Cytochrome c oxidase: one enzyme, two mechanisms?
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Animals↗
Biomedical subjects
R B Gennis
Publications and source records attributed to R B Gennis.
Localization of histidine residues responsible for heme axial ligation in cytochrome b556 of complex II (succinate:ubiquinone oxidoreductase) in Escherichia coli.
Complex II (succinate:ubiquinone oxidoreductase) from Escherichia coli contains four different subunits. Two of the subunits (SDHC and SDHD) are hydrophobic and anchor the two more hydrophilic (flavin and iron-sulfur) subunits (SDHA and SDHB) to the cytoplasmic membrane. Previous studies have shown that the complex of SDHC/SDHD is required to maintain the heme B component of the enzyme and that the heme B is ligated to the protein by two histidine ligands. In the current work, the histidines within SDHC and SDHD have been systematically mutated. SDHC-His91 and SDHD-His14 were eliminated as potential ligands by these studies. SDHC-His84 and SDHD-His71 have been identified as the most likely heme axial ligands in the E. coli enzyme, suggesting that the heme bridges these two subunits in the membrane. Furthermore, the results show that the four-subunit Complex II assembles and retains function despite the absence of the heme B prosthetic group in the membrane. The results do not rule out completely SDHC-His30 as a candidate for heme ligation, but do show that mutation at this position prevents assembly of Complex II in the membrane.
Aerobiosis↗
Substitution of lysine-362 in a putative proton-conducting channel in the cytochrome c oxidase from Rhodobacter sphaeroides blocks turnover with O2 but not with H2O2.
The recently reported X-ray structures of cytochrome oxidase reveal structures that are likely proton-conducting channels. One of these channels, leading from the negative aqueous surface to the heme a3/CuB bimetallic center, contains a lysine as a central element. Previous work has shown that this lysine (K362 in the oxidase from Rhodobacter sphaeroides) is essential for cytochrome c oxidase activity. The data presented demonstrate that the K362M mutant is impeded in the reduction of the heme a3/CuB bimetallic center, probably by interfering with the intramolecular movement of protons. The reduction of the heme-copper center is required prior to the reaction with dioxygen to form the so-called peroxy intermediate (compound P). This block can be by-passed to some extent by the addition of H2O2, which can react with the enzyme without prereduction of the heme-copper center and can then be reduced to water using electrons from cytochrome c. Hence, the K362M mutant, though lacking oxidase activity, exhibits cytochrome c peroxidase activity. Rapid mixing techniques have been used to determine the kinetics of this peroxidase activity at concentrations of H2O2 up to 0.5 M. The Km for peroxide is about 50 mM and the Vmax is 50 electrons s-1, which is considerably slower than the turnover that can be obtained for the oxidase activity of the wild-type enzyme (1200 s-1). The turnover of the mutant oxidase with H2O2 appears to be limited by the rate of reaction of the enzyme with peroxide to form compound P, rather than the rate of reduction of compound P to water by cytochrome c. The data require a reexamination of the proposed roles of the putative proton-conducting channels.
Catalysis↗
Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides.
In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I-362)) and threonine-359 (T(I-359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer. These residues have been proposed to be part of a proton-transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart. Mutation of K(I-362) and T(I-359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and approximately 35%, respectively, of those in the wild-type enzyme. The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of approximately 3 micros, not coupled to protonation reactions, is not affected in the mutant enzymes. However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of approximately 3 ms (at pH 9.0) is impaired in the KM(I-362) and TA(I-359) mutant enzymes. This is consistent with the slow reduction rate of heme a3 in the oxidized KM(I-362) enzyme because in the wild-type enzyme reduction of heme a3 is coupled to proton uptake. On the other hand, when reacting with O2, both the wild-type and mutant fully reduced enzymes become oxidized in approximately 5 ms, and proton uptake on this time scale is not affected. Hence, the results indicate that the KM(I-362) mutant enzyme is inactive because the proton-transfer pathway through K(I-362) and T(I-359) is involved in proton uptake during reduction of the oxidized binuclear center. Proton uptake during oxidation of the fully reduced enzyme takes place through a different pathway [through E(I-286) (Adelroth, P., et al. (1997) Biochemistry 36, 13824-13829)].
