 31., 1981). At low to moderate powers of 406.7 and 413.1 nm excitation (Figure 19) the Spectrum of the oxidized enzyme is observed. Only at 55 mW incident power do photoreduction effects, notably the increase in the intensity of bands at 1622 cm-1, l and 1520 cm-1, become evident. This work demon- 1610 cm" strates that the use of the oxidation state marker band as a judge of photoreduction is a poor criterion. Even with appreciable photoreduction (Figure 19d) there is little obvious change in this band. This observation, as well as the fact that photoreduction produces its first noticeable effects in the 1600 cm”1 region with 406.7 and 413.1 nm excitation, can be rationalized by reference to the excita- tion profile data presented above. Under conditions of partial photoreduction the predominant form of the enzyme is identified as the aerobic steady state: cytochrome g3 oxidized and cytochrome a reduced. The 32+ species has 1 an absorption peak at 443 nm which is 2015 cm- and 1634 cm-1 to the red of the 406.7 and 413.1 nm laser lines, respectively. Consequently, from Equation (2.7), we expect that vibrations in these frequency regions will be more strongly enhanced than those to lower frequency and that photoreduction will be apparent first in the high frequency Figure 19. 107 The incident laser power dependence of the resonance Raman Spectrum of resting cytochrome oxidase. The excitation frequency and power incident on the sample are indicated. In- strumental conditions: resolution, 6 cm-l; time constant 1 S; scan rate, 50 cm-l/min. From Babcock et a1, (1981). RAMAN Resting Cytochrome Oxidase ' Flow on a) 406.7, ISmW b) 406.7, 30mW INTENSITY I37_-—-—- 0’ 6'3 .08 8109.: c) 4:3,: ,30rnW N-w-Iln o'-‘° - T 'T ' ‘9 d) 4l3.l , 55mW I3 8 3a. 7 N EN T I9' n '° :0 Q 9 T I 1 l 1 l 1 J 1 i 1 l 1 I i I700 I600 I500 I400 I300 l200 I l00 |000 A22 (cm") Figure 19 109 modes. As more of the enzyme is reduced, this process also becomes evident in the 1360 cm.1 region (Figure 18b). These excitation profile arguments also rationalize earlier observations on the photoreduction of the oxidized enzyme (Salmeen et 31., 1978) in which excitation at 441.6 nm 2+ peak absorption) resulted in (72 cm"1 from the 443 nm 3 the observation of cytochrome 32+ modes throughout the Raman spectrum. B. Raman Spectra of Inhibitor Complexes of Cytochrome Oxidase and of its Oxygenated Form Figure 20 presents Raman spectra of several derivatives of cytochrome oxidase; the vibrational assignments from these data are summarized in Table 4. In the formate complex of the oxidized enzyme, cytochrome a§+-HCOOH is high-spin whereas cytochrome 33+ remains low-spin (Bab- cock et 31., 1976). There is thus no change in spin state in going from the resting enzyme to the formate complex and correspondingly the Raman spectra of these two oxidase species are similar (compare Figure 18a with Figure 20a). Upon reduction of cytochrome a in the presence of formate, the mixed valence Species, a2+ 33+ §+-HCOOH bands at 1676 cm- remain constant whereas the oxidized a 1 1 l -HCOOH, is formed (Fig- l 1 ure 20b). The 3 , 1572 cm” and 1478 cm"1 and 1507 cm’ 1 bands at 1648 cm- , 1590 cm- are replaced by bands at 1622, 1610, 1586 and 1518 cm- as reduction of Figure 20. 110 Resonance Raman spectra of the oxidized (a,d) and partially reduced (b,e) complexes of cyto- chrome oxidase with formate and cyanide. In c, the spectrum of oxygenated cytochrome oxi- dase, formed by air oxidation of the dithionite reduced enzyme, is shown. Instrumental con— 1 ditions: resolution, 6 cm ; time constant, 1 s; scan rate, 50 cm-l/min. lll . . H m 0 x I4 AWHH I my H . O YH 0V1; 0 N W0 C 0 C C .2 em . m. . XHH R” W. .W m m P. H "O m vmwT t o ) ‘1 ) N8—l y n .b C .0 FC % e 4 R. e P. x \.I a. . I i _ thT $09.. A! ooh. ~57 I wwwv wvm. 8.9% 8.9- .L Emzmeé 24241 m? (cm") Figure 20 112 .Aammav MUOUQmm 6cm CMSMHHMU EOHhe oeoe .meoe .omme .oome .eeee .eeme mme o «\e ~x2em22v+mm matte meoe .meoe .Neme .Nmee .meme oee o m\m Niomzoc+mm meme. oeoe .Nmoe .emme .Neee .eeme eee m Nxm ueo+mm meme. oooe .meee .mmme .omme .eom .mem mee m N +mm «Noe .emme .mmme mee o o +mm omoe .eece .omme .oome .eeee .meme ewe o ~\e +mm oeoe .eeoe .oome .oome .eeee .meme ewe o «\e zo.+mm oeoe .meoee .Neme .meee .meme eee o N\m mooom.+mm oeoe .meoee .Neme .meee .meme eee o ~\m +mm AHIEUV maceumune> ceumwuwuomemnu me .UHOOU Seam mweommm I I .mmeommm mmmpflxo meoenoouwo msowum> CH mm one o mEoenooumo mo mmeuummoem emceeumunfl> cam soHuMCHUHOOU .Hmoflumo .v magma 113 cytochrome 3 occurs. Upon CN- addition to the oxidized enzyme, the species, 33+ g§+-CN—, is formed and the 3 and 33 species occur in the low-Spin state. In the Raman spectrum of this derivative (Figure 20d), the high-spin §§+ band at 1572 cm.1 3+ and g3+-CN- 1 Show the low-spin ferric heme g marker band at 1590 cm— . is absent and both 3 The 1478 high-Spin band of the oxidized enzyme is also absent, having been replaced by the low-spin band at 1505 cm-1. The latter change is somewhat obscured by the l appearance of the 1472 cm- band which occurs in low-spin heme 33+ complexes (Callahan and Babcock, 1981). Upon re- duction of cytochrome 3 in the oxidized, cyanide complexed 2+ 23 (Figure 20e). As with the partially reduced formate 2+ . . + . enzyme, the mixed valence speCies, 3 -CN, 18 formed 1 Species, the 3 modes at 1626, 1610, 1520 and 1359 cm- appear. The low-spin §§+-CN- modes remain at 1673, 1641, 1589, 1504, 1475 and 1373 cm‘l. C. Discussion 1. gptical Properties of Cytochrome Oxidase The Raman data presented here and elsewhere (Salmeen gt gt., 1978; Babcock and Salmeen, 1979; Ondrias and Bab- cock, 1980), as well as the Raman observations made by Woodruff gt_gt. (1981), provide strong evidence in support of Vanneste's (1966) original decomposition of the optical 114 properties of cytochrome oxidase into separate cytochrome g and g3 contributions (Table 4). Thus laser excitation (441.6 nm) on the long wavelength side of the oxidized oxidase Soret band generates the Raman Spectrum of cyto- 3+ chrome g (Babcock and Salmeen, 1979). Laser excitation (413.1 nm, 406.7 nm) on the short wavelength Side produces the Raman spectrum of cytochrome 33+ term of Equation (2.7) and those vibrational modes of through the first cytochrome g3+ which are in resonance according to the second term of this equation. The cytochrome 33+ absorp- tion peak occurs at 427 nm, which is 790 cm.1 away from the 413.1 nm krypton line. Thus the observation that the low frequency modes in the Raman spectrum of the oxidized enzyme obtained with 413.1 nm excitation are those of cyto- chrome g§+ (Babcock and Ondrias, 1980; Woodruff gt gl,, 1981) can be rationalized. 2. Coordination Geometries of g and g1 Cytochrome g’is low-spin in both valence states (Tweedle gt gl., 1978) and consequently six coordination is expected. This expectation is borne out by a comparison between the Raman properties of heme g3+(NMeIm)2 and cytochrome 33+ in the 1500-1600 cm“1 region and is in agreement with earlier EPR (Blumberg and Peisach, 1979; Babcock gt gl., 1979) and MCD (Nozawa et a1., 1979) observations. An in- teresting anomaly for cytochrome 3 involves the behavior 115 of its formyl group. The formyl vibration is observed in the bis(imidazole)heme 33+ model compound at 1670 cm-1. For cytochrome 33+, however, the highest frequency mode is observed at 1650 cm-1 1 with a second vibration apparent at 1641 cm- (Figure 18a; Babcock and Salmeen, 1979). Thus, even though the bis(imidazole) heme g3+ species has the EPR crystal field parameters of cytochrome 33+, it does not have the vibrational properties of the tg’tttg_formyl group. Because cytochrome g§+ is high-Spin (Tweedle gt gt., 1978) and can exist as either five or six coordinate, the model compound data for heme g are extremely useful in the determination of its geometry. In the resting enzyme cyto- chrome g3+ 3 cm-1 in the high frequency region. A weak feature near 1615 cm.1 is also due to the 33+ contributes vibrations at 1676, 1572, and 1478 chromophore (Figure 18a). A comparison of these frequency positions with those of the high-spin five and six coordinate model compounds of Figure 17 and Table 4 clearly shows that the heme 33% (DMSO)2 complex reproduces the cytochrome 93+ spectrum well; the five coordinate model, heme g3+-C1 does so much less adequately. Thus we conclude that in the resting 3+ enzyme, the g3 species occurs in the high-spin, six co— ordinate state. The absorption spectrum of heme g3+ (DMSO)2 (Figure 11) also agrees well with Vanneste's spec- tral assignments of the Soret and visible region absorption 116 maxima. Table 4 also indicates that the frequency posi- 3 3 (NMeIm)2 compare well, confirming the assign- tions assigned to the g +-CN"' complex and those of the low- spin heme 33+ ment of cyanide derivative as six coordinate, low-spin. Finally g§+-HCOOH can be seen to occur as the six coordinate high-spin species consistent with the idea that formate serves as a ligand to the iron in this inhibitor complex (Nicholls, 1976). Our finding that cytochrome 33+ in the resting enzyme 3 is six coordinate has interesting ramifications with respect to the structure of the oxygen reducing site and the mode by which the prOposed exchange coupling between the iron of g3 and the associated copper (Van Gelder and Beinert, 1969; PaLmer, gt gt., 1976) is mediated. Two general models are available in the literature. These can be classified as "backside" bridging in which the c0pper of the heme 3 ring to which 02 binding occurs and "front-side" bridging in which the copper is coupled to the iron by a ligand which occupies the reduced protein dioxygen binding site (See Figure 21 a,b). A specific "back-side" model has been proposed in which the bridging ligand is a histidine residue (Palmer gt gt., 1976). This is represented in Figure 21c where a water molecule has been incorporated in the iron sixth ligand position to account for the Raman data. A "front-side" model has also been proposed which postulates a u-oxo bridging ligand (Blumberg and Peisach, 117 Resting Cytochrome gsrPossible Structures "i Cu2+ — B — FeM— L L — F'e3+— B-Cur' : \ g \ N “02 N ‘02 a) "back - side" b) "front-side" N "l 2+ . ' 3+ . : 3+ 2+ Cu -hls —-Re —H20 his—Fe \O/Cu I I N N c) Palmer 91 g! 0976) d) Blumbegg and Peisacn “979) Figure 21. Possible structures for the cytochrome a3 dioxygen reducing site. 118 1979) and is shown in Figure 21d; here a histidine residue is incorporated as the Sixth ligand to maintain the iron in the six coordinate state. Of these two structures, the "front-Side" bridging model has received experimental support from the fact that: l) imidazole bridging ligands are generally unable to support exchange coupling with magnitudes as large as those found in the enzyme (|2J|>2oo cm"l ) (Kolks gt gt., 1976; Landrum, gt gt., 1978; Haddad and Hendrickson, 1978; Petty gt gt., 1980), whereas u-oxo exchange couplings are well within this range (O'Keeffe gt gt., 1975) and 2) CO photolyzed from the g3 heme has been shown to bind to the Cua3 site (Alben gt gt., 1981). These results support the "front-side bridging model although u-oxo ligand structures generally force the iron to adopt a five coordinate, out-of-plane geometry (O'Keeffe et a1., 1975). Other ligands, par- ticularly carbonate, have been prOposed in lieu of the u-oxo bridge (Seiter, 1978). Since the resting form of the enzyme is formed on a time scale slow with respect to the turnover of the enzyme, this structure may not be im- portant in the catalytic process. Oxygenated cytochrome oxidase (Orii and King, 1976) which is formed on a much faster time scale may provide insight into the oxygen reduction mechanism. 1 The shoulder at the low frequency Side of the 1590 cm— band in oxygenated cytochrome oxidase in Figure 20c has 119 been resolved in higher resolution studies to be a peak at 1574 cm-1. This indicates a structure with approxi— mately the same core size as g§+ The dramatic reduction in intensity in the 1572-1574 cm- in the resting enzyme. 