The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

A Possible Role of Slips in Cytochrome C Oxidase in the Antioxygen Defense System of the Cell

Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.     

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.     

The proton pump of cytochrome c oxidase and its stoichiometry.

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikstr?m, M. F. K. (1977) Nature (Lond.) 266, 271--273].     

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. .     

Proton translocation by a native and subunit III-depleted cytochrome c oxidase reconstituted into phospholipid vesicles. Use of fluorescein-phosphatidylethanolamine as an intravesicular pH indicator

The existence of a proton pump associated with bovine cytochrome c oxidase (EC 1.9.3.1) has over the last few years been a matter of considerable dispute. In an attempt to resolve some of the problems with the measuring system we have synthesized fluorescein-phosphatidylethanolamine which when reconstituted with cytochrome c oxidase into phospholipid vesicles provided a reliable indicator of the intravesicular pH. It was observed that cytochrome c oxidase catalyzed the abstraction of almost 2 protons from the intravesicular medium/molecule of ferrocytochrome c oxidized. In parallel experiments whereby the extravesicular pH was measured with an electrode it was found that the enzyme appeared to be responsible for the appearance of almost 1.0 proton/molecule of ferrocytochrome c oxidized. Taken together these data unequivocally demonstrate that cytochrome c oxidase behaves as a proton pump. Furthermore, the other proton which was abstracted is believed to be used for the process of the reduction of oxygen. Similar experiments were performed with a cytochrome c oxidase preparation which was devoid of subunit III. Under these circumstances the enzyme appeared to be unable to translocate protons across the vesicular membrane but was competent to abstract protons from the intravesicular medium for the reduction of oxygen.     

Fungal respiration: a fusion of standard and alternative components

In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.     

Effects of detergents and cytochrome c binding on scalar and vectorial proton ejection by proteoliposomes containing cytochrome oxidase.

The detergent lauryl maltoside abolishes respiratory control and proton ejection by cytochrome c oxidase-containing proteoliposomes over a narrow concentration range. Expression of cryptic activity (inward-facing oxidase) is released over the same concentration range. Catalytic functions (Vmax. and Km) of the enzyme are not changed by the detergent. Lipid micelles containing detergent bind approximately the same amount of cytochrome c as do vesicles containing an equivalent amount of lipid. Uncoupler-insensitive proton release is seen when proteoliposomes are pulsed with ferrocytochrome c at low ionic strength. Such uncoupler-insensitive acidification is not seen at higher ionic strength, nor with oxygen pulses of anaerobic solutions previously incubated with cytochrome c. Vesicles at low ionic strength catalyse cytochrome c autoxidation; this process can mimic proton re-equilibration in systems that have pumped protons from inside to the bulk phase. Proton re-equilibration following a pulse of cytochrome c or oxygen is multiphasic. The slowest phases are attributed to vesicle heterogeneity, some internal alkali being retained within vesicles of low intrinsic proton permeability. This can be overcome by the addition of either very low levels of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or high levels of valinomycin.     

A cooperative model for proton pumping in cytochrome c oxidase

In this paper, the mechanism of proton pumping in cytochrome c oxidase is examined. Data on cooperative linkage of vectorial proton translocation to oxido-reduction of Cu(A) and heme a in the CO-inhibited, liposome-reconstituted bovine cytochrome c oxidase are reviewed. Results on proton translocation associated to single-turnover oxido-reduction of the four metal centers in the unliganded, membrane-reconstituted oxidase are also presented. On the basis of these results, X-ray crystallographic structures and spectrometric data for a proton pumping model in cytochrome c oxidase is proposed. This model, which is specifically derived from data available for the bovine cytochrome c oxidase, is intended to illustrate the essential features of cooperative coupling of proton translocation at the low potential redox site. Variants will have to be introduced for those members of the heme copper oxidase family which differ in the redox components of the low potential site and in the amino acid network connected to this site. The model we present describes in detail steps of cooperative coupling of proton pumping at the low potential Cu(A)-heme a site in the bovine enzyme. It is then outlined how this cooperative proton transfer can be thermodynamically and kinetically coupled to the chemistry of oxygen reduction to water at the high potential Cu(B)-heme a(3) center, so as to result in proton pumping, in the turning-over enzyme, against a transmembrane electrochemical proton gradient of some 250 mV.     

