The cytochrome c oxidase from Paracoccus denitrificans does not change the metal center ligation upon reduction

Cytochrome c oxidase catalyzes the reduction of oxygen to water. This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping"). The mechanism of proton pumping is still a matter of debate. Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase. Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K. No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed. The three histidine Cu(B) ligands are well defined in the oxidized and in the reduced states. These results are hardly compatible with the "histidine cycle" mechanisms formulated previously.     

Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: ferryl and oxy-ferrous species dominate

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.     

Cytochrome c oxidase: charge translocation coupled to single-electron partial steps of the catalytic cycle

The paper presents a survey of time-resolved studies of charge translocation by cytochrome c oxidase coupled to transfer of the 1st, 2nd 3rd and 4th electrons in the catalytic cycle. Single-electron photoreduction experiments carried out with the A-class cytochrome c oxidases of aa(3) type from mitochondria, Rhodobacter sphaeroides and Paracoccus denitrificans as well as with the ba(3)-type oxidase from Thermus thermophilus indicate that the protonmotive mechanisms, although similar, may not be identical for different partial steps in the same enzyme species, as well as for the same single-electron transition in different oxidases. The pattern of charge translocation coupled to transfer of a single electron in the A-class oxidases confirms major predictions of the original model of proton pumping by cytochrome oxidase [Artzatbanov, V. Y., Konstantinov, A. A. and Skulachev, V.P. "Involvement of Intramitochondrial Protons in Redox Reactions of Cytochrome a." FEBS Lett. 87: 180-185]. The intermediates and partial electrogenic steps observed in the single-electron photoreduction experiments may be very different from those observed during oxidation of the fully reduced oxidase by O(2) in the "flow-flash" studies. .     

Why is the reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR) not electrogenic?

The membrane-bound enzyme cNOR (cytochrome c dependent nitric oxide reductase) catalyzes the reduction of NO in a non-electrogenic process. This is in contrast to the reduction of O₂ in cytochrome c oxidase (CcO), the other member of the heme-copper oxidase family, which stores energy by the generation of a membrane gradient. This difference between the two enzymes has not been understood, but it has been speculated to be of kinetic origin, since per electron the NO reduction is more exergonic than the O₂ reduction, and the energy should thus be enough for an electrogenic process. However, it has not been clear how and why electrogenicity, which mainly affects the thermodynamics, would slow down the very exergonic NO reduction. Quantum chemical calculations are used to construct a free energy profile for the catalytic reduction of NO in the active site of cNOR. The energy profile shows that the reduction of the NO molecules by the enzyme and the formation of N₂O are very exergonic steps, making the rereduction of the enzyme endergonic and rate-limiting for the entire catalytic cycle. Therefore the NO reduction cannot be electrogenic, i.e. cannot take electrons and protons from the opposite sides of the membrane, since it would increase the endergonicity of the rereduction when the gradient is present, thereby increasing the rate-limiting barrier, and the reaction would become too slow. It also means that proton pumping coupled to electron transfer is not possible in cNOR. In CcO the corresponding rereduction of the enzyme is very exergonic.     

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.     

Understanding the mechanism of proton movement linked to oxygen reduction in cytochrome c oxidase: lessons from other proteins

Cytochrome c oxidase is a large intrinsic membrane protein designed to use the energy of electron transfer and oxygen reduction to pump protons across a membrane. The molecular mechanism of the energy conversion process is not understood. Other proteins with simpler, better resolved structures have been more completely defined and offer insight into possible mechanisms of proton transfer in cytochrome c oxidase. Important concepts that are illustrated by these model systems include the ideas of conformational change both close to and at a distance from the triggering event, and the formation of a transitory water-linked proton pathway during a catalytic cycle. Evidence for the applicability of these concepts to cytochrome c oxidase is discussed.     

Carbon monoxide-driven reduction of ferric heme and heme proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.     

Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.     

Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain

Antimycin, a specific and highly potent inhibitor of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain, does not inhibit reduction of cytochrome c1 by succinate in isolated succinate-cytochrome c reductase complex under conditions where the respiratory chain complex undergoes one oxidation-reduction turnover. If a slight molar excess of cytochrome c is added to the isolated reductase complex in the presence of antimycin, there is rapid reduction of one equivalent of c type cytochrome by succinate, after which reduction of the remaining c type cytochrome is inhibited. Antimycin fully inhibits succinate-cytochrome c reductase activity of isolated succinate-cytochrome c reductase complex in which the b-c1 complex undergoes multiple turnovers in a catalytic fashion. In addition, when antimycin is added to isolated reductase complex in the presence of cytochrome c plus cytochrome c oxidase, the inhibitor causes a "crossover" in the steady state level of reduction of the cytochromes b and c1 comparable to this classical effect in mitochondria. On the basis of these results, it is suggested that linear schemes of electron transfer are not adequate to account for the site of antimycin inhibition and the mechanism of electron transfer in the cytochrome b-c1 segment of the respiratory chain. The effects of antimycin are consistent with cyclic electron transfer mechanisms such as the protonmotive Q cycle.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy.

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.     

Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O(2) to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O(2) reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O(2) reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O(2) reduction, but a decrease in the apparent pK(a) of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.     

Functional intermediates in the reaction of membrane-bound cytochrome oxidase with oxygen.

