The reactions of hydrogen peroxide with bovine cytochrome c oxidase

Oxidised cytochrome c oxidase is known to react with two molecules of hydrogen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' species. These are widely used as model compounds for the equivalent P and F intermediates of the catalytic cycle. However, kinetic analysis of the reaction with H(2)O(2) in the pH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reaction with a single H(2)O(2). This species, termed F(&z.rad;), is spectrally similar, but not identical, to F. The reactions are equivalent to those previously reported for the bo type quinol oxidase from Escherichia coli (T. Brittain, R.H. Little, C. Greenwood, N.J. Watmough, FEBS Lett. 399 (1996) 21-25) where it was proposed that F(&z.rad;) is produced directly from P. However, in the bovine oxidase F(&z.rad;) does not appear in samples of the 607-nm form, P(M), produced by CO/O(2) treatment, even at low pH, although this form is shown to be identical to the H(2)O(2)-derived P state, P(H), on the basis of spectral characteristics and kinetics of reaction with H(2)O(2). Furthermore, lowering the pH of a sample of P(M) or P(H) generated at high pH results in F(&z.rad;) formation only on a minutes time scale. It is concluded that P and F(&z.rad;) are not in a rapid, pH-dependent equilibrium, but instead are formed by distinct pathways and cannot interconvert in a simple manner, and that the crucial difference between them lies in their patterns of protonation.     

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.     

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.     

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.     

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.     

Reaction of hydrogen peroxide with the rapid form of resting cytochrome oxidase.

The hydrogen peroxide binding reaction has been examined with alkaline-purified resting enzyme in order to avoid mixtures of low pH induced fast and slow conformers. At pH 8.8-9.0 (20 degrees C), the reactivity of resting enzyme was similar to the peroxide-free, pulsed conformer that has been characterized by other investigators. The reaction showed single-phase reactivity at 435 and 655 nm and required a minimum 8:1 molar excess of peroxide (over cytochrome a3) for quantitative reaction. At 16:1, the Soret band was stable for 1.0-1.5 h, but above 80:1, the band began showing generalized attenuation within 1-2 min. The peroxide binding reaction was also associated with an increase in absorbance at 606 nm which correlated with the rate of change at 435 and 655 nm. The observed rate constants at each of these wavelengths showed similar linear dependence on peroxide concentration, giving an average bimolecular rate constant of 391 M-1.s-1 and a Kd of 5.1 microM. The rise phase at 606 nm was observed to saturate at an 8:1 molar excess of peroxide but showed a slow, concentration-dependent first-order decay that gave a bimolecular rate constant and Kd of 38 M-1.s-1 and 20 microM, respectively. The decay was not associated with a change in the Soret absorption or charge-transfer regions, suggesting a type of spectral decoupling. An isosbestic point at 588 nm was consistent with the 606- to 580-nm conversion proposed by other investigators, although direct observation of a new band at 580 nm was difficult.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Calcium-bound structure of bovine cytochrome c oxidase

The crystal structure of bovine cytochrome c oxidase (CcO) shows a sodium ion (Na⁺) bound to the surface of subunit I. Changes in the absorption spectrum of heme a caused by calcium ions (Ca²⁺) are detected as small red shifts, and inhibition of enzymatic activity under low turnover conditions is observed by addition of Ca²⁺ in a competitive manner with Na⁺. In this study, we determined the crystal structure of Ca²⁺-bound bovine CcO in the oxidized and reduced states at 1.7 Å resolution. Although Ca²⁺ and Na⁺ bound to the same site of oxidized and reduced CcO, they led to different coordination geometries. Replacement of Na⁺ with Ca²⁺ caused a small structural change in the loop segments near the heme a propionate and formyl groups, resulting in spectral changes in heme a. Redox-coupled structural changes observed in the Ca²⁺-bound form were the same as those previously observed in the Na⁺-bound form, suggesting that binding of Ca²⁺ does not severely affect enzymatic function, which depends on these structural changes. The relation between the Ca²⁺ binding and the inhibitory effect during slow turnover, as well as the possible role of bound Ca²⁺ are discussed.     

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.     

Oxidative modification of cytochrome c by hydrogen peroxide

Oxidative alteration of mitochondrial cytochrome c has been linked to disease and is one of the causes of pro-apoptotic events. We have investigated the modification of cytochrome c by H2O2. When cytochrome c was incubated with H2O2, oligomerization of the protein increased and the formation of carbonyl derivatives and dityrosine was stimulated. Radical scavengers prevented these effects suggesting that free radicals are implicated in the H2O2-mediated oligomerization. Oligomerization was significantly inhibited by the iron chelator, deferoxamine. During incubation of deoxyribose with cytochrome c and H2O2, damage to the deoxyribose occurred in parallel with the release of iron from cytochrome c. When cytochrome c that had been exposed to H2O2 was analyzed by amino acid analysis, the tyrosine, histidine and methionine residues proved to be particularly sensitive. These results suggest that H2O2-mediated cytochrome c oligomerization is due to oxidative damage resulting from free radicals generated by a combination of the peroxidase activity of cytochrome c and the Fenton reaction of free iron released from the oxidatively-damaged protein.     

Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans

Determination of the three-dimensional structure of cytochrome c oxidase, the terminal enzyme of the respiratory chain, from Paracoccus denitrificans offers the possibility of site-directed mutagenesis studies to investigate the relationship between the structure and the catalytic function of the enzyme. The mechanism of electron-coupled proton transfer is still, however, poorly understood. The P(M) intermediate of the catalytic cycle is an oxoferryl state the generation of which requires one additional electron, which cannot be provided by the two metal centers. It is suggested that the missing electron is donated to this binuclear site by a tyrosine residue that forms a radical species, which can then be detected in both the P(M) and F(*) intermediates of the catalytic cycle. One possibility to produce P(M) and F(*) intermediates artificially in cytochrome c oxidase is the addition of hydrogen peroxide to the fully oxidized enzyme. Using electron paramagnetic resonance (EPR) spectroscopy, we assign a radical species detected in this reaction to a tyrosine residue. To address the question, which tyrosine residue is the origin of the radical species, several tyrosine variants of subunit I are investigated. These variants are characterized by their turnover rates, as well as using EPR and optical spectroscopy. From these experiments, it is concluded that the origin of the radical species appearing in P(M) and F(*) intermediates produced with hydrogen peroxide is tyrosine 167. The significance of this finding for the catalytic function of the enzyme is discussed.     

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.