The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

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.     

Kinetics of reduction of cytochrome c oxidase by dithionite and the effect of hydrogen peroxide

The reduction of cytochrome c oxidase by dithionite was reinvestigated with a flow-flash technique and with varied enzyme preparations. Since cytochrome a3 may be defined as the heme in oxidase which can form a photolabile CO adduct in the reduced state, it is possible to follow the time course of cytochrome a3 reduction by monitoring the onset of photosensitivity. The onset of photosensitivity and the overall rate of heme reduction were compared for Yonetani and Hartzell-Beinert preparations of cytochrome c oxidase and for the enzyme isolated from blue marlin and hammerhead shark. For all of these preparations the faster phase of heme reduction, which is dithionite concentration-dependent, is almost completed when the fraction of photosensitive material is still small. We conclude that cytochrome a3 in the resting enzyme is consistently reduced by an intramolecular electron transfer mechanism. To determine if this is true also for the pulsed enzyme, we examined the time course of dithionite reduction of the peroxide complex of the pulsed enzyme. It has been previously shown that pulsed cytochrome c oxidase can interact with H2O2 and form a stable room temperature peroxide adduct (Bickar, D., Bonaventura, J., and Bonaventura, C. (1982) Biochemistry 21, 2661-2666). Rather complex kinetics of heme reduction are observed when dithionite is added to enzyme preparations that contain H2O2. The time courses observed provide unequivocal evidence that H2O2 can, under these conditions, be used by cytochrome c oxidase as an electron acceptor. Experiments carried out in the presence of CO show that a direct dithionite reduction of cytochrome a3 in the peroxide complex of the pulsed enzyme does not occur.     

Effect of pH on the spectrum of cytochrome c oxidase hydrogen peroxide complex

Hydrogen peroxide binding to ferric cytochrome c oxidase in proteoliposomes brings about a red-shift of the enzyme Soret band and increased absorption in the visible range with two prominent peaks at approx. 570 and 607 nm. The molar absorptivity of the H2O2-induced difference spectrum is virtually pH-independent in the Soret band and at 570 nm, whereas the peak at 607 nm increases approx. 3-fold upon alkalinization in a narrow pH range 6.0-7.2, the effect being reversible. The pH profile of this transition indicates ionization of two acid-base groups with close pK values of 6.7. The lineshape of the peroxide compound difference spectrum is found to respond to pH changes inside the proteoliposomes. It is suggested that peroxide-complexed enzyme can undergo a pH-dependent transition to a form with increased extinction at 605-607 nm, possibly corresponding to the 420 nm (or 'pulsed') conformer of the ferric cytochrome oxidase formed as an early product of the enzyme oxidation. Accordingly, relaxation of the '420 nm' form to the resting state would be linked to an uptake of two protons from the M-aqueous phase. This protolytic reaction might be a partial step of the cytochrome oxidase proton pumping mechanism or it could serve to regulate interconversion between the active 'pulsed' and less active 'resting' states of the enzyme in the membrane.     

Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide.

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a process which is accompanied by the pumping of four protons across the membrane. Elucidation of the structures of intermediates in these processes is crucial for understanding the mechanism of oxygen reduction. In the work presented here, the reaction of H(2)O(2) with the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical based on comparison with other paramagnetic centers in COX was approximately 20%. Recent crystallographic data have shown that one of the Cu(B) ligands, His 276 (in the bacterial case), is cross-linked to Tyr 280 and that this cross-linked tyrosine is ideally positioned to participate in dioxygen activation. Here selectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed arise from a tyrosine radical species. It would seem also quite possible that this radical is an intermediate in the mechanism of oxygen reduction.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.     

Stoichiometry of carbon monoxide binding by cytochrome c oxidase.

The stoichiometry of carbon monoxide binding to beef heart cytochrome c oxidase has been reinvestigated both by titration of the reduced oxidase with CO and by measuring the amount of carboxyhemoglobin that is formed after adding oxyhemoglobin to a solution of the CO-enzyme complex. In the titration experiments the ratio of CO bounds to total heme a present was always less than 0.50 while in the experiments where oxyhemoglobin was added the results were variable and of lower accuracy. These observations do not agree with the recent conclusion of Volpe, J.A., O'Toole, M.C., and Caughey, W.S. (1975) Biochem. Biophys. Res. Commun. 62, 48-53 that CO is bound in a 1:1 ratio with heme a. An explanation for their results is suggested.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

Proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach

Cytochrome c oxidase is an efficient energy transducer that reduces oxygen to water and converts the released chemical energy into an electrochemical membrane potential. As a true proton pump, cytochrome c oxidase translocates protons across the membrane against this potential. Based on a wealth of experiments and calculations, an increasingly detailed picture of the reaction intermediates in the redox cycle has emerged. However, the fundamental mechanism of proton pumping coupled to redox chemistry remains largely unresolved. Here we examine and extend a kinetic master-equation approach to gain insight into redox-coupled proton pumping in cytochrome c oxidase. Basic principles of the cytochrome c oxidase proton pump emerge from an analysis of the simplest kinetic models that retain essential elements of the experimentally determined structure, energetics, and kinetics, and that satisfy fundamental physical principles. The master-equation models allow us to address the question of how pumping can be achieved in a system in which all reaction steps are reversible. Whereas proton pumping does not require the direct modulation of microscopic reaction barriers, such kinetic gating greatly increases the pumping efficiency. Further efficiency gains can be achieved by partially decoupling the proton uptake pathway from the active-site region. Such a mechanism is consistent with the proposed Glu valve, in which the side chain of a key glutamic acid shuttles between the D channel and the active-site region. We also show that the models predict only small proton leaks even in the absence of turnover. The design principles identified here for cytochrome c oxidase provide a blueprint for novel biology-inspired fuel cells, and the master-equation formulation should prove useful also for other molecular machines. .     

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Reaction of hydrogen peroxide and peroxidase activity in carboxymethylated cytochrome c: spectroscopic and kinetic studies

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H(2)O(2) proceeds through only one intermediate, compound I. The apparent rate constant (k(app)) for the reaction was found to be 17, 72 and 210 M(-1) s(-1) at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M(-1) s(-1) at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H(2)O(2)] s(-1)) is found to be larger than the corresponding value for native cytc (50 [H(2)O(2)] s(-1)) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H(2)O(2), and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 microM min(-1) mg(-1) at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (approximately 100%, approximately 50%, approximately 44%), HW (approximately 0%, approximately 44%, 41%) and 5C (approximately 0%, approximately 6%, 15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc.     

The oxidation of cytochrome c peroxidase by hydrogen peroxide. Characterization of products.

H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced.     

Role of the K-channel in the pH-dependence of the reaction of cytochrome c oxidase with hydrogen peroxide

The reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides with hydrogen peroxide has been studied at alkaline (pH 8.5) and acidic (pH 6.5) conditions with the aid of a stopped-flow apparatus. Absorption changes in the entire 350-800 nm spectral range were monitored and analyzed by a global fitting procedure. The reaction can be described by the sequential formation of two intermediates analogous to compounds I and II of peroxidases: oxidized COX + H2O2 --> intermediate I --> intermediate II. At pH as high as 8.5, intermediate I appears to be a mixture of at least two species characterized by absorption bands at approximately 607 nm (P607) and approximately 580 nm (F-I580) that rise synchronously. At acidic pH (6.5), intermediate I is represented mainly by a component with an alpha-peak around 575 nm (F-I575) that is probably equivalent to the so-called F* species observed with the bovine COX. The data are consistent with a pH-dependent reaction branching at the step of intermediate I formation. To get further insight into the mechanism of the pH-dependence, the peroxide reaction was studied using two mutants of the R. sphaeroides oxidase, K362M and D132N, that block, respectively, the proton-conducting K- and D-channels. The D132N mutation does not affect significantly the Ox --> intermediate I step of the peroxide reaction. In contrast, K362M replacement exerts a dramatic effect, eliminating the pH-dependence of intermediate I formation. The data obtained allow us to propose that formation of the acidic form of intermediate I (F-I575, F*) requires protonation of some group at/near the binuclear site that follows or is concerted with peroxide binding. The protonation involves specifically the K-channel. Presumably, a proton vacancy can be generated in the site as a consequence of the proton-assisted heterolytic scission of the O-O bond of the bound peroxide. The results are consistent with a proposal [Vygodina, T. V., Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998) Biochemistry 37, 3053-3061] that the K-channel may be involved in the delivery of the first four protons in the catalytic cycle (starting from reduction of the oxidized form) including proton uptake coupled to reduction of the binuclear site and transfer of protons driven by cleavage of the dioxygen O-O bond in the binculear site. Once peroxide intermediate I has been formed, generation of a strong oxene ligand at the heme a3 iron triggers a transition of the enzyme to the "peroxidase conformation" in which the K-channel is closed and the binuclear site becomes protonically disconnected from the bulk aqueous phase.