Flash photolysis of the membrane-bound cytochrome oxidase/carbon monoxide compound in the presence of oxygen at low temperatures and in the frozen state leads to the formation of three types of intermediates functional in electron transfer in cytochrome oxidase and reduction of oxygen by cytochrome oxidase. The first category (A) does not involve electron transfer to oxygen between -125 degrees and -105 degrees, and includes oxy compounds which are spectroscopically similar for the completely reduced oxidase (Cu1+alpha3(2+)-O2) or for the ferricyanide-pretreated oxidase (Cu2+alpha3(3+)-O2). Oxygen is readily dissociated from compounds of type A. The second category (B) involves oxidation of the heme and the copper moiety of the reduced oxidase to form a peroxy compound (Cu2+alpha 3(3+)-O2=or Cu2+alpha3(2+)-O2H2) in the temperature range from -105 degrees to -60 degrees. Above -60 degrees, compounds of type B serve as effective electron acceptors from cytochromes a, c, and c1. The third category (C) is formed above -100 degrees from mixed valency states of the oxidase obtained by ferricyanide pretreatment, and may involve higher valency states of the heme iron (Cu2+alpha3(4+)-O2=). These compounds act as electron acceptors for the respiratory chain and as functional intermediates in oxygen reduction. The remarkable features of cytochrome oxidase are its highly dissociable "oxy" compound and its extremely effective electron donor reaction which converts this rapidly to tightly bound reduced oxygen and oxidized oxidase.     

Inhibition of proton pumping in membrane reconstituted bovine heart cytochrome c oxidase by zinc binding at the inner matrix side

A study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn(2+) resulted in 50% decoupling of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn(2+) of proton release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn(2+) suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH(-) produced in the reduction of O(2) at the binuclear center are protonated to 2 H(2)O. This decoupling effect could be associated with Zn(2+) induced conformational alteration of an acid/base cluster linked to heme a(3).     

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

Exposure of bovine cytochrome c oxidase to high triton X-100 or to alkaline conditions causes a dramatic change in the rate of reduction of compound F.

The final step in the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a(3) in compound F, was investigated using a binuclear polypyridine ruthenium complex ([Ru(bipyridine)(2)](2)(1,4-bis[2-(4'-methyl-2, 2'-bipyrid-4-yl)ethenyl]benzene)(PF(6))(4)) as a photoactive reducing agent. In the untreated dimeric enzyme, the rate constant for reduction of compound F decreased from 700 s(-1) to 200 s(-1) as the pH was increased from 7.5 to 9.5. Incubation of dimeric enzyme at pH 10 led to an increase in the rate constant to 1650 s(-1), which was independent of pH between pH 7.4 and 10. This treatment resulted in a decrease in the sedimentation coefficient consistent with the irreversible conversion of the enzyme to a monomeric form. Similar results were obtained when the enzyme was incubated with Triton X-100 at pH 8.0. These treatments, which have traditionally been used to convert dimeric enzyme to monomeric form, have no effect on the steady-state activity. The data indicate that either the conversion of the bovine oxidase to a monomeric form or some structural change coincident with this conversion strongly influences the rate constant of this step in the catalytic cycle, perhaps by influencing the proton access to the heme-copper binuclear center.     

Cytochrome c oxidase: catalytic cycle and mechanisms of proton pumping--a discussion

Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water, a process in which four electrons, four protons, and one molecule of oxygen are consumed. The reaction is coupled to the pumping of four additional protons across the membrane. According to the currently accepted concept, the pumping of all four protons occurs after the binding of oxygen to the reduced enzyme and is exclusively coupled to the last two electron transfer steps. A careful analysis of the existing data shows that there is no experimental evidence for this paradigm. It is more likely that only three protons are pumped during the second half of the catalytic cycle of cytochrome c oxidase after the reaction with oxygen. In this article a variant of a recent mechanistic model of proton pumping by electrostatic repulsion is discussed. It is based on the electroneutrality principle in a way that in the catalytic cycle each electron transfer to the membrane-embedded electron acceptors is charge-compensated by uptake of one proton. The mechanism takes into account the findings with mutant cytochrome c oxidases and explains the results of many recent experiments, including the effects of hydrogen peroxide.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

The Alternative Oxidase: in vivo Regulation and Function

Abstract: This review focuses on the biochemical regulation and function of the alternative oxidase in vivo. About 10 years ago, two activation mechanisms were discovered in isolated mitochondria, namely activation by reducing sulfur bonds in the protein and activation by an allosteric effect of pyruvate. It was proposed that plants would have a regulatory mechanism to modify alternative oxidase activity in vivo. However, more recent studies have shown that these two activation mechanisms may not play such an important role in regulation of alternative oxidase activity in vivo after all. Pyruvate and reduction of the sulfide bonds in the protein are definitely required for alternative oxidase activity, but they do not appear to be regulating the activity in vivo.
Despite the energy wasting nature of the alternative oxidase, there was no obvious physiological function for the pathway for many years. It is now more clear that the alternative oxidase can prevent the production of excess reactive oxygen species radicals by stabilizing the redox state of the mitochondrial ubiquinone pool, while allowing continued activity of the citric acid cycle. This may be important under conditions when the NADH supply is relatively high (reductant overflow), or when the cytochrome pathway is restrained. The cytochrome pathway might be inhibited by naturally occurring cyanide, nitric oxide, sulfide, high concentrations of CO₂, low temperatures, or by limited phosphate supply.