Animals↗
Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits.
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.
Cytochrome b Group↗
Effects of mutation of the conserved lysine-362 in cytochrome c oxidase from Rhodobacter sphaeroides.
We describe the effects of a mutation, K362M, of the conserved lysine in cytochrome c oxidase from Rhodobacter sphaeroides, a residue located in a putative proton channel that may convey substrate protons to the binuclear center. Spectra of the "as prepared", ferricyanide-oxidized, and dithionite-reduced forms of the mutant protein confirm that the redox centers remain intact. Ligand binding kinetics of the ferricyanide-oxidized enzyme and of the dithionite-reducible fraction are similar to those of the wild type, indicating that the K channel is not the major route for CO, cyanide, formate, or peroxide entry into the structure. Protonation of the lysine residue is not redox-linked to heme a or CuB as judged from the essentially unaltered midpoint potentials of these centers in the cyanide-ligated enzyme. A difficulty in electron transfer from heme a to the binuclear center is indicated by the slow and only partial reduction of heme a3 by dithionite or ferrocytochrome c and by the presence of some reduced heme a in the as prepared mutant enzyme and under steady-state conditions. Further characterization of the K362M enzyme in the steady state shows that up to one electron, but not two, can enter the binuclear center easily. It is this inability to form the two-electron-reduced, oxygen-reactive R state that prevents activity. A model is proposed where the K channel serves as a dielectric well of high dielectric strength and low proton conductivity, rather than as a pathway for proton entry to the binuclear center. The function of this structure would be to decrease the cost of introducing a transiently uncompensated charge into the binuclear center prior to formation of a stable, charge-compensated R-state.
Amino Acid Sequence↗
Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen.
The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.
Amino Acid Substitution↗
A conserved glutamic acid in helix VI of cytochrome bo3 influences a key step in oxygen reduction.
We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine. Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation. Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.
Amino Acid Sequence↗
Factors determining electron-transfer rates in cytochrome c oxidase: studies of the FQ(I-391) mutant of the Rhodobacter sphaeroides enzyme.
The mechanisms of internal electron transfer and oxygen reduction were investigated in cytochrome c oxidase from Rhodobacter sphaeroides (cytochrome aa3) using site-directed mutagenesis in combination with time-resolved optical absorption spectroscopy. Electron-transfer reactions in the absence of O2 were studied after flash photolysis of CO from the partly-reduced enzyme and the reaction of the fully-reduced enzyme with O2 was studied using the so-called flow-flash technique. Results from studies of the wild-type and mutant enzyme in which phenylalanine-391 of subunit I was replaced by glutamine (FQ(I-391)) were compared. The turnover activity of the mutant enzyme was approximately 2% ( approximately 30 s-1) of that of the wild-type enzyme. After flash photolysis of CO from the partly-reduced mutant enzyme approximately 80% of CuA was reduced, which is a much larger fraction than in the wild-type enzyme, and the rate of this electron transfer was 3.2 x 10(3) s-1, which is significantly slower than in the wild-type enzyme. The redox potentials of hemes a and a3 in the mutant enzyme were found to be shifted by about +30 and -70 mV, respectively, as compared to the wild-type enzyme. During the reaction of the fully-reduced FQ(I-391) mutant enzyme with O2 a rapid kinetic phase with a rate constant of 1.2 x 10(5) s-1, presumably associated with O2 binding, was followed by formation of the P intermediate with electrons from heme a3 and CuB with a rate of approximately 4 x 10(3) s-1, and oxidation of the enzyme with a rate of approximately 30 s-1. The dramatically slower electron transfer between the hemes during O2 reduction in the mutant enzyme is not only due to the slower intrinsic electron transfer, but also due to the altered redox potentials. In addition, the results show that the reduced overall activity of the mutant enzyme is due to the slower electron transfer from heme a to the binuclear center during O2 reduction. The relation between the intrinsic heme a/heme a3 electron-transfer rate and equilibrium constant, and the electron-transfer rate from heme a to the binuclear center during O2 reduction is discussed.