1 band between oxygenated and oxidized cyt g3 may be caused by a large decrease in the extinction coefficient of this heme. The Soret absorption maximum of this Species shifts to 427 nm from N420 nm without an increase in intensity as is observed for the cyanide inhibited enzyme where cyt 33 is low-Spin. Thus the absorption properties of the oxy- genated enzyme seem to reflect predominantly the transition energies of cytochrome g with only a small contribution from cyt 33. A mixed-spin (S=3/2) species as suggested by Shaw gt gt., 1978, and Woodruff gt gt., 1982 may account for these observations. . + . In the reduced protein, only cytochrome g2 is para- 3 magnetic and occurs in the high-spin state. The MCD properties of this species are quite Similar to those of deoxyhemoglobin and deoxymyoglobin (Babcock gt gt., 1976) as are its ligand binding properties (Erecinska and Wilson, 2+ 1978). These observations suggest that g3 is five co- ordinate in the reduced enzyme in the absence of an added ligand. This conclusion is consistent with previous Raman results which identified Specific vibrations associated 2+ 3 215 cm- with g (Salmeen gt gt., 1978) including a vibration at 1. This mode has been assigned to the Fe-histidine 120 stretching mode by analogy to the 215 cm-1 vibration in de- oxyhemeglobin and deoxymyoglobin (Nagai gt gt., 1980; Hori and Kitagawa, 1980). An additional conclusion can be reached concerning the nature of the 33 Site from the Raman results presented here and in previous reports (Salmeen 2E.El°' 1978; Ondrias and Babcock, 1980; Babcock and Salmeen, 1979; VanSteelandt gt_gt., 1981). In both the oxidized and reduced states of the enzyme the formyl vibration of g3 is clearly enhanced and occurs at a frequency typical of a free, non-hydrogen bonded C=O. These observations indicate that the 33 site, at least in the vicinity of pyrrole ring IV, is hydrophobic and that bulk H20 is excluded. 3. Cytochrome g 3Catalytic Model This structural information concerning cytochrome g3 can be combined and a speculative model for dioxygen reduc- tion proposed. Figure 22 depicts a schematic of the g3 oxygen reducing site. The resting form of the enzyme is shown in the upper left hand corner where the Sixth ligand to cyt 33, B, mediates the strong exchange coupling between cyt g3 and CuEB. Reduction of the enzyme results in the structure in the upper right corner where cyt 33 is five coordinate and high-spin. It is this form, which is similar to the active site of hemoglobin, that binds oxygen (Figure 22, lower right). This enzyme-substrate complex, called 121 .muem mCHUSUOH cmmwxo mmMUon meoesooumu on» wo oeumfimnum cozoaoom No 5.8.1 inco:caomm oem 8.88m «o 5:326 .NN OHDOHL 122 Compound A (Chance gt _t., 1975) has been Shown to be spectroscopically similar to (NMeIm)2 heme gZ+-OZ (Bab- cock and Chang, 1979). Following partial O2 reduction a u-peroxy structure (lower left) has been postulated to be important in the catalytic cycle (Wikstrom gt gt., 1983). Further electron transfer, uptake of protons, and release of H20 eventually leads to the resting structure (upper left). However, the details of the reaction mechanism, have not been established. CHAPTER 5 THE ORIGIN OF THE CYTOCHROME A ABSORPTION RED-SHIFT I. Introduction The longer wavelength absorbance maxima of cytochrome oxidase relative to protoheme containing proteins results from its formyl-containing heme g chromophores (Figure l). The individual contributions of cytochromes g and g3 to the overall protein spectrum have been resolved by evidence from several laboratories (Wikstrom gt gt., 1976; Wilson, gt gl., 1978; Babcock and Salmeen, 1979; Scott and Gray, 1980; Halaka gt gt., 1981; Babcock gt gt., 1981; Woodruff gt 31., 1981) which support an independent chromophore model and indicate that the spectral deconvolution car- ried out by Vanneste (1966) provides reasonably accurate spectra of cytochromes g and 33. By using the information described in the previous chapter on the coordination geometries of cytochromes g and g3, it should be possible to prepare heme 3 model compounds which duplicate the optical properties of the gt_gttt chromophores. The results of our efforts to accomplish this are summarized in Table 5 along with Vanneste's spectral data on cytochromes g and 123 124 Table 5. Absorption Maxima and Formyl Stretching Fre- quencies of Cytochromes g and g3 with Corres- ponding Heme g Model Compounds. Soret V(Ci$) Heme g Species Solvent (nm) a(nm) cm cytochrome g§+ 414a 1676b heme g3+ (DMSO)2C CH2C12 410 1672 cytochrome g§+ 443a 1665d 2+ e heme g (2MeIm) CH2C12 442 1660 H20 434 1640 cytochrome g3+ 425a 595 16509 heme g3+ (NMeIm)2C CH2C12 422 588 1670 H20h 422 590 ---- cytochrome g2+ 444a 604 16109 heme 32+ (NMeIm)2e CH2C12 436e 588 1642 H20 436f 594 16339 From: aVanneste (1966); Ondrias and Babcock (1980); CCalla- han and Babcock (1981); dSalmeen gt gt. (1978) eVan- Steelandt-Frentrup gt gt, (1981); fBabcock gt gt. (1979b); h 9this work (see below); Babcock gt gt. (1979a). 125 g3. Heme g model compounds of the appropriate spin, co- ordination, oxidation state and solvent environment repro- duce well the spectral properties of the high-Spin species, cytochrome g3. Discrepancies arise, however, in the com— parison of cytochrome g and low-spin heme g. The decon- voluted a band and Soret maxima of ferric and ferrous cyto- chrome g are considerably red-shifted relative to oxidized and reduced bis-N-methylimidazole heme g, which by EPR standards, is an appropriate cytochrome g model (Blumberg and Peisach, 1979; Babcock 23,21-r 1979). Moreover, the model compound spectra are only Slightly sensitive to solvent and thus, the unusual red-Shift of cytochrome g, which was originally noted by Lemberg (1962), may involve a fairly complex chromophore/protein interaction. A second anomalous spectral characteristic of cyto- chrome g is apparent when the vibrational properties of its peripheral formyl group are compared with those of cytochrome g3, and their respective model compounds (Bab- cock and Salmeen 1979; Babcock gt gt., 1981). For high- Spin cytochrome g3, the same heme g models which reproduce the optical properties also mimic the formyl stretching fre- quency (Table 5), thus indicating that the position 8 aldehyde is free in a hydrOphobic environment (VanSteelandt- Frentrup et a1., 1981). On the other hand, the charac- teristic formyl vibration stretching frequencies observed for low-spin ferric and ferrous heme g model compounds are not apparent in the resonance Raman spectra of the 126 enzyme (Table 5). Thus the Raman data suggest a protein—induced altera- tion of the heme g formyl group in the cytochrome g bind— ing site which, in turn, may be linked to the absorption red-shift commented upon above. Such protein-chromophore interactions have been shown to be responsible for 12 yttg versus 12 ttttg spectral differences in other protein systems. For example, both the absorption red-shift and vibrational prOperties of retinal in rhodopsin and bacter- iorhodopsin have been accounted for to first order by the presence of a protonated Schiff base linkage between a lysine residue of the protein and the retinal aldehyde (Aton gt gt., 1977; Mathies gt gt., 1977; Marcus gt gt., 1979). Point charges in the vicinity of the chromophore have been advanced to explain further spectral differences (Honig gt gt., 1979; Sheves gt gt,, 1979). Similarly, in photo- synthetic systems, shifts in chlorophyll absorption spectra relative to model compounds have been attributed, in part, to perturbations induced by the protein environment (Davis, gt_gt., 1981). Finally, spectral differences between various formylated hemes when incorporated into apomyo- globin have been attributed to differences in local pro- tein environments (Tsubaki gt gt., 1980). Insight into such a protein-chromophore interaction in cytochrome oxidase can be obtained from data reported by Lemberg (1964) which showed that upon alkalinization 127 of cytochrome oxidase solutions the spectrum of the reduced enzyme shifts to shorter wavelength in two distinct steps. In the first, the absorption maxima move to (Soret, a) (436 nm, 596 nm) followed by a further blue shift to (428 nm, 575 nm). The latter species is clearly established as the Schiff's base adduct of the heme g aldehyde which is stable at pH levels greater than 12 (Lemberg, 1964, Take- mori and King, 1965). The basis of the initial absorption spectral shift to yield Soret and a maxima typical of low- spin ferrous heme g in an aqueous environment was attributed to an unspecified conformational change of the protein. This work was extended in Soret excitation resonance Raman experiments by Salmeen gt gt, (1978) who observed several changes in vibrational frequencies as the pH was raised and noted that the species absorbing at (436 nm, 595 nm), formed at pH 11.5, gives rise to a spectrum that is similar 2+(Im)2 in aqueous to the resonance Raman spectrum of heme g detergent solution. These pH induced effects on the optical properties of cytochrome oxidase have been reinvestigated by using resonance Raman, MCD and EPR spectroscopies as probes to understand the anomalous spectral features of cytochrome g. The magnetic techniques are useful in that changes in heme g spin state may be monitored. Resonance Raman spec- trosc0py provides similar information of coordination geometries through analysis of the structure sensitive vibrations of the porphyrin macrocycle (Callahan and 128 Babcock, 1981). Moreover, Raman detection of the formyl stretching vibration provides additional insight into the chromophore because this mode is sensitive to solvent ef- fects, covalent interactions and hydrogen bonding effects. By using this approach, we have been able to detect and assign the cytochrome g formyl stretching vibration. The combination of red-shifted absorption spectrum and altered formyl vibration is interpreted as arising from a pH de- pendent hydrogen bonding interaction between a protein residue, possibly tyrosine, and the cytochrome g formyl group. II. ng Dependent Spectral Shifts A. Oxidized Cytochrome Oxidase For oxidized cytochrome oxidase, the pH dependence of the resonance Raman spectrum is shown in Figure 23 and of the visible absorption spectrum in Figure 24. At pH 7.4, the high frequency vibrations of cytochrome g3+ occur at 1 1650, 1641, 1590, 1506, 1474 and 1373 cm_ and those of cytochrome 33+ are observed at 1676, 1615, 1572, 1477 and 3 1373 cm’1 (Babcock gt gt., 1981). At pH 11 vibrational band shifts are observed as follows: (a) a decrease in 3+ intensity of the cytochrome g3 1572 cm-1 1 creases in intensity at 1590 and 1641 cm- and (c) a decrease in intensity and frequency Shift in the g3 formyl Figure 23. 129 Resonance Raman spectra of oxidized cyto- chrome oxidase at several pH levels obtained with 413.1 nm excitation. Enzyme concen- tration was approximately 30-50 uM (heme g basis). Instrumental conditions: resolution, 6 cm-l; time constant ls, scan rate 50 cm-l/ min. 130 1934067 GIDHIOQ .50» I OXIDIZED CYTOCHROME OXIDASE 9) pH IIB ”le23 l500 I400 I300 I200 “00 IOOO Aficrfi' I I600 >tmzwe2. 24241 I700 Figure 23 131 .mwemEMm Hem MOM Amemmn m wfiwnv z: melee xemumEonummm mm3 coHumuucwocoo mahucm .mam>ma mm useemxam emuw>mm um mmwpexo mEoenoou>u pmNHUon mo muuommm :oHuQHOQO Haoeumo .em whomem ' 132 em whomem 5E5 £98.96; NO m0 1. 1- o.__ Io IIIII ~o_Io---- VN Iolll: wmo QMN_O_XO m p — b - n u — r a2 0 aouoqmsqv N._ 133 vibration from 1676 cm“1 to 1673 cm-1. The changes that occur in the optical absorption spectrum in this pH range (Figure 24) are (a) decreased absorbance at 655 nm, (b) increased absorbance at 600 nm and (c) a red-shift in the Soret maximum from 420 nm to 425 nm. The apparent mid— point for this shift is m9.5. These changes are consistent with a high to low-spin transition of the cytochrome g3 chromophore as the pH is increased. The core-size marker vibrations of a Six coordinate high-spin heme g model at 1572 and 1615 cm-1 disappear and are replaced by increased intensity in the corresponding modes of a six coordinate, l (Callahan 3+ 3 and the optical low-spin heme g species at 1590 and 1641 cm- and Babcock, 1981). The shift in the cytochrome g 1 to 1673 cm"1 formyl vibration from 1676 cm- absorption spectral shifts are also indicative of increased low-Spin character at alkaline pH (Callahan and Babcock, 1981; see below). EPR spectra of a similar series of enzyme samples (data not shown) taken under low power (2 mW), low temperature (10K) conditions Show a low-Spin heme absorption in the pH range 8.5-10.8 with g values of 2.58, 2.3 and 1.80, indicative of the low-spin hydroxide form of heme g3+ (Wever gt gt., 1977). The spin concentra- tion represented by this Signal increases with increasing pH up to 10 and subsequently decreases as the pH is in- creased further. Even at maximum intensity, however, it represents considerably less than one heme per cytochrome 134 oxidase. Similar behavior in the EPR spectrum was observed at moderately alkaline pH by Hartzell and Beinert (1974). No observable changes in the Raman intensity of the ano- malous cytochrome g3+ vibration at 1650 cm—1 occur in this pH range; only under strongly denaturing conditions is this vibration affected. At very high pH (>12), the Soret is broadened and blue- shifted to 413 nm. The