Explaining the enigmatic K(M) for oxygen in cytochrome c oxidase: a kinetic model

We present a mathematical model for the functioning of proton-pumping cytochrome c oxidase, consisting of cyclic conversions between 26 enzyme states. The model is based on the mechanism of oxygen reduction and linked proton translocation postulated by Wikstr?m and Verkhovsky (2007). It enables the calculation of the steady-state turnover rates and enzyme-state populations as functions of the cytochrome c reduction state, oxygen concentration, membrane potential, and pH on either side of the inner mitochondrial membrane. We use the model to explain the enigmatic decrease in oxygen affinity of the enzyme that has been observed in mitochondria when the proton-motive force is increased. The importance of the 26 transitions in the mechanism of cytochrome oxidase for the functional properties of cytochrome oxidase is compared through Metabolic Control Analysis. The control of the K(M) value is distributed mainly between the steps in the mechanism that involve electrogenic proton movements, with both positive and negative contributions. Positive contributions derive from the same steps that control enzyme turnover rate in the model. Limitations and possible further applications of the model are discussed.     

Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.     

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.     

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.     

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.     

The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase

EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn(2+) with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O-COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Neurophilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells

Photosystem II complex (PSII) of thylakoid membranes uses light energy to oxidise extremely stable water and produce oxygen (2H(2)O-->O(2)+4H(+)+4e(-)). PSII is compared with cytochrome c oxidase that catalyses the opposite reaction coupled to proton translocation. Cytochrome c oxidase has proton and water channels, and a tentative oxygen channel. I propose that functional PSII complexes also need a specific oxygen channel to direct O(2) from the water molecules bound to specific Mn atoms of the Mn cluster within PSII out to the membrane surface. The function of this channel will be to prevent oxygen being accessible to the radical pair P680(+)Pheo(-), thereby preventing singlet oxygen generation from the triplet P680 state in functional PSII. The important role of singlet oxygen in structurally perturbed non-functional photosystem II is also discussed.     

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.     

A stopped-flow study of the reaction of reduced cytochrome oxidase with oxygen

The reaction of a reduced cytochrome oxidase system consisting of beef heart cytochrome oxidase, cytochrome c, and ascorbate with molecular oxygen was kinetically and thermodynamically investigated using a stopped-flow, rapid wavelength-scanning technique. Processes for oxidation of ferrocytochrome a, bound ferrocytochrome c, and free ferrocytochrome c have been identified, and their rate constants have been determined. Values of the activation energy for these reactions indicate that the oxidation of bound ferrocytochrome c is a simple chemical electron-transfer process and that oxidations of ferrocytochrome a and free ferrocytochrome c are complex processes involving changes in protein conformation.     

Cooperativity and flexibility of the protonmotive activity of mitochondrial respiratory chain

Functional and structural data are reviewed which provide evidence that proton pumping in cytochrome c oxidase is associated with extended allosteric cooperativity involving the four redox centers in the enzyme . Data are also summarized showing that the H+/e- stoichiometry for proton pumping in the cytochrome span of the mitochondrial respiratory chain is flexible. The DeltapH component of the bulk-phase membrane electrochemical proton gradient exerts a decoupling effect on the proton pump of both the bc1 complex and cytochrome c oxidase. A slip in the pumping efficiency of the latter is also caused by high electron pressure. The mechanistic and physiological implications of proton-pump slips are examined. The easiness with which bulk phase DeltapH causes, at least above a threshold level, decoupling of proton pumping indicates that for active oxidative phosphorylation efficient protonic coupling between redox complexes and ATP synthase takes place at the membrane surface, likely in cristae, without significant formation of delocalized DeltamuH+. A role of slips in modulating oxygen free radical production by the respiratory chain and the mitochondrial pathway of apoptosis is discussed.