Electron Transport↗
Subunit II of the cytochrome bo3 ubiquinol oxidase from Escherichia coli is a lipoprotein.
The purified Escherichia coli cytochrome bo3 ubiquinol oxidase contains four subunits that are each integral components of the cytoplasmic membrane. The molecular weight of each of the subunits has been determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI). The observed molecular weight of subunit II (CyoA) is considerably less than the calculated value from the deduced amino acid sequence, indicating possible posttranslational processing. The similarity of a portion of the sequence near the N-terminus of CyoA with the sequences of known prokaryotic membrane-bound lipoproteins suggested that CyoA is proteolytically processed to generate an N-terminus at Cys25, and that Cys25 is covalently modified by the addition of lipids. This would be consistent with the observed molecular mass, and was confirmed by demonstrating the incorporation of radioactive palmitic acid into subunit II of the cytochrome bo3 oxidase. Site-directed mutagenesis replacing Cys25 by alanine prevents the processing, generating a precursor form of CyoA with a higher molecular mass. The C25A mutant of CyoA still assembles as an active quinol oxidase capable of supporting growth of the cells by aerobic respiration. Hence, this unusual processing of a cytoplasmic membrane protein, which is already anchored to the membrane by two transmembrane helices, is not essential for either assembly or function.
Aerobiosis↗
The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer.
The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2-) to the oxidized form (Fe3+OH-). This redox step requires the delivery of a "chemical" H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660-669]. The D channel is likely to be involved in the uptake of both "chemical" and "pumped" protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.
Animals↗
One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase.
The cytochrome bo3 ubiquinol oxidase from Escherichia coli is a member of the heme-copper superfamily of proton-pumping respiratory oxidases. An improved preparative protocol was desired that would minimize the potential damage during protein isolation of labile mutants of the oxidase. Variants of the oxidase containing a histidine tag at the carboxy-terminus of either subunit I, II or III were constructed. The constructs with the histidine tag on either subunit I or II successfully allowed the enzyme to be isolated with high purity in one step using Ni2+ affinity chromatography. The enzyme with the histidine tag on subunit II is particularly useful insofar as the enzyme isolated in this manner has little, if any, heterogeneity resulting from the presence of heme O in the low spin heme-binding site, i.e., cytochrome oo3 is minimized. The enzyme can be prepared in virtually any quantity very rapidly and is suitable for biophysical characterization. Cytochrome bo3 was prepared in either Triton X-100, sucrose monolaurate, or dodecyl maltoside. The enzyme isolated in the presence of either sucrose monolaurate or dodecyl maltoside contains approximately one equivalent of associated ubiquinone, whereas this is absent when Triton X-100 is used. However, the UV/vis absorbance and steady-state kinetic properties of the enzyme are virtually identical regardless of which detergent is used. These data are consistent with previous reports that cytochrome bo3 contains an equivalent of 'tightly associated' ubiquinone, but clearly demonstrate that this quinone can be removed without damaging the enzyme and is not critical to the maintenance of the native structure of the oxidase.
Benzoquinones↗
FTIR study of conformational substates in the CO adduct of cytochrome c oxidase from Rhodobacter sphaeroides.