visible region Shows no distinct maxima although there is an increased absorbance at 635 nm. The major vibrational frequencies observed at pH 12 for the oxidized enzyme are 1635, 1583, 1492 and 1373 cm-1. These band positions are similar to those observed 3+ (Callahan and for five coordinate, high-spin heme g Babcock, 1981). Electron paramagnetic resonance Spectra of alkaline pH enzyme samples result in a gradual decrease and disappearance in the low-spin cytochrome g3+ reson— ance at g=3, with an apparent pK in the range 10-10.5, in agreement with the RR observations which Show the ab- sence of any low-spin Species at pH 12. Although the Raman and Optical absorption spectra at pH 12 suggest five co- ordinate high-spin heme g3+ species, no high—spin EPR signal is observed at alkaline pH. This suggests severe protein denaturation in this pH range with release and subsequent aggregate, possibly u—oxo dimer, formation by the free heme g chromophores. Supporting evidence for this configuration of the heme g chromophores lies in the 135 Similarity of the optical absorption prOpertieS of oxidized cytochrome oxidase at pH 12 and the previously reported data for heme g u-oxo dimers (Caughey gt gt., 1975). B. Reduced Cytochrome Oxidase Reduced cytochrome oxidase also shows a series of ab- sorption shifts as the pH of the medium is raised (Figure 25). The Soret and a maxima gradually shift from (Soret, a), (443 nm, 604 nm) at pH 7.4 to (436 nm, 595 nm) at pH 11.5 (Table 6). In the Soret region, cytochromes g and g3 make roughly equal contributions to the absorption at neutral pH (Vanneste, 1966). In the a band region, however, cyto- 2+ is the dominant absorber. Thus, the shift of the chrome_g oxidase a maximum from 604 nm to 596 nm as the pH of the reduced enzyme is raised implies that cytochrome g is being perturbed, as noted originally by Lemberg and Pilger (1964). The pH dependence of the half bandwidth at half- height of the visible absorption band is shown in Figure 27. This titration curve has a pK of approximately 10.5. The half-bandwidths of ferrous low-spin heme g model compounds vary with solvent from approximately 300 cm-1 1 in an aqueous environment in non-polar solvent to 500 cm- (Babcock gt gt., 1979). Similarly, the two extremes of the titration curve correspond to half-bandwidths of ap- proximately 300 to 500 cm.1 for the a band of cytochrome oxidase at neutral and alkaline pH. This observation 136 .2: mum >emmeexoummm mum mcoflumnucwocoo mewncm .mHm>mH mm Hmum>mm pm mmmnflxo mEounooumo twosome mo mnuowmm coaumuomnm HmuHumO ASS 265.963 Ono com com com 09v 00¢ a d .mm mesmem aouoqmsqv 137 Table 6. Absorption Maxima and Individual Chromophore Formyl Vibrational Assignments of Reduced Cyto- chrome Oxidase at Neutral and Alkaline pH. _ 2+ _ 2+ Soret v (GO->133 v (C—O-)l_q pH (nm) at (nm) (cm ) (cm ) 7.4 443 604 1665 1610 9.5 441 601 1633 1610 11.5 436 596 1633 1633 12.5 428 575 ---- _-__ 138 suggests that a solvent environment change occurs at the low-spin heme chromophore, cytochrome g2+ as the pH is increased. Because of the overlapping absorption spectra of the oxidase heme g chromophores and because alkaline modification of cytochrome g2+ was suspected as well (Sal- meen gt gt., 1978), three other spectroscopic probes have been used to identify more conclusively the shifts arising from the individual heme centers. The MCD spectrum of reduced cytochrome oxidase has 2+ 2+ distinct contributions from cytochromes g and g3 , offering a probe of the structural changes which occur at thus each of the individual chromophores at alkaline pH. An intense (As/T = 79.3 (M-cm-T)-1 at 446.7 nm) asymmetric A- and C- term MCD spectrum is observed for the native enzyme (Figure 26), which arises mainly from high-Spin 2+ 3 for low-spin species, cytochrome g2+ and heme g2+(NMeIm)2 1 cytochrome g (Babcock gt gt,, 1978). Spectra obtained are less intense (As/T = 35.0 and 27 (M-cm-T)- at 452 nm, respectively) and more symmetric (Babcock gt gt., 1979). In addition to the MCD intensity differences, the various coordination and spin-states of cytochromes g2+ and g§+ and isolated heme g complexes have characteristic Soret region trough/peak ratios: cytochrome g§+, 0.5; high-Spin heme g2+ in ethylene glycol, 0.5; cytochrome g2+, 0.75; and low-Spin heme g2+(NMeIm)2, 1.0. These features can be used to monitor the properties of cytochromes g and g3 139 Figure 26. MCD Spectra of reduced cytochrome oxidase at pH 7.4 and 11.5. Enzyme concentration of enzyme is 10 uM. A e/H (M - cm-Tesla).l 80 03 CD A (D N O 140 1 7 MCD Reduced cyt. ox. — pH 7.4 --- pH ”.0 450 Mnm) Figure 26 560 141 as the pH is changed. Figure 26 reproduces MCD spectra of reduced cytochrome oxidase at neutral and at alkaline pH. The variation of the Soret MCD trough/peak ratio versus pH is summarized for the reduced enzyme in Figure 27. The initial value of 0.5 increases gradually to a value of 0.7 in the pH range 9.5-10. This MCD ratio of 0.7 and the absorption spectrum of reduced cytochrome oxidase at pH 10 are similar to the Spectral properties obtained for the fully reduced enzyme plus cyanide (Bab- cock gt gt., 1976). The minor absorption band in the reduced enzyme optical spectrum at 565 nm, which is absent in inhibitor complexes that convert cytochrome g3 to a low— spin Species (HCN, CO), is also absent at pH 10 (Figure 25). These two pieces of data suggest that cytochrome g§+ undergoes a high- to low-Spin transition in the pH range 8.5-11.5. The trough/peak ratio further increases to a value of 1.0 at pH 11.5. This pH dependent step is res- ponsible for the shift from a trough/peak ratio characteris- tic of cytochrome g2+ (0.7) to a ratio typical of a low- spin heme g2+ model compound (1.0). This final species also has absorption maxima corresponding to those of isolated low-spin heme g (Soret, a), (436 nm, 596 nm), respectively. Further increases in this MCD parameter at strongly alkaline pH'S (m12) can be attributed to Schiff's base formation at the hemes g aldehydes as determined by the characteristic a band absorption maximum of 575 nm. 142 .m.m H mm How paymesoemo umcp we mafia weHOm one uv>\eouov> mo Oeumu muemcwuce prHHmEhoc mnu ucmmmemmu mmHmCMHHu ammo .prme ABC n.0vv\mmvv xmmm\nmsouu no: mo musflom dump ucwmwummn mmaouflo Umaaem .m.oe n so how oouoeooeco acre he ocee oeeom one .ucoeoc met: no ocuoez Imam: pawn o thHHmEHoc mum mmaoueu ammo .mmmpexo meonzooumu twosome mo mumumEmemm emuuoomm Hmem>mm MOM mm Mo :oHuUSSM a mo mm>eso coeuwnuefi .hm mesmem 143 em whom: Io m. __ o. m m e 4 _ e _ . _ e _ e H» . 4 00 T- 4 q 0 O O \C" \\ n “0 «.01 o o “knee .. moo \ .\ \ \ Il' V.0 T 1 o .\ ‘\ \ a J \\ QC j. 0 . \ 4 4 m.o 0 \ All QC e... o . \ A d 4 I: I. II ‘\ O._ T: O v Q < ”N O.— . u . .689 .e .o. m xo +7588. 6&5 o no. "xo. 6 o Home? are 3.8868: 8.2 6 out. _ _ mmqoxo. mzomropxo awesome 144 Attempts to calculate a titration curve of the MCD trough/ peak ratio from contributions of the individual chromo- phores were unsuccessful in mimicking the observed results. This may be a reflection of the heterogeneity of sites (Brudvig gt gt., 1981) or of the time dependence of the alkaline pH effects (Lemberg and Pilger, 1964). Visible excitation resonance Raman spectroscopy pro- vides a second probe and a more exact separation of the 2+ and g§+ to the selective enhancement of the vibrations of one cytochrome g pH dependent spectral shifts owing chromophore over the other. Figure 28 Shows the reson- ance Raman spectra of reduced cytochrome oxidase and its partially reduced inhibitor complexes (Figure 28a, b and c) obtained with a band excitation at 605 nm. The Raman Spectra are similar regardless as to whether cytochrome g3 is ferrous, five coordinate and high-spin (Figure 28a) or ferric, six coordinate and low-spin (Figure 28b) or high-spin (Figure 28c) (Bocian gt gt., 1979). Because of the well documented dependence of resonance Raman band position and intensity upon heme coordination geometry and extinction coefficient in the Herzberg-Teller scatter- ing region (Spaulding gt gt., 1975; Spiro, gt gt., 1979) we would expect these alterations in cytochrome g3 spin and valence states to be reflected by shifts in the Raman Spectrum if it were a strong absorber in the a band region. However, the visible excitation resonance Raman spectra of Figure 28 are essentially identical, independent of Figure 28. 145 Visible excitation resonance Raman spectra of reduced cytochrome oxidase a), and partially reduced inhibitor complexes b) and c). The Spectrum in c) was obtained with a flowing sample arrangement. Enzyme concentration was approximately 200 uM. The sample conditions in d) are W200 uM heme g, 0.5 M 2-methylimidazole, in 0.07 M CTAB, 0.1 M sodium phosphate, 0.001 M EDTA, pH 7.4, with sodium dithionite as re- ductant. Instrumental conditions: resolution 5 cm-l; a)-c) time constant ls, scan rate 50 cm-l/min; conditions in d) time constant 2.5 S, scan rate 20 cm-l/min. Roman Intensity 146 I j I r j 1 Cytochrome Oxidase -VisibIe Excitation kex=605 nm a)reduced,pH 7.4 - o - . ‘1 § $ ’5‘. a ' §§ If, I gga ' 0 - 8 2 __ '. 8 - 2 ' f’ a 3 ' a e d 8 . ' 2- . . .§ 3 ' I“ DID. reduced + KCN c)p.reduced +HC00+I -£'.I EH -3}; a w - . ' ' § ? 7e .. I $3" g d)heme 92* (2MeIm) #1 l l J I 1 I700 I600 I500 I400 I300 I200 II00 I000 A7 (cm") Figure 28 147 oxidation, coordination or spin-state of cytochrome g3, and therefore we attribute the vibrations observed to cytochrome g and make the corollary conclusion that it is the dominant absorber in this region. Furthermore, the 2+ prominent vibrations of heme g (2MeIm), (Figure 28d) at 1533, 1555 and 1605 cm-1, a model for the coordination and Spin-state of cytochrome g§+ (VanSteelandt gt gt., 1981), are not observed in the RR spectra of reduced cyto- chrome oxidase; thereby providing additional evidence that vibrations of cytochrome g2+ alone are observed under these conditions. Visible excitation Raman spectra, then, can be used to monitor the pH dependence of a single heme chromophore, cytochrome g2+. Resonance Raman spectra ob- tained with visible excitation of reduced cytochrome oxi- dase at several pH levels are shown in Figure 29. The changes observed are (a) a reduction in intensity of the l and 1329 cm-1 bands and (b) a decrease in in- 1 1569 cm" tensity and shift in frequency of the 1114 cm- vibration to 1109 cm-1. These pH effects titrate over the pH 10- 11.5 range. Since the vibrations observed with visible excitation of the reduced enzyme at neutral pH arise solely from cytochrome g2+ and noting the fact that no new vibrations are observed with visible excitation at alkaline pH, this suggests that thespectral shifts occurring with a pK W 10.5 arise from a pH dependent modification of cytochrome g2+. Figure 29. 148 Visible excitation resonance Raman spectra of reduced cytochrome oxidase at several pH levels, with excitation wavelength as noted in the figure. Enzyme concentration was 200- 300 uM (heme g basis). Instrumental condi- tions: resolution 5 cm-l, time constant 2.5 s, scan rate 20 cm_l/min. Roman Intensity 149 I 1 I j TI T Reduced Cytochrome Oxidase-Visible Excitation _ a)pH 24 0| _ .- 0 2 o ' T _ OI . V (Tl _ a: 'a ' u-9 w? 8’ g fi§8 E. ' § ' 2" s 3 ' ' 'I’ I - a V 8 °.. ' '7‘ . 3 o a I ' 3: g I I ' I I I I I I b) pH 9.0 : A“ 'SOZNTI' : I I E5 8 ' .5. ,. t t -2. ' 5' 3 ‘6‘ ' 8 I» 7‘ - ' I I § ” H = ' I ' g 7‘ 8,: I l I 5 c) pH IO.5 ' 1.;598nm L J J j I 1 I700 I600 I500 I400 l300 I200 IIOO IOOO Az'z'Icm") Figure 29 150 To document the pH dependencies of the two heme cen- ters further, Soret excitation resonance Raman spectros- copy has been employed as a third technique. The selec- tive enhancement of the vibrations of a single chromo- phore by proper choice of excitation frequency for the partially reduced inhibitor complexes of cytochrome oxidase is not feasible at alkaline pH because of the unfavorable pH values of HCN and HCOOH. For this reason, we are limited to the study of the fully reduced enzyme. Although complicated by the fact that vibrations of both chromo- phores are enhanced, Soret excitation resonance Raman spectra can yield significant structural information. The Soret resonance Raman Spectra of reduced cytochrome oxidase at several alkaline pH levels are shown in Figure 30. At pH 7.4, with excitation at 406.7 nm, the charac- 2+ at 1622, 1610, 1586, 2+ 3 are observed (Table 7). The teristic vibrations of cytochrome g 1 1569, 1520 and 1358 cm_ and of cytochrome g at 1665, 1610, 1569 and 1358 cm'1 changes that occur as the pH is raised to 9.5 are (a) decreases in intensity at 1665, 1610 and 1115 cm-1, (b) an 1 increase in intensity at 1633 cm- and (c) a Shift in 1 intensities in the 1220-1250 cm- region. Since the formyl stretching vibration of cytochrome g§+ at 1665 cm.1 is well separated from the other ring vibrations, its intensity ratioed to the intensity of v4 at 1358 cm-1 establishes a titration curve for the pH dependent changes *4. “I (J (D ’m—J Ut H m O H (D {‘t‘ (D ’0 xc tation resonance Raman spectra of reduced cytochrome oxidase at neutral and alka- lin pH (a-e). En2"me concentration was ap— proximatel" 40 23. Sample conditions in f) are 550 LE heme a, 0.7 g N-methyl imidazole, 0.07 2'! TAB, 0.001 )4 EDTA, 0.1 M sodium phos- pnate, pH 7.4. Instrumental conditions: resolu- . - 1 . tion 6 cm ; a)-e) time constant 2.5 s, scan rate 20 cm-l/min; f) time constant 15, scan rate 50 cm-l/min. 152 Roman Intensity Reduced Cytochrome OxIdase Aex‘ 406.7nm d)pl-I I07 a) pH ".6 ”home or (NMeIm )2 (no) I700 I600 I500 I400 IEIOO I2100 A17 (cm") IIOO IOOO Figure 30 153 Table 7. Reduced Cytochrome Oxidase Soret Excitation Heme Chromophore Vibrational Assignments.a ------ cytochrome g2+------ ------cytochrome g§+-—---- A? (cm-l) Assignment (#) AU (cm-l) Assignment (#) 1665 v(C=O) 1622 Blg' 010 1610 Blg' v10 1610 H-bonded v(C=O) 1586 A , 0 lg 2 1575 Alg’ 02 1569 Blg' v11 or 1565 319' v11 or b b Eu' v38 Eu’ V37 1520 Alg’ 03 1473 Alg' v3 1358 Alg’ v4 1358 Alg' v4 aSymmetries and mode numbers from Abe gt gt., 1978 for further documentation of these assignments, see Salmeen gt gt., 1978. bSee Choi gt gt., 1982. 