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.     

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.     

Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae

Cytochrome oxidase (COX) is considered to integrate in a single enzyme two consecutive mechanistically different redox activities--oxidase and peroxidase--that can be catalyzed elsewhere by separate hemoproteins. From the viewpoint of energy transduction, the enzyme is essentially a proton pumping peroxidase with a built-in auxiliary eu-oxidase module that activates oxygen and prepares in situ H2O2, a thermodynamically efficient but potentially hazardous electron acceptor for the proton pumping peroxidase. The eu-oxidase and peroxidase phases of the catalytic cycle may be performed by different structural states of COX. Resolution of the proton pumping peroxidase activity of COX and identification of individual charge translocation steps inherent in this reaction are discussed, as well as the specific role of the two input proton channels in proton translocation.     

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.     

Proton uptake controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the "peroxy" to "oxoferryl" transition, internal transfer of the fourth electron from CuA to heme a has the same rate as proton uptake from the bulk solution (8,000 s-1). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a CuA ligand, was replaced by leucine, which resulted in an increased redox potential of CuA. During reaction of the reduced mutant enzyme with O2, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s-1), whereas electron transfer from CuA to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from CuA to heme a were blocked, the results from this study show that the CuA --> heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a3-CuB before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.     

Photosynthetic electron transport involved in PxcA-dependent proton extrusion in Synechocystis sp. Strain PCC6803: effect of pxcA inactivation on CO2, HCO3-, and NO3- uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2, 5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3- uptake activity was very low in the pxcA mutant at low [Na+] (approximately 100 microM). At pH 6.5, the pxcA strain did not take up CO2 or NO3- at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3-, and NO3- is discussed.     

Decoupling of the bc1 complex in S. cerevisiae; point mutations affecting the cytochrome b gene bring new information about the structural aspect of the proton translocation

Four mutations in the mitochondrial cytochrome b of S. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e- ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Rago et al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12-42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e- ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at the bc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/2e- ratio and the transmembrane potential at the bc1 complex level. The results suggest that a partial decoupling of the bc1 complex is induced by the cytochrome b point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild type bc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.     

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

The post-translational modification in cytochrome c oxidase is required to establish a functional environment of the catalytic site

Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the CuB-cytochrome a3 binuclear catalytic site and forms a hydrogen bond with the hydroxy group on the farnesyl side chain of the heme. In addition, through a post-translational modification, Y288 is covalently linked to one of the histidine ligands that is coordinated to CuB. In the Y288F mutant enzyme, the "as-isolated" preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome a3 heme, which is largely six-coordinate low-spin in both oxidation states of the mutant, cannot be reduced by cytochrome c, but only by dithionite, possibly due to a large decrease in its reduction potential. It is postulated that the Y288F mutation prevents the post-translational modification from occurring. As a consequence, the catalytic site becomes disrupted. Thus, one role of the post-translational modification is to stabilize the functional catalytic site by maintaining the correct ligands on CuB, thereby preventing nonfunctional ligands from coordinating to the heme.     

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.     

New insights into the catalytic cycle of flavocytochrome b2

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1. Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [Ki = 30 mM; Lederer, F. (1978) Eur. J. Biochem, 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.     

Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase

Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.     

The water channel of cytochrome c oxidase: inferences from inhibitor studies

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.     

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.     

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.     

Retransplantation of patients with severe posttransplant hepatitis B in the first allograft

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.     

The mechanism of proton pumping by cytochrome c oxidasex127e comments

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the "peroxy" forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a3 propionates and are driven to the outside ("pumped") by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P --> F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F' with an alternative route to O.