Expression of the Escherichia coli bo-type ubiquinol oxidase with a chimeric subunit II having the CuA-cytochrome c domain from the thermophilic Bacillus caa3-type cytochrome c oxidase

The C-terminal periplasmic domain of subunit II of the Escherichia coli bo-type ubiquinol oxidase was replaced with the counterpart of the thermophilic Bacillus caa3-type cytochrome c oxidase containing the CuA-cytochrome c domain by means of gene engineering techniques. The chimeric terminal oxidase was expressed by a pBR322 derivative in a terminal oxidase deficient mutant of E. coli, although the amount of the chimeric enzyme was smaller than that of the Escherichia coli bo-type ubiquinol oxidase expressed by the original cytochrome bo-expressing plasmid. The chimeric enzyme showed much higher TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity than the wild-type cytochrome bo, but lower activity than the thermophilic Bacillus caa3-type cytochrome c oxidase. The chimeric subunit II was confirmed to bind to heme C. These results suggest that the CuA-cytochrome c domain grafted to this membrane anchor can facilitate electron transfer from reduced TMPD to low-spin protoheme b in subunit I.     

Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein

The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.     

Cloning of the cta operon from alkaliphilic Bacillus firmus OF4 and characterization of the pH-regulated cytochrome caa3 oxidase it encodes

We have cloned and sequenced the DNA of alkaliphilic Bacillus firmus OF4 encompassing the cta operon that encodes a pH-regulated cytochrome caa3 oxidase. The gene organization is identical with that of the homologous Bacillus subtilis caa3 oxidase locus (van der Oost, J., von Wachenfeld, C., Hederstedt, L. & Saraste, M. (1991) Mol. Microbiol. 5, 2063-2072). The deduced amino acid sequences of the four putative structural subunits (CtaC-F) indicate substantial similarity to caa3-type oxidases from other Bacillus species and to other members of the family of mitochondrial-type aa3 oxidases. A marked paucity of basic residues was noted in the cytochrome c-containing domain of CtaC, which faces the highly alkaline external milieu. We have also purified the enzyme as a three-subunit complex, with possible trace amounts of a fourth subunit. N-terminal sequence analysis of the two largest subunits confirmed them to be encoded by the cloned cta genes. An additional, minor caa3 component with distinctive chromatographic properties was noted during purification. Analysis of mRNA with a ctaD probe revealed an abundant 4-kilobase message of the right size to encode CtaC-F. The cellular content of this message varied with growth pH. Cells grown at pH 10.5 contained 2 to 2.5 times more message than those grown at pH 7.5, in good correspondence with the relative amounts of caa3 oxidase found in the cells. The ctaB gene, immediately upstream from the ctaC-F genes, was found to be transcribed onto a low abundance 5-kilobase message, which is likely also to encode CtaC-F. Levels of this message were not affected by growth pH.     

Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon. Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach. We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme. Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site. Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.     

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.     

Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center

The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer kinetics during the reduction and turnover of the cytochrome caa3 complex from Bacillus subtilis

The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes. The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II. Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex. Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a. Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase. The cytochrome c from B. subtilis is the most efficient cytochrome c of those tested. Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present. The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD. This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3. It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself. Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.     

How replacements of the 12 conserved histidines of subunit I affect assembly, cofactor binding, and enzymatic activity of the Bradyrhizobium japonicum cbb3-type oxidase

Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb3-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the following cofactors: the low spin heme B and the binuclear high spin heme B-CuB center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidines of FixN in cofactor binding and function of the Bradyrhizobium japonicum cbb3-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Th?ny-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394, 349-352). Four of the remaining histidines were not essential for activity or assembly (positions 226, 246, 333, and 457); by contrast, histidines 331, 410, and 418 were required both for activity and stability of the enzyme. The last group of mutant enzymes, H420A, H280A, H330A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was added to the C terminus of subunit I. The His6-tagged cbb3-oxidase complexes were purified 20-fold by a three-step purification protocol. With the exception of the H420A mutant oxidase, the mutant enzymes H280A, H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics like the wild-type His6-tagged enzyme. The His6-tagged H420A mutant oxidase differed from the His6-tagged wild-type protein by showing altered visible light spectroscopic characteristics. No stable mutant oxidase lacking copper or heme B was obtained. This strongly suggests that copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.     