154 of cytochrome g§+ (Figure 27). This band decreases in intensity with an apparent pK = 9.3 corresponding to the pH range of the initial changes observed by MCD. After complete disappearance of the native cytochrome g2+ 3 vibration (1665 cm-1 band) at pH 10, further band changes in formyl the high frequency region are observed. These consist of an additional decrease in intensity in the 1610 cm-1 band with a concomitant increase at 1633 cm-1; the shoulder at 1570 cm”1 is no longer strongly observed at pH 11.5. The vibrational spectrum of reduced cytochrome oxidase at pH 11.5 is essentially identical with that of a low- spin ferrous heme g model compound in an aqueous environ- ment (Figure 30f) and the band at 1633 cm"1 is characteris- tic of the formyl vibration of heme g under these condi- tions. This second set of vibrational shifts occur in a pH range comparable to the range of cytochrome g2+ pH dependent shifts as determined by visible excitation Raman data. As the pH is increased above 11.5, vibrations characteristic of the Schiff's base species are detected (Salmeen, gt gt., 1978). III. pg Dependent Structural Changes A. Reduced Cytochrome Oxidase The original optical/Raman work on cytochrome oxidase at high pH (Salmeen et a1., 1978) was somewhat paradoxical 155 in that the major apparent alteration as detected by Raman spectroscopy appeared to occur at cytochrome g3 while the primary optical Shift appeared to involve cytochrome g (Lemberg and Pilger, 1964). The present, more detailed study resolves this paradox and shows that for reduced cytochrome oxidase both heme g chromOphores are gradually modified in structure by alkaline pH. Moreover, these alterations Show different pH dependencies which allow us to determine the structural changes responsible for the spectral shifts. The MCD, visible and Soret excitation Raman data identify three pH dependent steps: a change in cytochrome g3 which occurs with a pK $9.3, an alteration of cytochrome g with a pK m10.5, and at pH >11.5, formation of the Schiff's base adducts of both chromOphores. For the pH range 8.5-10, the optical absorption, MCD and Soret excitation resonance Raman data suggest a high- to low-spin transition and a solvent environment change at the g3 Site. It has been reported that redox titrations of cytochrome oxidase, monitored by MCD, display unusual behavior above pH 9 (Carithers and Palmer, 1981). This shift in thermodynamic behavior may be a reflection of the structural changes induced in cytochrome g3 by mildly alkaline conditions. Cytochrome g is affected by somewhat more alkaline conditions. With pK m10.5, its spectral characteristics Shift to those of an isolated low-Spin heme g2+ model 156 compound in an aqueous environment. Soret excitation resonance Raman spectra offer the most insight into the structural changes which occur during this process (Figure 30). One of the major band shifts that occurs in this pH range is the decrease in intensity of the anomalous cyto- l . . and concomitant increase 1 chrome g2+ vibration at 1610 cm- at 1633 cm'l. From the fact that the 1633 cm“ 2+ band arises from a low-spin heme g formyl vibration exposed to an aqueous environment (Figure 30f) and from the mirrored shifts in the 1610 and 1633 cm.1 vibrations, we assign the 1610 cm.-1 band observed in the native protein to a per- turbed formyl vibration of the cytochrome g chromophore. The largest optical absorption changes, from (441 nm, 601 nm) to (436 nm, 595 nm), also occur in this pH range, which indicate that the optical properties of cytochrome g2+ and the physical state of its formyl group are linked. In view of the Significant perturbation of heme optical properties induced by peripheral aldehydes (Gouterman, 1959), such a linkage is not surprising. The argument above indicates that the absorption red- shift of cytochrome g tg’ytyg at neutral pH relative to its model compounds and the structural alteration of its formyl group are related. This observation, coupled with earlier results which showed that low-Spin heme g models accurately produce other vibrations of cytochrome g, particularly its core Size marker bands (Callahan and Babcock, 1981), 157 provides criteria with which to judge possible models for the structure of the cytochrome g Site. For example, the presence of nearby polarizing amino acids capable of forming n complexes with the heme g system may be ex- pected to alter the spectral properties of cytochrome II-l.I by analogy with the absorption red-Shift observed by Mauzerall (1965) for n complexes between aromatic rings and uroporphyrin. However, Shelnutt (1981) has Shown that formation of a 6 complex is accompanied by vibrational frequency changes of several wavenumbers in the high fre- quency core-Size marker bands. Such frequency Shifts are not apparent in the cytochrome g Raman spectrum. In ad- dition, metalloporphyrin g complexes with n acceptors Show only small spectral shifts which are unable to account for the differences between isolated heme g and tg ytyg cyto- chrome g Spectral prOperties. A second possible model in- volves the occurrence of strained or hindered axial ligands to the cytochrome g iron which may perturb the optical properties of the chromophore. Carter gt gt. (1981) have shown that hindered axial ligands shift heme EPR ligand field parameters in a characteristic manner and used their observation to rationalize the liganding in mitochondrial t cytochromes. Such an explanation is unlikely for cyto- chrome g owing to our previous EPR results on the chromo- phore and its models (Babcock gt gt., 1979a). Moreover, Raman core size marker bands are perturbed by sterically 158 hindered axial ligands (Frentrup, J., unpublished); such perturbations are not observed in the Raman spectrum of the tg,ytyg chromophore. A third possible means to account for the red-Shifted cytochrome g absorption Spectrum is suggested by studies on rhodopsin and bacteriorhodopsin (Honig, gt gt., 1979; Sheves gt gt., 1979) and involves the specific arrangement of point charges in the heme g bind- ing site of cytochrome g. The extension of the external point charge model to tetrapyrrole-based systems has resulted in reports of a range of absorption spectral shifts (Davis gt gt., 1981; Eccles and Honig, 1982), but such a model would not be able to account for the altered formyl vibrational frequencies in cytochrome g without additional 39.222 assumptions. Point charge effects, however, may be important for the Spectroscopy of cyto- chrome g3 in isolated cytochrome oxidase and could account for the effects of Ca2+ on the absorption properties of various heme g Species reported by Wikstrdm and coworkers (Saari gt gt}, 1980). It appears, therefore, that a perturbation to the cyto- chrome g ring system which only indirectly influences the formyl group will not account for the combined optical, Raman and EPR data; rather, a Specific interaction at the carbonyl seems necessary in order to rationzalize the spectroscopic results. Structural modifications of the cytochrome g fonmyl group which may explain the observed 159 phenomena include the following: (a) protonated Schiff's base formation at the peripheral formyl group, (b) non- planarity of the position 8 aldehyde with the porphyrin n-system, and (c) hydrogen bonding to the peripheral al- dehyde. The previously suggested structure of a protonated Schiff base between the cytochrome g3+ aldehyde and an 6- amino group of a lysine residue of the protein (Ondrias and Babcock, 1980) is not supported by model compound studies (Ward gt gt., 1983) and can therefore be eliminated as a likely explanation. Non-planarity of the peripheral aldehyde and porphyrin ring n-systems also seems unlikely for two reasons. First, the fact that we observe a high frequency vibration from a perturbed formyl group indicates that the carbonyl and porphyrin n-systems must have some degree of overlap for resonance enhancement to occur. Sec- ondly, it is difficult to rationalize a red-shifted heme absorption Spectrum in both the Soret and a-band regions when the perturbation invoked decreases the extent of conjuga- tion of the porphyrin n-system. In order to effect both a red-shifted absorption spec- trum and a decreased formyl stretching frequency, a greater electron-withdrawing capability at the peripheral formyl group is needed. A hydrogen bonding interaction in which the formyl C=O acts as the proton acceptor provides a reasonable structure within which such effects could occur. The de- crease in carbonyl stretching frequency upon hydrogen 160 bond formation is well known and, because the visible and Soret absorption bands of heme g are n+n* transitions, an absorption red-shift is predicted to result from hydro— gen bonding (Pimentel and McClellan, 1960). Moreover, because the hydrogen bond is a specific perturbation to the formyl group, the major porphyrin ring vibrations would not be modified to any great extent, in agreement with earlier observations. Therefore, we conclude that a hydrogen bond between an amino acid residue and the peripheral alde— hyde can explain the spectra of cytochrome g and the ob- served pH dependent behavior. We suggest that tyrosine may be the proton-donating group involved in the hydrogen bond interaction with the cytochrome g peripheral aldehyde based on the ability of phenol as hydrogen donor to low— Spin heme g model compounds to mimic the cytochrome g3+ absorption spectrum. A schematic of the proposed structure of the heme g binding site in cytochrome g at neutral pH is given in Figure 31. The pH dependence of the hydrogen bonded form of cytochrome g presented above could arise from either a titration of the proton donor group or a disruption of the hydrogen-bonded structure by an alkaline pH-induced conformational change of the protein. Since we can monitor only the hydrogen acceptor (C=O) stretching frequency by RRS and not the donor (R-H) stretching frequency we are unable to distinguish experimentally between these two 161 .mem>me mm Hmum>mm um mononmoeouno m mew: Uwospmu Ugo powepexo wnu mo mmmcmno Housuosuum pmmomoum mo oeumfimsom .em occeee 162 / Gnu-'- em ounces . omI we Io +nm 66952.6 mmao>CH we odoum Haeuom Hmumcoflumm one .mmuem aceummee Hmexm 039 on» mcflhmdooo mmcflpfiumfls cuez mumcepuooo xem me some m mew: one .mmmpexo msounoouwu :e m mfiouzooumo How musuosuum muflm m>Huum omumasumom .mm ounces Figure 34. 