A carboxyl-terminal hydrophobic region of yeast cytochrome c1 is necessary for functional assembly into complex III of the respiratory chain

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

Replacement of putative axial ligands of heme iron in yeast cytochrome c1 by site-directed mutagenesis

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.     

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

Sequencing the gene encoding desulfovibrio desulfuricans ATCC 27774 nine-heme cytochrome c.

Contradicting early suggestions, the sequencing of the gene encoding the Desulfovibrio desulfuricans (ATCC 27774) nine-heme cytochrome c proves that this cytochrome is not the product of the degradation of the 16-heme containing cytochrome c [Coelho et al. (1996) Acta Cryst. D52, 1202-1208]. However, preliminary data indicate that the cytochrome gene is part of an operon similar to the DvH hmc operon, which contains the gene coding for the 16-heme cytochrome c [Rossi et al. (1993) J. Bacteriol. 175, 4699-4711]. Also, the amino acid sequence deduced from the DNA sequence shows four residues in the C-terminal not predicted in the amino acid sequence obtained by X-ray methods [Matias et al. (1999) Structure 7, 119-130].     

The role of extra fragment at the C-terminal of cytochrome b (Residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.     

Molecular analysis of the cytochrome bc 1-aa 3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c 1

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.     

Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.     

Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase

The cyt-2-1 mutant of Neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (Mitchell, M.B., Mitchell, H.K., and Tissieres, A. (1953) Proc. Natl. Acad. Sci. U.S.A. 39, 606-613; Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W., and Neupert, W. (1988) J. Biol. Chem. 263, 9388-9394). By rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a N. crassa genomic library using sib selection. Analysis of the DNA sequence of the cyt-2+ gene revealed an open reading frame of 346 amino acids that has homology to the yeast cytochrome c heme lyase. The open reading frame is interrupted by two short introns. Codon usage and Northern hybridization analysis suggest that the cyt-2 gene is expressed at low levels. The cyt-2-1 mutant allele was cloned from a partial cyt-2-1 gene bank using the wild-type gene as a probe. Sequence analysis of the mutant gene revealed a 2-base (CT) deletion that alters the reading frame for 21 codons before generating an early stop codon in the protein-coding sequence. It was previously suggested that the cyt-2-1 mutation inactivates one of two regulatory circuits controlling the production of cytochrome aa3. The finding that the cyt-2-1 mutation affects the coding sequence for cytochrome c heme lyase provides a direct explanation for the deficiency of cytochrome c in the mutant and suggests that the lack of cytochrome aa3 is a regulatory response to the deficiency of cytochrome c.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Isolation and structure of a rat cytochrome c gene

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.     

Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus

Cytochrome oxidase from T. thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R. M., Choc, M. G., Tarr, G. E., Findling, K. L., and Fee, J. A. (1983) J. Biol. Chem. 258, 112-123). We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T. thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD). These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions. Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme. The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3. When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized. The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme.     

Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.     

Reaction mechanism of bovine heart cytochrome c oxidase

The 1.9 A resolution X-ray structure of the O2 reduction site of bovine heart cytochrome c oxidase in the fully reduced state indicates trigonal planar coordination of CuB by three histidine residues. One of the three histidine residues has a covalent link to a tyrosine residue to ensure retention of the tyrosine at the O2 reduction site. These moieties facilitate a four electron reduction of O2, and prevent formation of active oxygen species. The combination of a redox-coupled conformational change of an aspartate residue (Asp51) located near the intermembrane surface of the enzyme molecule and the existence of a hydrogen bond network connecting Asp51 to the matrix surface suggest that the proton-pumping process is mediated at Asp51. Mutation analyses using a gene expression system of the Asp51-containing enzyme subunit yield results in support of the proposal that Asp51 plays a critical role in the proton pumping process.     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.