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).     

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.     

Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen

Cytochrome c oxidase is a membrane-bound enzyme that catalyses the reduction of O2 to H2O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo-ferryl (P(r)-->F) and the oxo-ferryl to oxidised (F-->O) transitions. The Zn2+ binding resulted in a decrease of the F-->O rate from 820 s(-1) (no Zn2+) to a saturating value of approximately 360 s(-1) with an apparent K(D) of approximately 2.6 microM. The P(r)-->F rate (approximately 10[(4) s(-1)] before addition of Zn2+) decreased more slowly with increasing Zn2+ concentration and a K(D) of approximately 120 microM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the P(r)-->F and F-->O transitions are associated with proton uptake through the D-pathway, a Zn2+-binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co-ordinate Zn2+.     

Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase

Cytochrome c oxidase couples electron transfer from cytochrome c to O 2 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with O 2, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.     

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.     

Membrane potential-controlled inhibition of cytochrome c oxidase by zinc

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K(I) of < or = 5 microm when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. The external zinc inhibition is removed by EDTA and is not competitive with cytochrome c binding but is competitive with protons. Only Cd(2+) of the many metals tested (Mg(2+), Mn(2+), Ca(2+), Ba(2+), Li(2+), Cs(2+), Hg(2+), Ni(2+), Co(2+), Cu(2+) Tb(3+), Tm(3+)) showed inhibitory effects similar to Zn(2+). Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.     

Stopped-flow studies of cytochrome oxidase reconstituted into liposomes: proton pumping and control of activity

The transient kinetics of proton pumping and the electron transfer properties of cytochrome oxidase inserted into small unilamellar vesicles have been investigated by stopped-flow spectrophotometry. In the presence of valinomycin, proton pumping and cytochrome c oxidation by cytochrome oxidase are synchronous up to rate constants of approximately 9 sec-1. Moreover, the enzyme depleted of subunit III ("three-less oxidase") was also shown to pump protons, although with a significantly smaller stoichiometry. Thus, subunit III is not the only (or even the main) proton channel, although it may be involved in the regulation of activity. The kinetics of cytochrome c oxidation by COV in the absence and in the presence of ionophores have been investigated. Analysis of the time course of the process in the transient and steady state phases indicates that the onset of control by the electrochemical gradient follows the transfer of four electrons, i.e., one complete turnover of the oxidase. Two possible alternative interpretations for the control of the turnover phase are presented and 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.     

Inhibition of proton transfer in cytochrome c oxidase by zinc ions: delayed proton uptake during oxygen reduction

We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn(2+) the transition from the "peroxy" (P(R)) to the "ferryl" (F) intermediate has a time constant of approximately 100 micros and it is associated with proton transfer from the bulk solution with an intrinsic time constant of <100 micros, but rate limited by the P(R)-->F transition. While in the presence of 100 microM Zn(2+) the P(R)-->F transition was slowed by a factor of approximately 2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn(2+)) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of approximately 2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn(2+) was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn(2+) was observed on the F-->O transition rate when measured in D(2)O, because in D(2)O, the transition is internally slowed to approximately 10 ms, which is already slower than with bound Zn(2+). Together with earlier results showing that both the P(R)-->F and F-->O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn(2+) binds to and blocks the entrance of the D-pathway.     

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.     

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer.

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.     

Membrane potentials in reconstituted cytochrome c oxidase proteoliposomes determined by butyltriphenyl phosphonium cation distribution

Equilibration of the butyltriphenyl phosphonium (BTPP+) cation into cytochrome c oxidase reconstituted proteoliposomes was measured potentiometrically. The maximum membrane potential (delta psi) generated by oxidase activity was estimated to lie between -65 and -90 mV, vesicle interior negative, when internal BTPP+ binding is taken into account. Formation of delta psi was completely prevented by valinomycin and carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone but only 10% inhibited by levels of N',N'-dicyclohexylcarbodiimide that abolish proton pumping by the oxidase. delta psi is thus maintained by at least one charge transfer process that does not involve proton movement. A nonlinear relationship was obtained between oxidase activity and steady-state delta psi. The value of delta psi estimated by BTPP+ distribution was lower than that calculated using the optical probes safranine and a carbocyanine dye. Possible reasons for this discrepancy are discussed.     