172 High frequency Soret excitation Raman spectra of cytochrome oxidase and low-spin heme g model compounds. In (a) and (c), the beef heart en- zyme, dissolved in 0.05 M Hepes, 0.5% lauryl maltoside, pH 7.4, was used. In (d) cyto- chrome oxidase (glgg3) from Thermus thermgphilus in the Hepes/maltoside buffer was used. In (b) and (e) the bis-(N-methyl imidazole) heme g complex was dissolved in methylene chloride. The carbonyl stretching frequency for cyto- chrome g3 is indicated by t in (a), (c) and (d). RAMAN INTENSITY 173 I 1 A .l )\ = 406.7nm ex a)0xidized toohrome oxidase B.H.) b)Herne 93+ (NMeIm)2' CI” CHZCIZ c)Reduced cytochrome oxidase(B.H.) d)Reduced CIOO3 (H88) (X“=44I.6 hm) e)I-Ieme 92* (NMelm)2 CHéflz l 1 I600 Aficn‘i') Figure 34 1 l500 174 indicates that the carbonyl group of g3 is isolated in a hydrophobic environment in both of its valence states (VanSteelandt-Frentrup gt gt., 1981; Babcock gt gt., 1981). The formyl vibrational frequencies for cyt g (Callahan and Babcock, 1983) and its model compounds are indicated by vertical lines in the figure. For both valence states, the tg gttg carbonyl shows a significant frequency de- crease compared to its corresponding model compound. This decrease amounts to 20 cm-1 cm.1 for the reduced center. The Thermus protein shows an for oxidized cyt g and to 35 analogous decrease which, along with similar data for rat liver cytochrome oxidase (Babcock gt gt., 1981) indicates that the lower tg‘gttg frequency is not a peculiarity of the beef enzyme. Shifts in carbonyl vibrational frequencies of this magni- tude are commonly observed upon hydrogen bond formation (Murthy and Rao, 1968). Moreover, because the visible and Soret optical absorption bands of heme g are n-n* transitions which involve the formyl as part of the system, an absorption red shift Should accompany hydrogen bond formation. Both the magnitude of the formyl vibra- tional frequency decrease and of the absorption red— shift Should increase as the strength of the hydrogen bond increases (Pimentel and McClellan, 1960). The pro- portionality between carbonyl frequency decrease and ab- sorption red-shift appears to hold for cyt g in its two 175 valence states. For the ferric form, the a band absorption maximum is shifted by 10 nm (284 cm-1) to 598 nm relative to its nonhydrogen bonded model and the carbonyl frequency 1 (Table 8). For the ferrous form, the decrease is 20 cm- absorption red-shift relative to the nonhydrogen bonded model is greater, 17 nm (480 cm-1) and likewise the vibra- tional frequency is decreased by 35 cm-l. To explore the coupling between the carbonyl vibrational frequency, the optical absorption red-Shift and the hydrogen bond strength in cyt g in more detail, optical and Raman spectra for several low-spin heme g and c0pper porphyrin g model compounds have been recorded in different solvents and in the presence of various hydrogen donors. Copper porphyrin g was used to obtain spectrosc0pic parameters for the chromophore in the presence of more acidic donors. These cannot be used with heme g(NMeIm)2 because they protonated Ne of the iron ligand, which results in the formation of the high-Spin heme g complex. Deuterated rather than protonated phenol was used as a donor in order to avoid overlap of its vibrations with those of the por- phyrin macrocycle in the high frequency region. Table 8 summarizes the results in terms of a band maximum, carbonyl frequency and hydrogen bond strength (see below) for the models and for cyt g. In Figure 35, the absorption red- shift is plotted as a function of both the carbonyl fre- quency decrease and the calculated hydrogen bond strength. 176 III II oooe III com I Neommo SH 6 Seuwnonom +mso ea c o m m m.m mm mmce mem mom mo eocot +Ieo no he AEHozzc+mm came a N m.e me mmoe eom eom m I o m ce esemzzv+mm mews m III II meme III emm m Weommo ce naeozzv o mace e +~ m.m mm oeoe owe eoo +mm oeohcooueo o o.o v come om mmm m eommcuas ce esemzzv 6 mean m +m e.~ oe emoe com mom mocmtuooHowtoehp ce Aseozzc+mm mew: e e.m we Nmoe oom mom co eocmro+meommo N I Ce AEszzv+mm wEwm m III II oeoe III mmm m WHUNmO CH AEHQZZV m GETS N +m o.m om omoe emm mom +mm oeoenooueo e mace . Aemuxv AHIEUV AHIEUV HIEUV AEGV mmeommm oz mmm< OHUD< OHCI JD< xmmx Am An A0 An em .mmflommm m deuznmuom +m50 can m mEmm popcom cmmoepmm mo woeumeewuomemco oemoomouuommm .m magma 177 new UnsooEoo nouooeone onu new moeocosooum onenuuouum Hmnoneoo CH oonoHoMMHp wonosooum poo onooosoo UoUMOHUCH one How oEonE COeuQHOmno Hooeumo UnonIo an oonouommep monosooum .AH.mv nOeuosom Eoum commasoaoo mo numnouum neon comouwmno .moeuomm poonon comoupmntcon mnflnnommounoo onu U .mocosvoum mcwnououum ewnonuoo H>Eeomo .moHuomm cocoon comouownIco: mcHUCOQmoHHoo onn Q .EUEonE GOHUQHOmno HMOHUQO pcontoo oo eocoto+~eo~mo o.m em Nome new mom I :e o neuwnmuom +NDO we o.m em meme eom eoo eococmonoeroIo+Neo~mo CH o neu>nmuom +Nso me o.m we meme new mom HocmnuoouoHnOfluu Ce o neumnmuom so me . +m m.m mm ovwe mew mew neon oenoooouosawfluu+maommo an m ceuwnmuom so He . +~ oHOE A Evy A Eon Eov easy moHoomm .oz neooxv at at HI mmm< OHCD< OHCW ow< xowe no no no .poscHucoo .m oenoe 178 Figure 35. Absorption red-shift for cytochrome g, heme g or Cu porphyrin g Species as a function of hydrogen bond enthalpy as calculated from Equation 6.1. The points are numbered ac- cording to the compounds listed in Table l. 0 IOOO 2000 I T T I I- 2+ - Cyt Q “\5/ II / / / 4004 1 / / 9/ _ .. Cyt 93+ I3 .. [£5122 ‘7;\‘V/' I4 / / 200- 8 /4 3 - / / / / // 5 2 J 1L 1 J 0 2 4 6 -AHHB (kcals/mole) Figure 35 180 The approximately linear relationship apparent in the figure is typically what one observes in correlating hydrogen bonding phenomena in a series of loosely related compounds (Arnett, gt gt., 1974). B. Quantification of Hydrogen Bond Energies From the formyl frequency shift observed upon hydrogen bond formation, an estimate of the strength of the hydrogen bond for the heme g species under consideration can be made. This relies upon a variation of the Badger Bauer rule, which has been widely applied to vibrational data obtained for the hydrogen donor involved in the bond (gggt, Pimentel and McClellan, 1960). The corresponding relation- ship for the acceptor is (Zadorozhnyi and Ischencko, 1965) _ = ’Kc=oAHHB (6 '1) where AHHB is the hydrogen bond enthalpy, K portionality constant, C=O is a pro- tho is the vibrational frequency of the free (nonhydrogen bonded) acceptor. By using a series of aldehydes, ketones and carboxylic acids as acceptors and phenols or alcohols as donors, a value of Kc=O = 4 10-3 mole/kcal has been estimated (Zadorozhnyi and Ischenko, 1965). Realizing that this value is likely to hold only roughly for heme g species, particularly for the diverse 181 class of donors in Table 8 (Arnett gt gt., 1974), it has been used to estimate AHHB for the various complexes. These are given in Table 8 and are used in Figure 35 to obtain the correlation between hydrogen bond strength and absorption red-shift. When the hydrogen bond enthalpy is expressed in wavenumbers the slope of the least squares line drawn in the figure is 0.28. This value is within the range one expects for the proportionality between hydrogen bond strength and the n—n* transition red-Shift which results (Pimentel and McClellan, 1960). From the data in Table 8 and Figure 35 it is apparent that both ferric and ferrous cyt g have optical, vibrational and hydrogen bond enthalpy characteristics which are con- sistent with the model compounds. An important conclusion follows from this observation because it indicates that the major protein-induced modification of the chromOphore oc- curs by hydrogen bonding to the formyl group. Other per- turbations, for example, shifts in the electrostatic po- tential at the heme (Warshel and Weiss, 1981) or point charge effects (Honig gt gt., 1979) may influence the optical Spectrum as well but these effects appear to be small compared to the hydrogen bonding interaction. More- over, the hydrogen-bonding effects are more pronounced for cyt g2+ than for cyt g3+. Because the hydrogen bond strengthens upon reduction, the redox potential of cyt g is more positive than it would be in the absence of such an interaction. Modulation of the hydrogen bond strength 182 tg’gttg for either oxidized or reduced cyt g may provide an explanation for the unusual redox properties of the heme g components of cytochrome oxidase (gtgt, Babcock gt gt., 1978). The chemical basis for the difference in hydrogen bond enthalpy for the two cyt g valence states is straightforward. Sheridan, Allen and their coworkers have investigated re- dox dependencies in hydrogen bond strength for both heme and FeS proteins (Valentine gt gt., 1979; Sheridan gt gt., 1981). In heme proteins, where they explored the iron ligand, histidine, as a proton donor, the ferric state is stabilized by hydrogen bond formation. In FeS proteins, where the metal ligand is a hydrogen acceptor, they noted stronger hydrogen bonds for the reduced cluster. The por- phyrin g ligand to iron in cyt g resembles the FeS protein case in that it is a proton acceptor and we eXpect stronger hydrogen bonding in the ferrous state, ttgt, upon reduction of the iron the electron density on the carbonyl oxygen increases and it becomes a better proton acceptor. Cyt g behaves this way tg_gttg and it is also observed when heme g model compounds are considered. For example, the hydrogen bond formed between phenol and low-Spin heme g is stronger by No.8 kcal/mole when the iron is in the ferrous, as opposed to the ferric, valence state (Table 8). The increased negative charge at the carbonyl oxygen in ferrous heme g is also apparent in that g 25 cm-1 decrease in 183 carbonyl stretching frequency, presumably due to popula- tion of the n* antibonding orbital, is observed for fer- rous heme g models compared to ferric heme g in the absence of hydrogen bonding effects (Figure 34). Given the approximate nature of Equation (6.1), the cal— culated hydrogen bond strength change which occurs on redox cycling of cyt g is in the neighborhood of 2-2.5 kcal/mole (90-110 mV). This energy is comparable to the electrochemical proton gradient against which protons are translocated in mitochondria (estimated to lie between 160 and 230 mV with a best value of around 200 mV (Wik- strém gt gt., 1981), particularly if the hydrogen bond strength change is augmented by other redox-linked pro- cesses, gtgt, a Shift in electrostatic potential at the heme (Warshel and Weiss, 1981) or additional hydrogen bond contributions from the cyt g axial histidines (Valentine gt gt., 1979; Babcock gt gt,, 1979). Alternatively, one could envision a situation in which both cyt g as discussed below, and Cua, as suggested by Chan gt gt (1979), operated as proton pumps in which the duty cycle for each would be one/half. Before discussing possible pump models for cyt g however, it is necessary to consider the relationship between the hydrogen bond enthalpy changes observed and the free energy actually made available for proton transloca- tion. In the early literature on hydrogen bond formation, increases in hydrogen bond strength (decreases in AH?) 184 for a series of hydrogen bonded structures appeared to be 0 F change remained somewhat constant (Pimentel and McClellan, accompanied by decreases in AS so that the free energy 1960). More recently, and arguing from a much larger data set, Arnett and coworkers (1974) concluded that such a generalization is not justified in that a hydrogen bond reaction series may be essentially isoentropic yet Show large changes in AHg. In these cases, the change in AH? will be reflected in a change in AGg. Such a situation is likely to occur in cyt g owing to the fact that the major contribution to the decrease in AS? for a reaction series where it is observed is usually attributed to loss of trans- lational entropy upon complexation. Because the proton donor to the cyt g formyl is most likely immobilized by its polypeptide environment, the translational entrOpy will be small regardless of the hydrogen bond strength. Thus we expect that changes in cyt g hydrogen bond enthalpy will be accompanied by a free energy change of a comparable mag- nitude and that this will be available for proton pumping. III. H+ Pump Models From a variety of models in which the redox driven change in cyt g hydrogen bond strength could be used to translocate protons, two will be considered here. In the first, the free energy change at the formyl serves as a switch which produces a conformational change in a different 185 region of the protein which allows proton conduction. In the second, the proton hydrogen-bonded to the carbonyl is an in- tegral component of a proton wire and is actually pumped during the redox cycle. Relevant to both of these models is the relationship between hydrogen bond strength and hydrogen bond geometry discussed by Valentine gt gt. (1979). The geometry is determined by two factors: the distance, t, between the heavy atoms involved in the hydrogen bond and the angular deviation of the bond, 6 from linearity. With these definitions, the hydrogen bond strength is propor- tional to cose/r. A. Conformational Change Model In the first model, the reduction of cyt g leads to a decrease in either 5 or 8 (or both). The change in local structure about the heme g chromophore is transmitted to the proton translocating section of the protein where a conformational change resulting in proton translocation occurs. Supporting this model is the observation that the major conformational change which occurs upon reduction of cytochrome oxidase is controlled by the redox state of cyt g (Cabral and Love, 1972). A second piece of poten- tially supporting evidence comes from the observation that dissociation of cytochrome oxidase subunit III abolishes the pH dependence in the midpoint potential of cyt g in the cyanide inhibited enzyme (Penttilé and Wikstrém, 1981). 