Fourier transform infrared (FTIR) spectroscopy of cytochrome c oxidase from Rhodobacter sphaeroides reveals multiple CO stretch bands that are associated with different conformational substates of the enzyme. Here we report the temperature dependence of the infrared bands for the CO bound to the Fea3 heme iron and to CuB. We have also studied the kinetics of ligand return from Fea3 to CuB using temperature derivative spectroscopy (TDS). Two classes of substates (alpha/beta) can be distinguished from their different properties with regard to the width of the IR band, the temperature dependence of the peak position, and the peak of the enthalpy distribution. The pronounced temperature dependence of the stretch frequencies in the beta conformation and the lack thereof in the alpha conformation implies very different dynamic behavior in the active site and reflects structural differences between the two conformations, most likely a shift of the position of CuB in response to a change in its stereochemical environment. Similar conformational changes will be necessary during the catalytic cycle of the enzyme when dioxygen is bound in the active site.
Binding Sites↗
Kinetics of electron and proton transfer during the reaction of wild type and helix VI mutants of cytochrome bo3 with oxygen.
Site-directed mutagenesis was used to investigate the mechanism of electron and proton transfer in the ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The reaction between the fully reduced form of the enzyme and dioxygen was studied using the flow--flash method. After rapid mixing of CO-bound enzyme with an O2-containing solution, CO was photodissociated, and the subsequent electron- and proton-transfer reactions were measured spectrophotometrically, the latter using a pH-indicator dye. In the wild-type, pure bo3 enzyme, without bound quinones, we observed a single kinetic phase with a rate constant of about 2.4 x 10(4) s-1, associated with formation of the ferry1 oxygen intermediate, followed by proton uptake from solution with a rate constant of about 1.2 x 10(4) s-1. Enzyme in which heme o instead of heme b was incorporated into the low-spin site displayed a slower ferry1 formation with a rate constant of about 3.6 x 10(3) s-1. Upon replacement of the acidic residue glutamate 286 in helix VI of subunit I with a nonprotonatable residue, electron transfer was slightly accelerated, and proton uptake was impaired. Mutations of other residues in the vicinity of E286 also resulted in a dramatic decrease of proton uptake, suggesting that the environment of this residue is important for efficient proton transfer. In the closely related cytochrome aa3 from P. denitrificans, the corresponding residue (E278) has been suggested to be part of a proton-transfer pathway [Iwata, S., Ostermeier, C., Ludwig, B., & Michel, H. (1995) Nature 376, 660-669]. The results are discussed in terms of a model for electron-proton coupling during dioxygen reduction.
Carbon Monoxide↗
Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.
Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.
Carbon Monoxide↗
Polar residues in helix VIII of subunit I of cytochrome c oxidase influence the activity and the structure of the active site.
The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a3-CuB center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate-determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral properties of the heme a3-CuB center. Mutation of T359, below heme a3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, approximately 15 A below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However, the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.
Amino Acid Sequence↗
A pH-dependent polarity change at the binuclear center of reduced cytochrome c oxidase detected by FTIR difference spectroscopy of the CO adduct.
A pH-dependent polarity change at the heme-copper binuclear center of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been identified by low-temperature FTIR difference spectroscopy. "Light"-minus-"dark" FTIR difference spectra of the fully reduced CO-enzyme adduct were recorded at a range of pH, and the dominance of different populations of bound CO, alpha and beta, was found to vary with pH. An apparent pKa of about 7.3 for the transition was obtained. The alpha and beta forms are differentiated by different polarities at the heme-copper binuclear center of the enzyme, sensed by the stretching frequencies of CO bound either to the heme alpha 3 Fe or to CuB. Several site-directed mutants in the vicinity of the heme-copper center are shown to favor either the alpha or the beta forms of the enyzme, suggesting that what is being monitored is an equilibrium between two conformations of the reduced form of the oxidase. Recent resonance Raman evidence has been presented demonstrating that the alpha and beta forms of the R. sphaeroides oxidase exist at room temperature; therefore, the pH-dependent change in the polarity in the vicinity of the heme-copper center may be functionally significant.
Amino Acid Sequence↗
Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase.
Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.
Cytochrome a Group↗