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.     

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. .     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.     

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of < or = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)-->F3-->O4. However, proton pumping was impaired specifically during the P3-->F3, but not during the F3-->O4 transition at Zn2+ concentrations of < or = 25 microM. Furthermore, proton pumping during the P3-->F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3-->F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3-->F3 transition.     

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa(3)-type quinol oxidase from the archaeon Acidianus ambivalens and the ba(3)-type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H(+)/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba(3) oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.     

Understanding the cytochrome c oxidase proton pump: thermodynamics of redox linkage.

The cytochrome c oxidase complex (CcO) catalyzes the four-electron reduction of dioxygen to water by using electrons from ferrocytochrome c. Redox free energy released in this highly exergonic process is utilized to drive the translocation of protons across a transmembrane electrochemical gradient. Although numerous chemical models of proton pumping have been developed, few attempts have been made to explain the stepwise transfer of energy in the context of proposed protein conformational changes. A model is described that seeks to clarify the thermodynamics of the proton pumping function of CcO and that illustrates the importance of electron and proton gating to prevent the occurrence of the more exergonic electron leak and proton slip reactions. The redox energy of the CcO-membrane system is formulated in terms of a multidimensional energy surface projected into two dimensions, a nuclear coordinate associated with electron transfer and a nuclear coordinate associated with elements of the proton pump. This model provides an understanding of how a transmembrane electrochemical gradient affects the efficiency of the proton pumping process. Specifically, electron leak and proton slip reactions become kinetically viable as a result of the greater energy barriers that develop for the desired reactions in the presence of a transmembrane potential.     

Effect of membrane potential and pH gradient on electron transfer in cytochrome oxidase

Steady-state spectra of cytochrome oxidase in phospholipid vesicles were obtained by using hexaammineruthenium(II) and ascorbate as reductants. Cytochrome a was up to 80% reduced in the steady state in coupled vesicles. Upon addition of nigericin or acetate, which decrease delta pH, resulting in an increase in delta psi, cytochrome a became more oxidized in the steady state with no change in the rate of respiration. On the other hand, uncouplers or valinomycin plus nigericin, which lower both delta psi and delta pH, stimulated respiration 2-8-fold and also lowered the steady-state level of reduction of cytochrome a. These experiments indicate that electron transfer between cytochromes a and a 3 is sensitive primarily to the pH gradient. Studies with the reconstituted and the soluble enzyme at various pH values indicated that the pH on the matrix side of the membrane, rather than delta pH, controlled the steady-state level of reduced cytochrome a. Hexaammineruthenium(II) substituted for cytochrome c in measurements of proton pumping by cytochrome oxidase. Dicyclohexylcarbodiimide, which eliminated proton pumping by cytochrome oxidase, decreased the effect of ionophores on the steady-state level of reduced cytochrome a.     

The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation

In previous studies the allosteric inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP-ratios via binding of the nucleotides to the matrix domain of subunit IV was demonstrated. Here we show that the allosteric ATP-inhibition of the isolated bovine heart enzyme is switched on by cAMP-dependent phosphorylation with protein kinase A of subunits II (and/or III) and Vb, and switched off by subsequent incubation with protein phosphatase 1. It is suggested that after cAMP-dependent phosphorylation of cytochrome c oxidase mitochondrial respiration is controlled by the ATP/ADP-ratio keeping the proton motive force Deltap low, and the efficiency of energy transduction high. After Ca(2+)-induced dephosphorylation this control is lost, accompanied by increase of Deltap, slip of proton pumping (decreased H(+)/e(-) stoichiometry), and increase of the rate of respiration and ATP-synthesis at a decreased efficiency of energy transduction.