186 The possible mechanics of proton conduction as related to a conformational change have been considered in detail by Nagle and Morowitz (1978). The mechanism by which the hydrogen bond geometry change at the heme g site is trans- mitted through the protein to the pumping section may bear some resemblance to that proposed by Perutz and Brunori (1982) for the control of the Root effect in fish hemo- globins. B. Mydrogen Bond Chain Model The second model represents a direct mechanism for proton translocation in that the formyl hydrogen bonded proton is pumped during the redox cycle. This kind of mechanism requires that covalent bonds involving hydrogen and the polypeptide heavy atom in the formyl hydrogen bond are made and broken during proton translocation. Because resonance Raman can provide vibrational information on only the chromophore moiety of a protein, we have no direct insight as to the identity of the proton donor to the cyt g carbonyl. However, we suspect that it is an -OH group from either an alcohol or a carboxylic acid side chain. An -NH2 group is much less likely owing to the facility with which amines form Schiff base linkages with heme g_(Ward et a1., 1983) and -SH groups can be eliminated owing to the extremely weak hydrogen bonds they form (Perutz and Brunori, 1982). 187 With these considerations, the mechanism in Figure 36, which is similar in certain aspects to the switch model for bacteriorhodopsin proposed by Nagle and Mille (1981), can be postulated. In 36a, the stable form of ferric cyt g is shown where a fairly weak (3 kcal/mole) hydrogen bond exists between the peripheral C=O and the -OH donor. As indicated in the figure, the weak hydrogen bond assumes a nonlinear geometry (8 ¢ 00). Two proton-donating groups, Rr-Hd and Rl-Hb, are located in the immediate vicinity of the cyt g carbonyl. Rr-Hd is connected to the right-hand side of the membrane by an asymmetric hydrogen-bonded chain and Rl-Hb is Similarly connected to the left Side of the membrane (Nagle and Morowitz, 1978; Nagle and Mille, 1981). On the right hand side Rr-Hd is shown in the oxidized, resting form of the enzyme (Figure 36a) with Hd hydrogen- bonded to the oxygen of the donor group to cytochrome g. This interaction determines the configurational energy of the hydrogen bond chains, and in Figure 36a, the interac- tion, R-O----H holds the right hand side proton chain d-Rr' in its high energy conformation. The left hand side proton chain is in its low energy form. The concept of hydrogen bonded chains has been developed by Nagle and coworkers (Nagle and Morowitz, 1978; Nagle and Mille, 1981). Such a chain could conceivably span protein subunits so the involvement of Subunit III in cytochrome oxidase proton translocation may be accommodated by this model as well. 188 .mmoem m50eeo> one oceeso mn0eeoa neone >eeenooe 0e nlo meoeeoa neHB noemeeum team oeo coeeoo mnemEdm one Ce po>eo>ne odomoenwn onB .GOHeoHDmemdoo m e>o oouepexo .oenoem one oeoeonomoe 0e .oeoHUoEeoene no mo Any neHB .mmoo Ioem moem o3e m we mesooo n0eeopex0om .meo>eeuommoe .N man a mmoem oceedo esooo noenz oeseoseem Ge mooconu one oeooepne Any new no. Se m3oeem one .oeem one e0 ance voodooe oanoem one .on Ce weesmoe noen3 mmoooum moem 03e o no csonm we sceeosoom .emInm... mo poeonmemop we now H>Euom one e0 ewoe one 0e mesooo sceeoedoemcou mouono eoBoH wee 2e neono poocon nomoeo>n o u....emIUm mo poeonmemop we can moonm H>Ee0e one e0 enmee one 0e musuuo n0eeoesmeenoo Sonono nonmen wee ce neono Cocoon comoepmn 4 .erMOHUne we oeem m emu one e0 Ee0e noneoexo oenoem one .on CH .eroueUCe we oocoeo> some one “O H Ox. om an noeooepce we aeoe05.m oEon m oEOHnooemo one .omop Iexo oEoen00e>c ne dado noeoem no>euptxoooe one How Emenonooe oenemmom m .om oeooem 189 Eton 68:68 639.... no +Nmu om oscoem m+ no ©+ Eton 68.96.2858 thou o oEoEooSO toe EmEoncmE ocean. cocoon. a. 190 Proton pumping proceeds from left to right. Reduction of the iron results in a two step change in the hydrogen bond geometry and produces the stable reduced form of cyt g shown in Figure 36c. In the first step, process 1, re- duction of the iron leads to greater electron density at the carbonyl oxygen. As a result, the C=O----HC-O hydrogen bond strengthens as it shifts to a more linear configuration The O----Hd-Rr hydrogen bond breaks and the right side of the chain relaxes to its lower energy configuration. Proton release on the right side may occur at this step, but it is not necessary. In the second step, the -OHC oxygen, which has become a stronger proton acceptor as a result of the strengthening of the OHC----O=C hydrogen bond (Huyskens, 1977), stabilizes the higher energy configuration in the left side chain as the Rl-Hb----O hydrogen bond forms Situa- tions similar to this, 142;! ones in which an amino acid Side chain acts as both a hydrogen donor and a hydrogen ac- ceptor, do not appear to be unusual. Serine in fish hemo- globins (Perutz and Brunori, 1982) and tyrosine in HiPIP (Sheridan gt gt., 1981) provide two examples. Moreover, the two processes are correlated in that as the donor hydro- gen bond strengthens the acceptor hydrogen bond will also strengthen (Huyskens, 1977). The result of reduction, then, produces the configuration shown in Figure 36c. Upon oxidation of cyt g the two step process Shown in 3 and 4 returns the Site to its resting oxidized conformation. In 191 step 3, the OHC----O=C hydrogen bond weakens as the ROHC group shifts to its relaxed configuration. As this occurs, the Hc----Rr bond forms and strengthens. The Hb----O hydro- gen bond continues to strengthen as a result of the process and becomes covalent. The result is the state shown in Figure 36d. Although localized positive and negative charges are shown on Rr and R1, respectively, they need not occur if the covalent bond breaking/bond making pro- cesses are closely coupled. In the final step in the pro- cess, 4, the R1 group picks up a proton from the left (Ha) to become neutral and returns to the resting low energy conformation. On the right, the RiH loses a proton to 2 the right and is stabilized in the higher energy conforma- tion by the O-°--H-Rr hydrogen bond. The essential feature of the model of Figure 36 is the redox-linked switch from high energy configuration of the asymmetric hydrogen-bonded chain on the right and low energy configuration on the left to the opposite conformation, ttgt, low energy on the right and high energy on the left. Re- laxation of this state as oxidation occurs resets the system for a second cycle of proton translocation. One further point should be emphasized for this model. The 2-2.5 kcal/ mole associated with cytochrome g hydrogen bond strength changes is not enough energy to make the proton pump sig- nificantly irreversible under steady state conditions in mitochondria. For this to occur, other redox coupled 192 events (e.g., those suggested above) must also contribute to the overall free energy required to make the pump thermo- dynamically feasible. The Situation thus becomes analogous to Warshel and Weiss' (1981) view of hemoglobin where both chemical bond strength change (242:! "strain") and electro- static processes were proposed to contribute to the free energy difference between the R-state and the T-state. Within this context, the model of Figure 36 postulates the switching element and a source for part of the energy required to drive a proton pump in cytochrome oxidase. CHAPTER 7 SUMMARY AND FUTURE WORK I. Summary Heme structures important for electron transfer and proton transfer in the enzyme cytochrome oxidase have been dis- cussed. The lack of EPR Signals from the g Site of the 3 protein, and the overlapping absorption properties of the two heme g chromophores have made cytochrome g3 almost in— accessible to spectrosc0pic probes. Although optical dif- ference and MCD spectroscopies have provided some in- sight into the properties of cytochromes g3, resonance Raman spectroscopy has been shown to supply detailed structural information about this redox center. In ad- dition to the structural information available with this technique, the frequency dependence of the resonance en- hancement provides direct information about the electronic transitions of the chromophores. In the resting enzyme, cytochrome g3 has been shown to be Six coordinate and high-spin. The identity of the sixth ligand is not known, but u-oxo bridge (Blumberg and Peisach, 1979) and sulfur bridge (Powers gt gt., 1981) 193 194 structures have been proposed. The oxygenated enzyme, a late intermediate in oxygen reduction, displays very little absorption intensity in the Soret region and a Raman spectrum similar in band frequencies to the resting form. These properties may be a reflection of an intermediate spin (S=3/2) state of cyt g3 in the oxygenated enzyme (Shaw gt gt., 1978; Woodruff gt gt., 1982) although the appropr- iate heme g model compounds have not been fully investi- gated. In all liganded or native forms of the enzyme studied, the formyl of cytochrome g3 is observed to be in a hydrophobic environment. The apolar nature of the g3 Site may be important for the ready solubility of molecular oxygen and for fine-tuning the redox potential of this site. The alkaline pH dependence of the enzyme reported in Chapter 5 shows spectral shifts induced in the g3 site at pH 9.0. Similar studies at acidic pH levels allow the specification of pH 6.5 - 8.5 as the pH range for the enzyme in its native Spectral and hence structural, form. The red-shifted cytochrome g absorption spectrum and anomalous C=O stretching frequency have been interpreted as arising from a hydrogen bonding interaction between a nearby amino acid (possibly tyrosine) and the peripheral aldehyde of the cytochrome g ring. The linear relationship that exists between a band red-shift and the downshift in C=O stretching frequency allows the quantification of the hydrogen bond strengths found tg ytyg, The hydrogen bond strengths differ between ferrous and ferric cyt g by 195 2 - 2.5 kcal/mole. This strengthening of the hydrogen bond upon reduction of the enzyme may be used to drive redox-linked events such as proton pumping. Two models, one a conformational change model and the other which in- volves hydrogen bond chains, have been presented. II. Future WOrk This work has focussed on the high frequency region (>1000 cm-l) of the resonance Raman spectra of cytochrome oxidase and its derivatives. The low frequency region of the Spectrum still needs to be understood. Mostly bend- ing vibrations and out-of-plane bending modes and stretch- ing frequencies are expected in the 100-1000 cm-1 region. WOodruff EE.El° (1980) have proposed that several low fre- quency vibrations of oxidized cytochrome oxidase are due to Cu23 since their band positions are similar to those ob- served in type 1 copper—protein Raman Spectra. Their argu- ments that these vibrations do not correspond to cytochrome g3 vibrations are not valid, but some of the low frequency vibrations may reflect the cyt g3 - Cu interaction. Ex- 9—3 citation frequencies at the maximum or Slightly to the red of the cyt g3 electronic transitions are needed for selec- tive enhancement of the g3 low frequency vibrations. The cyt g3 - Cuit-3 exchange coupling can be eliminated or modi- fied by generating a form of the enzyme with cyt g3 oxidized 196 and Cuil—3 reduced. This can be done by chemical oxidation (Eiflir ferricyanide or porphyrexide) of the reduced enzyme or by formation of the 1/4 reduced enzyme (Brudvig gt gt,, 1980). Cytochrome g3 out-of-plane vibrations would be ex- pected to Shift as the structure around Cui3 is changed. Experimentally, this requires an anaerobic flow system to keep the enzyme in its modified form and to avoid photo- reduction of cytochrome g. A more direct probe of the cyt g3 — Cu site involves i3 excitation into the 655 nm absorption band of the oxidized enzyme. This band is present in the optical spectrum when the exchange coupling between the two metal centers is strong. Vibrations of both the heme and copper centers would be expected to be enhanced if this electronic transi- tion were a copper based charge transfer band. Because of 1 -1) the low extinction of the band (%3 mM- cm , this experi- ment requires high concentrations, low laser powers (xex = 647.1 nm, Kr+ laser) and a flowing sample arrangement. (Pre- liminary attempts have not yielded any results in two dif- ferent laboratories). To study the reaction intermediates of cytochrome oxi- dase, in addition to the low-temperature triple trapping method of Chance gt gt., (1975) Raman experiments on ener- gized cytochrome oxidase vesicles could be performed. Wik- Str6m (1980) has Shown that forms of the enzyme with spectra similar to the low temperature intermediates are generated 197 in ATP energized cytochrome oxidase vesicles. He attrib— utes this to partial reverse electron flow through the en- zyme, and, if this interpretation is correct then changes in structure at the catalytic site could be monitored. ATP has also been shown to red-shift the absorption spectrum of cytochrome g in mitochondria (Erecinska gt gt., 1972), so if the hydrogen bonded carbonyl of cytochrome g is involved in the H+—pumping mechanism of the enzyme, shifts in the 1610 cm'1/1650 cm‘l bands would be expected. As stated before, when dealing with the oxidized form of cytochrome g laser excitation induces photoreduction of this heme center. Procedures that can be utilized to minimize the problem are: 1) use of flowing sample arrangement, 2) removal of the putative flavin contaminant by using cyto- chrome g affinity chromatography (Thompson and Ferguson- Miller, 1983) or 3) the use of low temperatures combined with visible excitation (Bocian gt gt., 1978). The role of cytochrome g in the H+-pumping function of the enzyme needs to be more firmly established. The pH dependent midpoint potential of cytochrome g is one of the major arguments for the involvement of this center in the H+-pumping mechanism. Cytochrome g displays a -20 mV/pH unit redox potential dependence (Artzatbonov gt gt., 1978; Carithers and Palmer, 1981) which is quite different from the -60 mV/pH unit dependence expected if one H+/e- were taken up in the reduction reaction. A -20 mV/pH unit 198 potential dependence can be explained if the pKa of the protonated group in the oxidized form differs by only 1-2 pK units from the reduced form. The protonatable groups are probably amino acid residues on the surface of the protein that have redox-linked pKa's similar to the Bohr effect in hemoglobin. The identity of these amino acids and their function for proton translocationhas yet to be established. As stated in Chapter 1, DCCD binding to subunit III or removal of subunit III blocks proton translocation (Wikstrdm gt gt., 1983). Resonance Raman experiments that monitor the structure of cyt g, in particular its formyl stretching vibration, in the DCCD bound or subunit III-less forms of the enzyme might depict an inactive hydrogen bonded struc- ture. Preliminary experiments of this type (ttgt, DCCD bound cytochrome oxidase) reveal small shifts in the car- bonyl stretching frequency of the oxidized and reduced enzyme. These Shifts are not fully understood and should be investi- gated further. Small but reproducible shifts in the C=O stretching frequency of cytochrome g2+ are also observed when the deuterium is substituted for hydrogen in the aqueous sol- vent (DZO/H20)' D20 incorporation was used to investigate whether the hydrogen involved in the hydrogen bond to the peripheral aldehyde was exchangeable. Because deuterium bonds generally are weaker than hydrogen bonds (McDougall 199 and Long, 1962) a change in v(C=O) is expected if deuterium is incorporated. Thereijsa specific effect on only the 1610 - 1620 cm-1 region (predominantly cyt g) of the re- duced enzyme in D20. Because the shifts observed are rather small, Raman difference spectroscopy (Shelnutt gt gt., 1979) or high resolution conventional RRS must be used. If the shifts observed are a reflection of deuterium substitution for hydrogen in the cytochrome g formyl group hydrogen bond, this can help interpret similar shifts ob- served in DCCD binding to the H20 solubilized enzyme. The study of bacterial oxidases, because of the Simpler subunit structure, may provide insight into the energy transduction mechanism of this enzyme. The resonance Raman spectral differences of mitochondrial cytochrome oxidase, and of the two subunit oxidases from Paracoccus denitri— ficans and Rhodopsuedomonas §phaeroides which pump protons with a ratio of 2H+/e-, (Wikstrom _e_t a” 1983), 40.6 H+/e- (Solioz gt gt., 1982) and zero H+/e- (Gennis, gt gt., 1982), respectively, may reveal the structures necessary for proton pumping. The special feature of bacterial pro- teins is that modified amino acids can be added to the growth medium and therefore small polypeptide changes can be made. If hydrogen bond chains are important in the proton pumping mechanism, the elimination or variation of certain acidic or basic amino acids could inhibit this function of the enzyme. 200 Several spectroscopic questions remain outstanding con- cerning cytochrome oxidase and its heme g chromophores. The Split Soret band observed for cyt g at low tempera- tures (77 K) (Nicholls and Chance, 1974) may be caused by the additional electron withdrawing capacity of the hydro— gen bonded formyl group relative to the unperturbed formyl. The extent of splitting of the Soret maxima for a series of chlorins has been shown to be related to the electron- withdrawing strength of the peripheral substituents (Ward, 1983). Low temperature optical spectra of hydrogen bonded heme g model compounds can be recorded in order to test this hypothesis for heme g species. Although heme g has a vinyl group at position 4 of the porphyrin macrocycle, no vibration has been assigned to the C=C stretch. In protoheme containing species this vibration is observed at W1620 cm-l. For heme g either the vinyl group is not coupled to the 6 system of the ring or it is coupled with other normal modes of the ring and therefore not a group frequency. A similar observation has been made for the vinyl group of chlorophyll g (Lutz gt gt., 1982) and the explanation given was that the vinyl group lies out of the plane of the ring. The use of dye lasers with visible excitation of the heme g chromophores allows the acquisition of data for excitation profiles. If any charge transfer bands or transitions due to cyt g3 are present under the strong 201 cyt g absorption in this region, vibrations that follow a different frequency dependence than those of cytochrome g will be observed. This could supply out-of-plane axial ligand vibrational information or cytochrome g3 specific information. Excitation profiles also contain informa- tion about the electronic states of the species monitored. The symmetry of the ring and the degree of splitting of the x, y degenerate transition moments are possible pieces of information available. With tunability in both the visible and Soret regions, with elimination of the photoreduction problem and with high resolution data acquisition, resonance Raman spectros- copy, in conjunction with other spectrosc0pic techniques (optical, MCD and EPR) will continue to be a useful probe of the chromophores involved in the catalytic function of cytochrome oxidase. REFERENCES REFERENCES Abe, M., Kitagawa, T. and Kyogoku, Y. (1978) J. Chem. Phys. gt, 4526-4534. Adar, F. (1975) Arch. Biochem. Biophys. 70, 644-650. Adar, F. and Erecinska, M. (1979) Biochem. 8, 1825-1829. _— -—_. Adar, F. and Yonetani, T. (1978) Biochim. Biophys. Acta 502, 80-86. Alben, J. O., Moh, P. P. Fiamingo, F. G., and Attschuld, R. A. (1981) Proc. Nat'l. Acad. Sci. USA it, 234-237. Antalis, T. M. and Palmer, G. (1982) J. Biol. Chem. 2 7, 6194-6206. Artzatbanov, V. Y., Konstantinov, A. A., and Skulachev, V. P. (1978) FEBS Lett. gt, 180-185. Arnett, E. M., Mitchell, E. J. and Murty, T. S. S. R. (1974) J. Am. Chem. Soc. 22, 3875-3891. Asher, S. A. (1981) in Methods in Enzymology, Vol. 76, Academic Press, New York, pp. 371-413. Aton, B., DoukaS, A. G., Callender, R. H., Becher, B., Ebrey, T. G. (1977) Biochem. t2, 2995-2999. Azzi, A. (1980) Biochim. Biophys. Acta 594, 231-252. ——-—0— Babcock, G. T. and Chang, C. K. (1979) FEBS Lett. 7, 358-362. Babcock, G. T. and Salmeen, I. (1979) Biochem. tg, 2493-2498. Babcock, G. T., Vickery, L. E. and Palmer, G. (1976) g. Biol. Chem. 251, 7907-7919. Babcock, G. T., Vickery, L. E. and Palmer, G. (1978) g. Biol. Chem. 253, 2400-2411. Babcock, G. T., Ondrias, M. R., Gobeli, D. A., Van Steelandt, J. and Leroi, G. E. (1979) FEBS Lett. 108, 147-151. 202 203 Babcock, G. T., Van Steelandt, J., Palmer, G., Vickery, L. B., and Salmeen, I. (1979) in Cytochrome Oxidase (King, T. E. et al., eds) Elsevier/North Holland BIomedical Press, pp. 105-115. Babcock, G. T., Callahan, P. M., Ondrias, M. R. and Salmeen, I. (1981) Biochem. g2, 959-966. Baum, J. C. and McClure, D. S. (1979) J. Am. Chem. Soc. 101, 2340-2343. Beinert, H. and Shaw, R. W. (1977) Biochim. Biophys. Acta 504, 187-199. Bickar, D., Bonventura, J. and Bonaventura, C. (1982) Biochem. it, 2661-2666. Bisson, R., Jacobs, B. and Capaldi, R. (1980) Biochem. t2, 4173-4178. _— Blair, D. F., Bocian, D. F., Babcock, G. T. and Chan, S. I. (1982) Biochem. gt, 6928-6935. Blokzijl-Homan, M. F. J. and Van Gelder, B. F. (1971) Biochim. Biophys. Acta 234, 493-498. Blumberg, W. E. and Peisach, J. (1979) in Cytochrome Oxidase (King, T. E. et al., eds), Elsevier/North-Holland Bio- medical Press, pp. 153-159. Bocian, D. F., Lemley, A. T., Petersen, N. O., Brudvig, G. W. and Chan, S. I. (1979) Biochem. =3, 4396-4402. Brudvig, G. W., Stevens, T. H., and Chan, S. I. (1980) Biochem. t2, 5275-5285. Brudvig, G. W., Stevens, T. H., Morse, R. H. and Chan, S. I. (1981) Biochem. 32! 3912-3921. Cabral, F. and Love, B. (1972) Biochim. Biophys. Acta 238, 181-186. Cabral, F. and Love, B. (1972) Biochim. BiOphys. Acta 83, 181-184. Callahan, P. M. and Babcock, G. T. (1981) Biochem. 29, 952- 958. _— Callahan, P. M. and Babcock, G. T. (1983) Biochem. g;, 452- 461. _— Capaldi, R. A. (1982) Biochem. Biophys. Acta 94, 291-306. ————— _— 204 Carithers, R. P., and Palmer, G. (1981) J. Biol. Chem. 25 , 7967-7976. Carter, K. A., Tsai, A. and Palmer, G. (1981) FEBS Lett. 132, 243-246. Casey, R. P. and Azzi, A. (1983) FEBS Lett. 154, 237-242. Casey, R. P., Thelen, M. and Azzi, A. (1980) J. Biol. Chem. 255, 3994-4000. Caughey, W. S., Smythe, G. A., O'Keeffe, D. H., Maskasky, J. E. and Smith, M. L. (1975) J. Biol. Chem. 250, 7602- 7622. Chan, S. I., Bocian, D. F., Brudvig, G. W., Morse, R. H., Stevens, T. H. (1978) in Frontiers gt Biological Ener- getics, (Dutton, P. L., et al. eds) Vol. 2, Academic Press, New York, pp. 883-888. Chan, S. I., Bocian, D. F., Brudvig, G. W., Morse, R. H. and Stevens, T. H. (1979) DevelOpments tg Biochemistry t, Cytochrome Oxidase, (King, T. B., Orii, Y., Chance, B. and Okunuki, K., eds) Elsevier/North Holland, Amster— dam, pp. 177-188. Champion, P. M. and Albrecht, A. C. (1979) J. Chem. Phys. it, 1110-1121. Chance, B. and Williams, G. R. (1955) J. Biol. Chem. 217, 409-427. Chance, B. and Williams, G. R. (1956) Adv. Enzymology tt, 65-134. Chance, B., Saronio, C. and Leigh, J. S. (1975) J. Biol. Chem. 250, 9226-9237. Choi, S., Spiro, T. G., Langry, K. C., Smith, K. N., Budd, D. L. and LaMar, G. N. (1982), J. Am. Chem. Soc. 104, 4345-4351. Clark, R. J. H. and Stewart, B. (1979) Structure and Bonding Clore, G. M. and Chance, B. (1978) Biochem. J. 173, 811-820. Clore, G. M., Andreasson, L.-E., Karlsson, B., Asa, R. and Malmstrom, B. G. (1980) Biochem. J. 185, 139-154. Collins, D. M., Countryman, R. and Hoard, J. L. (1972) J. Am. Chem. Soc. it, 2066-2072. 205 Criddle, R. S. and Bock, R. M. (1959) Biochim. Biophys. Res. Comm. t, 138-142. Davis, R. C., Ditson, S. L., Fentiman, A. F. and Pearlstein, R. M. (1981) J. Amer. Chem. Soc. 103, 6823-6826. Dockter, M. E., Steinemann, A. and Schatz, G. (1978) 3. Biol. Chem. 253, 311-317. Dolphin, D. H., Sams, J. R. and Tsin, T. G. (1977) Inorg. Chem. 16, 711-717. ___ —— Eccles, J. and Honig, B. (1982) Biophys. J. 37, 228a. Erecinska, M. and Wilson, D. F. (1978) Arch. Biochem. Biophys. 151, 304-315. Erecinska, M., Wilson, D. F., Sata, N. and Nicholls, P. (1972) Arch. Biochem. Biophys. 151, 188-193. Fee, J. A. Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Nat'l. Acad. Sci, USA 11, 147- 151. __' Felton, R. H. and Yu, N.-T. (1978) in The Porphyrins (Dolphin, D., ed), Vol. 3, Part A. Academic Press, New York, pp. 347-393. Ferguson-Miller, S. (1983), personal communication. Frey, T. G., Chan, S. H. P. and Shatz, G. (1978) J. Biol. Chem. 253, 4389-4395. ' Friedman, J. M. and Hochstrasser, R. M. (1973) Chem. Phys. t, 457-467. _ Friedman, J. M., Rousseau, D. L. and Adar, F. (1977) Proc. Nat'l. Acad. Sci. USA tt,2607-27ll. Fuhrop, J. H. and Smith, K. M. (eds), in Laboratory Methods tg Porphyrin and Metallopotphyrin Research (1975), Elsevier/North Holland, Amsterdam, p. 42. Gennis, R. B., Casey, R. P., Azzi, A. and Ludwig, B. (1982) Eur. J. Biochem. 