The role of the D- and K-pathways of proton transfer in the function of the haem-copper oxidases

The X-ray structures of several haem-copper oxidases now at hand have given important constraints on how these enzymes function. Yet, dynamic data are required to elucidate the mechanisms of electron and proton transfer, the activation of O(2) and its reduction to water, as well as the still enigmatic mechanism by which these enzymes couple the redox reaction to proton translocation. Here, some recent observations will be briefly reviewed with special emphasis on the functioning of the so-called D- and K-pathways of proton transfer. It turns out that only one of the eight protons taken up by the enzyme during its catalytic cycle is transferred via the K-pathway. The D-pathway is probably responsible for the transfer of all other protons, including the four that are pumped across the membrane. The unique K-pathway proton may be specifically required to aid O-O bond scission by the haem-copper oxidases.     

Proton translocation by cytochrome c oxidase: a rejoinder to recent criticism

Ten years ago, intermediate reaction steps in the catalytic cycle of cytochrome c oxidase were titrated with phosphorylation potential in isolated mitochondria, and the results were interpreted as evidence for thermodynamic linkage of proton translocation exclusively to the oxidative reaction steps of the catalytic cycle [Wikstr?m, M. (1989) Nature 338, 776-778]. Michel has recently argued that this work was flawed, and proposed a mechanism in which one of the four steps of proton translocation is linked to the reductive phase of the catalytic cycle [Michel, H. (1999) Biochemistry 38, 15129-15140]. Here, the original data are scrutinized and related to information that has accumulated since this work was published. The analysis shows that the main conclusions from this work still hold. Michel's mechanism of proton translocation is briefly discussed, and found to be at odds with some experimental observations.     

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.     

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.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the "prepumped" proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different Em values of the electron acceptor in the binuclear site.     

Cytochrome c Oxidase as a Proton-Pumping Peroxidase: Reaction Cycle and Electrogenic Mechanism

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

Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy-coupled interconversion

Several issues relevant to the current studies of cytochrome c oxidase catalytic mechanism are discussed. The following points are raised. (1) The terminology currently used to describe the catalytic cycle of cytochrome oxidase is outdated and rather confusing. Presumably, it would be revised so as to share nomenclature of the intermediates with other oxygen-reactive heme enzymes like P450 or peroxidases. (2) A "catalytic cycle" of cytochrome oxidase involving complete reduction of the enzyme by 4 electrons followed by oxidation by O(2) is a chimera composed artificially from two partial reactions, reductive and oxidative phases, that never operate together as a true multi-turnover catalytic cycle. The 4e(-) reduction-oxidation cycle would not serve a paradigm for oxygen reduction mechanism and protonmotive function of cytochrome oxidase. (3) The foremost role of the K-proton channel in the catalytic cycle may consist in securing faultless delivery of protons for heterolytic O-O bond cleavage in the oxygen-reducing site, minimizing the danger of homolytic scission reaction route. (4) Protonmotive mechanism of cytochrome oxidase may vary notably for the different single-electron steps in the catalytic cycle.     

Filling the catalytic site of cytochrome c oxidase with electrons. Reduced CuB facilitates internal electron transfer to heme a3

In the reductive phase of its catalytic cycle, cytochrome c oxidase receives electrons from external electron donors. Two electrons have to be transferred into the catalytic center, composed of heme a(3) and Cu(B), before reaction with oxygen takes place. In addition, this phase of catalysis appears to be involved in proton translocation. Here, we report for the first time the kinetics of electron transfer to both heme a(3) and Cu(B) during the transition from the oxidized to the fully reduced state. The state of reduction of both heme a(3) and Cu(B) was monitored by a combination of EPR spectroscopy, the rapid freeze procedure, and the stopped-flow method. The kinetics of cytochrome c oxidase reduction by hexaamineruthenium under anaerobic conditions revealed that the rate-limiting step is the initial electron transfer to the catalytic site that proceeds with apparently identical rates to both heme a(3) and Cu(B). After Cu(B) is reduced, electron transfer to oxidized heme a(3) is enhanced relative to the rate of entry of the first electron.     

Proton pumping by cytochrome oxidase: progress, problems and postulates

The current status of our knowledge about the mechanism of proton pumping by cytochrome oxidase is discussed. Significant progress has resulted from the study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proton translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative substrate provides a very useful experimental tool to explore the enzymology of this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transfer steps during turnover. It is proposed that the initial step in the reaction involves a proton transfer to the active site that is important to convert metal-ligated hydroxide to water, which can more rapidly dissociate from the metals and allow the reaction with dioxygen which, we propose, can bind the one-electron reduced heme-copper center. Coordinated movement of protons and electrons over both short and long distances within the enzyme appear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl radical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons.     

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. .     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

Cytochrome c oxidase: 25 years of the elusive proton pump

Since its discovery [Nature 266 (1977) 271], the function of cytochrome c oxidase (and other haem-copper oxidases) as a redox-driven proton pump has been subject of both intense research and controversy, and is one of the key unsolved issues of bioenergetics and of biochemistry more generally. Despite the fact that the mechanism of proton translocation is not yet fully understood on the molecular level, many important details and principles have been learned. In the hope of accelerating progress, some of these will be reviewed here, together with a brief presentation of a novel proton pump mechanism, and of the emergence of a molecular basis for control of its efficiency.     

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.     

Proton pathways, ligand binding and dynamics of the catalytic site in haem-copper oxygen reductases: a comparison between the three families

Haem-copper oxygen reductases are the widest spread enzymes involved in aerobic respiratory chains, in Eukarya, Bacteria and Archaea. However, both the catalytic mechanism for oxygen reduction and its coupling to proton translocation remain to be fully understood. In this article we analyse the experimental data gathered in recent years for haem-copper reductases presenting features distinct from the mitochondrial-like enzymes. These features further support the classification of several families of haem-copper oxygen reductases based on their proton pathways and previously proposed by us [Biochim. Biophys. Acta 1505 (2001) 185], and allow to identify the minimal essential elements for these enzymes.     

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).     

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.     

Transmembrane charge separation during the ferryl-oxo -> oxidized transition in a nonpumping mutant of cytochrome c oxidase

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).     

Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides with a view to evaluating the role of this residue in redox-linked proton translocation. The mutation E286D did not have any dramatic effects on enzyme properties and retained 50% of wild-type catalytic activity. For E286Q a fraction of the binuclear center was trapped in an unreactive, spectrally distinct form which is most likely due to misfolded protein, but the majority of E286Q reacted normally with formate and cyanide in the oxidized state, and with carbon monoxide and cyanide in the dithionite-reduced form. The mutation also had little effect on the pH-dependent redox properties of haem a in the reactive fraction. However, formation of the P state from oxidized enzyme with hydrogen peroxide or by aerobic incubation with carbon monoxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic steady state in the presence of ferrous cytochrome c was characterized by essentially fully reduced haem a and ferric haem a3, suggesting that the mutation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a3 following anaerobiosis, there was no indication of accumulation of significant amounts of P state. We propose that the glutamate is implicated in several steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.     

Concerted involvement of cooperative proton-electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu(A) and heme a3/Cu(B) are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e- cooperative linkage at Fe(a3)/Cu(B) is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e- linkage at heme a (and Cu(A)). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu(A) and heme a3/Cu(B) in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R-->O transition), reductive (O-->R transition) and a full catalytic cycle (R-->O-->R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e- linkage at heme a/Cu(A) and heme a3/Cu(B) with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.     

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.