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.     

Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity

Purified mitochondrial cytochrome c oxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear heme a3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a3 nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreased Vmax and a raised Km for oxygen. Under these conditions, the redox state of cytochrome a is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 microM peroxynitrite to 10 microM fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochrome a3, partial reduction of cytochrome a, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 microM) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.     

Expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.     

The Bradyrhizobium japonicum coxWXYZ gene cluster encodes a bb3-type ubiquinol oxidase

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium, has a complex respiratory electron-transport chain, capable of functioning throughout a wide range of oxygen tensions. It does so by synthesizing a number of terminal oxidases, each appropriate for different environmental conditions. We have previously described the cloning of the large catalytic subunit, coxX, from one of the terminal oxidases from B. japonicum [Surpin, M.A., Moshiri, F., Murphy, A.M. and Maier, R.J. (1994) Genetic evidence for a fourth terminal oxidase from Bradyrhizobium japonicum. Gene 143, 73-77]. In this work, we describe the remaining subunits of this terminal oxidase complex, which is encoded by the coxWXYZ operon. The polypeptide encoded by coxW does not contain any amino acid residues that are known to bind the CuA atom of cytochrome c terminal oxidases, but contains residues thought to be involved in ubiquinol binding. Terminal oxidase cyanide inhibition titration pattern comparisons of the wild type with a coxWXYZ insertion mutant indicated the new oxidase is expressed microaerobically. However analysis of hemes extracted from microaerobically incubated cells revealed the absence of heme O in this strain (from both the wild type and the mutant) of B. japonicum. Therefore, coxWXYZ most likely encodes a microaerobically-expressed bb3-type ubiquinol oxidase.     

Solution structure of thermostable cytochrome c-552 from Hydrogenobacter thermophilus determined by 1H-NMR spectroscopy

The solution structure of a thermostable cytochrome c-552 from a thermophilic hydrogen oxidizing bacterium Hydrogenobacter thermophilus was determined by proton nuclear magnetic resonance spectroscopy. Twenty structures were calculated by the X-PLOR program on the basis of 902 interproton distances, 21 hydrogen bonds, and 13 torsion angle constraints. The pairwise average root-mean-square deviation for the main chain heavy atoms was 0.91 +/- 0.11 A. The main chain folding of the cytochrome c-552 was almost the same as that of Pseudomonas aeruginosa cytochrome c-551 that has 59% sequence identity to the cytochrome c-552 but is less thermostable. We found several differences in local structures between the cytochromes c-552 and c-551. In the cytochrome c-552, aromatic-amino interactions were uniquely formed between Arg 35 and Tyr 32 and/or Tyr 41, the latter also having hydrophobic contacts with the side chains of Tyr 32, Ala 38, and Leu 42. Small hydrophobic cores were more tightly packed in the cytochrome c-552 because of the occupancies of Ala 5, Met 11, and Ile 76, each substituted by Phe 7, Val 13, and Val 78, respectively, in the cytochrome c-551. Some of these structural differences may contribute to the higher thermostability of the cytochrome c-552.     

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.     

A novel chloride-binding site modulates the heme-copper binuclear center of the Escherichia coli bo-type ubiquinol oxidase

Cytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping. Previous studies [Y. Orii, T. Mogi, M. Sato-Watanabe, T. Hirano, and Y. Anraku (1995) Biochemistry 34, 1127-1132] suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer. To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties. UQO-412, which exhibits the Soret peak at 412 nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift. UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center. Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a K(d) value of 3.5 mM. Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable. The present studies demonstrated that the E. coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center. Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion.     

Lifelong severe verrucosis associated with human papillomavirus type 2: report of a case with a 38-year follow-up

Sulfite ion (HSO3-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO3- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO3-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO3- on cytochrome c oxidase activity were studied with the plasma membranes of HSO3(-)-resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO3-. To investigate the inhibition mechanism of HSO3- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO3-. In contrast, the same concentration of HSO3- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO3- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.     

Mechanism of inhibition of electron transfer by amino acid replacement K362M in a proton channel of Rhodobacter sphaeroides cytochrome c oxidase

The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.