125, 189-195. Georgevich, G., Darley-Usmar, V. M., Malatesta, F., and Capaldi, R. A. (1983) Biochem., in press. Gouterman, M. (1959) J. Chem. Phys. it, 1139-1161. Gouterman, M. (1961) J. Mol. Spec. 6, 138-163. 206 Haddad, M. S. and Hendrickson, D. N. (1978) Inorg. Chem. t1, 2622-2630. ’— Halaka, F. G. (1981) Ph.D. Thesis, Michigan State University. Halaka, F. G., Babcock, G. T. and Dye, J. L. (1981) J. Biol. Chem. 256, 1084-1087. Halaka, F. G., Barnes, Z. K., Babcock, G. T. and Dye, J. L. (1982) J. Biol. Chem., submitted. Hartzell, C. R. and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338. Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Nat'l. Acad. Sci. USA it, 2477-2481. Heitler, W. (1954) in The Quantum Theory gt Radiation, 3rd edition, Oxford University Press (Clarendon), New York. Henderson, R., Capaldi, R. A. and Leigh, J. S. (1977) J. Mol. Biol. 112, 631-648. — Hinkle, P. and Mitchell, P. (1970) J. Bioenerg. t, 45-60. Hinkle, P. C., Kim, J. J. and Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawino- wicz, M. A., Arnaboldi, M. and Motto, M. G. (1979) J. Am. Chem. Soc. 101, 7084-7086. Hori, H. and Kitagawa, T. (1980) J. Am. Chem. Soc. 02, 3608-3613. Huong, P. V. and Pommier, J. C. (1977) C. R. Acad. Sci. Ser C. 28 , 519. Huyskens, P. L. (1977) J. Am. Chem. Soc. 9, 2578-2582. Keilin, D. (1966) in The History gt Cell Respiration and Cytochromes, (prepared by J. Keilin), Cambridge Uni- versity Press, Cambridge. Kitagawa, T., Ozaki, Y. and Kyogoku, Y. (1978) in Advances tg Biophysics (Kotani, M., ed), Vol. 11, University Park Press, Baltimore, MD, pp. 153-196. Kolks, G., Frihart, C. R., Rabinowitz, H. N. and Lippard, S. J. (1976) J. Am. Chem. Soc. 23, 5720-5721. Kumar, C., Nagui, A., Chance, B., Ching, Y., Powers, L. and Hartzell, C. R. (1981) Biochem. J. gt, 409a. 207 Landrum, J. T., Reed, C. A., Hatano, K. and Scheidt, W. R. (1978) J. Am. Chem. Soc. 100, 3232-3233. Lemberg, R. (1962) Nature (London) 193, 373-374. Lemberg, R. (1964) Proc. Roy Soc. 3159, 429-435. Lemberg, M. R. (1969) Phys. Rev. 49, 48-121. Lemberg, R. and Barrett, J. (1973) in Cytochrome, Academic Press, New York. Lemberg, R. and Pilger, T. B. G. (1964) Proc. Roy Soc. London, Ser. B. 159, 436-448. Lemberg, R., Bloomfield, B., Caiger, P. and Lockwood, W. (1955) Australian J. EXptl. Biol. 3, 435-444. .—-—- _— Lehninger, A. L., Ul Hassan, M., and Sudduth, H. C. (1954) J. Biol. Chem. 210, 911-922. , Rector, C. W. and Platt, J. R. (1950) 18, 1174-1181. Longuet-Higgins, H. J. Chem. Phys. Ludwig, B. (1980) Biochim. Biophys. Acta 5 4, 177-189. Lutz, M., Hoff, A. J. and Brehamet, L. (1982) Biochim. Bio- phys. Acta 679, 331-341. Maley, G. F. and Lardy, H. A. (1954) J. Biol. Chem. 210, 903-909. Malmstrém, B. G. (1979) Biochim. Biophys. Acta 49, 281-303. Maltempo, M. M. (1976) Biochim. Biophys. Acta 434, 513-518. Marcus, M. A., Lemley, A. T. and Lewis, A. (1979) J. Raman Spec. g, 22-25. Maroney, P. M. and Hinkle, P. (1983) J. Biol. Chem., in press. Mashiko, T., Kastberm, M. B., Spartalian, K., Scheidt, R. W. and Reed, C. A. (1978) J. Am. Chem. Soc. 100, 6354-6355. Mathies, R., Freedman, T. B. and Stryer, L. (1977) J. Mol. Biol. 109, 367-372. Mauzerzall, D. (1965) Biochem. 4, 1801-1810. McClain, M. W. and Harris, R. A. (1977) in Excited States (Lin, E. C., ed), Academic Press, New York, pp. 1-56. 208 McDougall, A. O. and Long, F.A.J. (1962) Chem. Soc. 66, 429-433. Mitchell, P. (1961) Nature 191, 144-148. Mitchell, P. and Moyle, J. (1983) FEBS Lett. 151, 167-178. —_ _— Moffitt, W. (1954) J. Chem. Phys. 2, 320-333. Murthy, A. S. N. and Rao, C. N. R. (1968) Appl. Spec. Rev. 2. 69-191. Nafie, L. A., Pezolet, M. and Peticolas, W. L. (1973) Chem. Phys. Letts. 20, 563-568. Nagai, K., Kitagawa, T. and Morimoto, H. (1980) J. Mol. Biol. 136, 271-289. Nagle, J. F. and Mille, M. (1981) J. Chem. Phys. _3, 1367- 1372. _ Nagle, J. F. and Morowitz, H. J. (1978) Proc. Nat'l. Acad. Sci. USA 12' 298-302. Nicholls, P. (1976) Biochim. Biophys. Acta 43 , 13-29. Nicholls, P. and Chance, B. (1974) in Molecular Mechanisms gt Oxygen Activation (Hayaishi, 0., ed), Academic Press, New York, pp. 479-534. Nicholls, D. and Locke, R. (1981) in Chemiosmotic Proton Circuits tg Biological Membranes (Skulachev, V. P. and Hinkle, P. C., eds), Addison-Wesley, Reading, MA. Nozawa, T., Orii, Y., Kaito, A., Yamamoto, T. and Hatano, M. (1979) in Develgpments tg Biochem. Vol. t, Cytochrome Oxidase (King, T. E., Orii, Y., Chance, B. and Okunuki, K., eds), Elsevier, Amsterdam, pp. 117-128. Ohnishi, T., Blum, H., Leigh Jr., J. S. and Salerno, T. C. (1979) in Membrane Bioenergetics (Lee, C. P. et a1., eds), Addison-Wesley, Reading, MA, pp. 21-30. Ohnishi, T., LoBrutto, R., Salerno, J. C., Bruckner, R. C. and Frey, T. G. (1983) J. Biol. Chem. 247, 14821-14825. O'Keeffe, D. H., Barlow, C. H., Smythe, G. A., Fuchsman, W. H., Moss, T. H., Lilienthal, H. R. and Caughey, W. S. (1975) Bioinorg. Chem. 2, 125-147. Ondrias, M. R. and Babcock, G. T. (1980) Biochem. Biophys. Res. Commun. 2;, 29-35. 209 Orii, Y. and King, T. E. (1976) J. Biol. Chem. 2 l, 7487- 7493. Palmer, G., Babcock, G. T. and Vickery, L. E. (1976) Proc. Nat'l. Acad. Sci. USA it, 2206-2210. Peisach, J. (1978) in Frontiers gt Biological Energetics (Dutton, P. L. et a1., eds) Vol. 2, Academic Press, New York, pp. 873-881. Pentilla, T. (1983) Eur. J. Biochem., in press. Pentilla, T. and Wikstrom, M. (1981) in Vectorial Reactions tg Electron and Ion Transport tg Mitochondria and Bacteria (Palmieri, F. et al., eds), Elsevier/North Holland, Amsterdam, pp. 71-80. Perrin, M. H., Gouterman, M. and Perrin, C. L. (1969), g. Chem. Phys. 22' 4137-4150. Person, P. and Zipper, H. (1964) Biochim. Biophys. Acta 2;, 605-607. Perutz, M. F. and Brunori, M. (1982) Nature 299, 421-426. Petty, R. H., Welch, B. R., Wilson, L. J., Bottomley, L. A. and Kadish, K. M. (1980) J. Am. Chem. Soc. 102, 611- 620. Pimentel, G. C. and McClellan, A. L. (1960) in The Hydrqgen Bond, W. H. Freeman and Company, San Francisco, pp. 157-164. Platt, J. R. (1956) in Radiation Biology (Hollander, A., ed) Vol. III, Chapter 2, McGraw—Hill, New York. Powers, L., Chance, B., Ching, Y. and Angiolillo, P. (1981) Biophys. J. ti, 465-498. Prochaska, L. J., Bisson, R., Capaldi, R. A., Steffens, G. C. M., and Buse, G. (1981) Biochim. Biophys. Acta 637, 360-373. Reed, C. A. and Landrum, J. T. (1979) FEBS Lett. 06, 265- 267. Reinhammer, B., Malkin, R., Jensen, P., Karlsson, B., Andreasson, L.-E., Asa, R., Vannggrd, T. and Malmstrom, B. G. (1980) J. Biol. Chem. 255, 5000-5003. Remba, R. D., Champion, P. M., Fitchen, D. B., Chiang, R. and Hager, L. P. (1979) Biochem. tt, 2280-2290. 210 Reynafarje, B., Alexandre, A., Davies, P. and Lehninger, A. L. (1982) Proc. Nat'l. Acad. Sci. USA 12' 7218-7222. Rosevear, P., Van Aken, T., Baxter, J. and Ferguson-Miller, S. (1980) Biochem. t2, 4108-4115. Rousseau, D. L., Friedman, J. M. and Williams, P. F. (1978) in Topics tg Current Physics, Vol. 11, Chapter 6, Springer, New York, pp. 202-251. Saari, H., Pentilla, T. and Wikstrdm, M. (1980) Biomembranes tt, 325-338. Salmeen, I., Rimai, L., Gill, D., Yamamoto, T., Palmer, G., Hartzell, C. R. and Beinert, T. H. (1973) Biochem. Bio- phys. Res. Commun. 2, 1100-1107. Salmeen, I., Rimai, L. and Babcock, G. T. (1978) Biochem. ti, 800-806. Saraste, M., Penttila, T. and Wikstrdm, M. (1981) Eur. J. Biochem. 115, 261-268. Scholler, D. M. and Hoffman, B. M. (1979) J. Am. Chem. Soc. 101, 1655-1662. Scott, R. A. and Gray, H. B. (1980) J. Amer. Chem. Soc. 102, 3219-3224. Sebald, W., Machleidt, W. and Wachter, E. (1980) Proc. Nat'l. Acad. Sci. USA ll, 785-789. Seiter, C. H. A. (1978) in Frontiers gt Biological Energetics (Dutton, P. L., Leigh, Jr., J. S. and Scarpa, A., eds) Academic Press, New York, pp.798-804. Seiter, C. H. A. and Angelos, S. G. (1980) Proc. Nat'l. Acad. Sci. USA ll, 1806-1808. Shaw, R. W., Hansen, R. E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637-6640. Shaw, R. W., Hansen, R. E. and Beinert, H. (1979) Biochim. Biophys. Acta 548, 386-396. Shelnutt, J. A. (1980) J. Chem. Phys. 2, 3948-3958. Shelnutt, J. A. (1981) J. Am. Chem. Soc. 103, 4275-4277. Shelnutt, J. A. (1981) J. Chem. Phys. 4, 6644-6657. 211 Shelnutt, J. A., O'Shea, D. C., Yu, N.-T., Cheung, L. D. and Felton, R. H. (1976) J. Chem. Phys. gt, 1156-1165. Shelnutt, J. A., Cheung, L. D., Chang, R. C. C., Yu, N.-T. and Felton, R. H. (1977) J. Chem. Phys. gt, 3387-3398. Shelnutt, J. A., Rousseau, D. L., Dethmers, J. K. and Mar- goliash, E. (1979) Proc. Nat'l. Acad. Sci. USA lg, 3865- 3869. ‘— Sheridan, R. P., Allen, L. C. and Carter Jr., C. W. (1981) J. Biol. Chem. 256, 5052-5057. Sheves, M., Nakanishi, K. and Honig. B. (1979) J. Amer. Soc. Sievers, G., Osterlund, K. and Ellfolk, N. (1979) Biochim. Biophys. Acta 581, 1-14. Simpson, W. T. (1949) J. Chem. Phys. 7, 1218-1221. Smith, D. W. and Williams, R. J. P. (1970) in Structure and Bonding, Vol. 17, Springer-Verlag, Berlin, pp. 1-45. Solioz, M., Carafoli, E. and Ludwig, B. (1982) J. Biol. Chem. 257, 1579-1582. Sone, N. and Yanagita, Y. (1982) Biochim. Biophys. Acta 682, 216-226. Spaulding, L. D., Chang, C. C., Yu, N.-T., and Felton, R. H. (1975) J. Am. Chem. Soc. 2;, 2517-2525. Spiro, T. G. (1974) Accts. Chem. Res. 7, 339-345. Spiro, T. G., Stong, J. D. and Stein, P. (1979) J. Am. Chem. Soc. 101, 2648-2655. Steffens, G. J. and Buse, G. (1979) HOppe-Seyler's Z. Physiol. Chem. 360, 613-619. Stevens, T. H., Martin, C. T., Wang, H., Brudvig, G. W., Scholes, C. P., and Chan, S. I. (1982) J. Biol. Chem. 275, 12106-12113. Stevens, T. H., Brudvig, G. W., Bocian, D. F. and Chan, S. I. (1979) Proc. Nat'l. Acad. Sci. USA lg, 3320-3324. Sutherland, J. C., Vickery, L. E. and Klein, M. P. (1974) Rev. Sci. Instrum. gt, 1089-1094. Takemori, S. and King, T. E. (1965) J. Biol. Chem. 240, 504-513. 212 Thompson, D. and Ferguson-Miller, S. (1983) Biochem., in press. Tsubaki, M., Nagai, K. and Kitagawa, T. (1980) Biochem. _2, 379-385. _— Tweedle, M. F., Wilson, L. J., Gardia-Iniguez, L., Babcock, G. T. and Palmer, G. (1978) J. Biol. Chem. 253, 8065- 8071. Tzagoloff, A. (1982) in Mitochondria, Plenum Publishing Corp., New York. Valentine, J. S., Sheridan, R. P., Allen, L. C. and Kahn, P. C. (1979) Proc. Nat'l. Acad. Sci. USA 76, 1009-1013. Vanderkooi, J. M., Landesberg, R., Hayden, G. W. and Owen, C. S. (1977) Eur. J. Biochem. gt, 339-347. Vanneste, W. H. (1966) Biochem. g, 838-848. Van Gelder, B. F. and Beinert, H. (1969) Biochim. Biophys. Acta 189, 1-24. Van Steelandt-Frentrup, J., Salmeen, I. and Babcock, G. T. (1981) J. Am. Chem. Soc. 103, 5981-5982. Ward, B., in preparation. Ward, B., Callahan, P. M., Young, R., Babcock, G. T. and Chang, C. K. (1983) J. Am. Chem. Soc. 105, 634-636. Warshel, A. and Weiss, R. M. (1981) J. Am. Chem. Soc. 1 3, 446-451. Weiss, C. (1972) J. Mol. Spec. 4, 37-80. Wever, R., Van Ark, G. and Van Gelder, B. F. (1977) FEBS Lett. gg, 388-390. Wikstrom, M. K. F. (1977) Nature (London) 66, 271-273. Wikstrdm, M. (1981) Proc. Nat'l Acad. Sci. USA it, 4051-4054. Wikstrdm, M. and Krab, K. (1979) Biochim. Biophys. Acta 549, 177-222. Wikstrom, M. K. F., Harmon, H. J., Ingledew, W. J. and Chance, B. (1976) FEBS Lett. gg, 259. Wikstrom, M., Krab, K. and Saraste, M. (1981) in Cytochrome Oxidase - A Synthesis, Academic Press, London. 213 Wikstrém, M., Krab, K. and Saraste, M. (1981) Ann. Rev. Biochem. =2, 623-655. Wikstrém, M., Saraste, M. and Penttila, T., (1983) personal communication. Wilson, D. F., Lindsay, J. G. and Brocklehurst, E. S. (1972) Biochim. Biophys. Acta 256, 277-286. Wilson, M. T., Lalla-Maharajh, W., Darley-Usmar, V., Bona- ventura, J., Bonaventura, C. and Brunori, M. (1980) J. Biol. Chem. 255, 2722—2728. Winter, D. B., Bruynickx, W. J., Foulke, F. G., Grinich, N. P. and Mason, H. S. (1980) J. Biol. Chem. 255, 11408-11414. Woodruff, W. H., Dallinger, R. F., Antalis, T. M. and Palmer, G. (1981) Biochem. gg, 1332-1338. Woodruff, W. H., Kessler, R. J., Ferris, N. S., Dallinger, R. F., Carter, K. R., Antalis, T. M. and Palmer, G. (1982) in "Electrochemical and Spectrochemical Studies of Biological Redox Components" (Kadish, K. M., ed), Advances in Chemistry Series 201, American Chemical Society pp. 625-659. Yamamoto, T., Palmer, G., Gill, D., Salmeen, I. and Rimai, L. (1973) J. Biol. Chem. 248, 5211-5213. Yamanaka, T., Kamita, Y. and Kukumori, Y. (1981) J. Biochem. (Tokyo) gg, 265-273. Zadorozhnyi, B. A. and Ischenko, I. K. (1965) QBt. Spectry. 12: 306-308. Zerner,M., Gouterman, M. and Kobayashi, H. (1966) Theoret. Chim. Acta (Berlin) g, 363-400. Zobrist, M. and LaMar, G. N. (1978) J. Am. Chem. Soc. 100, 1944-1946.