The mechanism of proton pumping by cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the “peroxy” forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a₃ propionates and are driven to the outside (“pumped”) by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P → F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F′ with an alternative route to O.     

The proton pumping pathway of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O(2) in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O(2) reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O(2) reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping.     

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located approximately 30 A from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from approximately 9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P --> F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.     

Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase

Molecular oxygen acts as the terminal electron sink in the respiratory chains of aerobic organisms. Cytochrome c oxidase in the inner membrane of mitochondria and the plasma membrane of bacteria catalyzes the reduction of oxygen to water, and couples the free energy of the reaction to proton pumping across the membrane. The proton-pumping activity contributes to the proton electrochemical gradient, which drives the synthesis of ATP. Based on kinetic experiments on the O–O bond splitting transition of the catalytic cycle (A → P_R), it has been proposed that the electron transfer to the binuclear iron–copper center of O₂ reduction initiates the proton pump mechanism. This key electron transfer event is coupled to an internal proton transfer from a conserved glutamic acid to the proton-loading site of the pump. However, the proton may instead be transferred to the binuclear center to complete the oxygen reduction chemistry, which would constitute a short-circuit. Based on atomistic molecular dynamics simulations of cytochrome c oxidase in an explicit membrane–solvent environment, complemented by related free-energy calculations, we propose that this short-circuit is effectively prevented by a redox-state–dependent organization of water molecules within the protein structure that gates the proton transfer pathway.Life on Earth is supported by a constant supply of energy in the form of ATP. Cytochrome c oxidase (CcO) in the respiratory chains of mitochondria and bacteria catalyzes the exergonic reduction of molecular oxygen (O₂) to water and uses the free energy of the reaction to pump protons across the membrane (1–3). The oxygen reduction reaction takes place at a highly conserved active site formed by two metal sites, heme a₃ and Cu_B (Fig. 1 A and B), called the binuclear center (BNC). The electrons donated by the mobile electron carrier cytochrome c reach the BNC via two other conserved metal centers, Cu_A and heme a (Fig. 1A). The protons required for the chemistry of O₂ reduction to water, and for proton pumping, are transported with the assistance of side chains of polar amino acids and conserved water molecules in the protein interior (4–6) (Fig. 1A). Two such proton transfer pathways have been described in the mitochondrial and bacterial A-type oxidases (to distinguish between different types of oxidases, see ref. 7), namely, the D and K channels (8, 9), the names of which are based on the conserved amino acid residues Asp91 and Lys319, respectively (Fig. 1A, amino acid numbering based on the bovine heart CcO). The D channel is responsible for the translocation of all of the pumped protons, and for the transfer of at least two of the four protons required for oxygen reduction chemistry, whereas the K channel supplies one or two protons to the BNC during the reductive phase of the catalytic cycle (8, 9). The D channel terminates at a highly conserved glutamic acid residue, Glu242, from where the protons are either transferred to the BNC for consumption, or to the proton-loading site (PLS) for pumping across the membrane (Fig. 1A). In 2003, Wikström et al. postulated a molecular mechanism in which water molecules in the nonpolar cavity above Glu242 would form proton-transferring chains, the orientation of which depends upon the redox state of the enzyme (10). They proposed that the reduction of the low-spin heme would result in transfer of a proton via a preorganized water chain from Glu242 to the d-propionate (Dprp) of the high-spin heme, whereas in the case when the electron has moved to the BNC, the water chain would reorientate and conduct protons from Glu242 to the BNC (Fig. 1A, and see below). Even though there is little direct experimental support available for such a water-gated mechanism, a recent FTIR study indeed suggests changes in water organization upon changes in the redox state of the enzyme (11). Many of the elementary steps that were postulated in the water-gated mechanism have gained support from experiments in the recent past (12, 13).Open in a separate windowFig. 1.(A) A three-subunit (SU) CcO. SU I (blue), II (red), and III (orange) are displayed as transparent ribbons. The D and K channels of proton transfer are marked with blue arrows. Crystallographic water molecules present in these proton channels are shown in purple. Electron transfer (red arrow) takes place from Cu_A (orange) via heme a (yellow) to the binuclear center comprising heme a₃ (yellow)–Cu_B (orange). Protons are transferred from Glu242 (E242) either to the PLS or to the binuclear center (black arrows). Lipid bilayer (silver lines), water (gray dots), and sodium (light yellow) and chloride (cyan) ions are also displayed. (B) The catalytic cycle of CcO. The states of heme a₃, Cu_B, and the cross-linked tyrosine are displayed. Each light orange rectangle corresponds to a state of the BNC, the name of which is displayed in red (Upper Right). Pumped protons are shown in blue, black H⁺ indicates uptake of a proton for water formation, and e⁻ indicates transfer of an electron from the low-spin heme a. Catalysis of O₂ reduction occurs clockwise.It is generally thought that the proton pump of CcO operates via the same mechanism in each of the 4 one-electron reduction steps of the catalytic cycle (Fig. 1B). However, kinetic data on two different transitions (A → P_R and O_H → E_H) have suggested dissimilarities in some of the elementary steps (12, 13). Fully reduced enzyme reacts with oxygen and forms an oxygenated adduct A in ca. 10 µs, followed by splitting of the O–O bond leading to formation of the P_R intermediate (in ∼25 μs) that is linked to loading of the PLS with a proton (3, 12). O–O bond splitting from state A in the absence of electrons in heme a or Cu_A yields the stable state P_M without proton transfer to the PLS (3, 12). Therefore, it is the electron transfer from heme a into the BNC accompanying O–O bond scission during A → P_R that is linked to the proton transfer to the PLS. The structure of the P_R intermediate is well characterized with ferryl heme a₃, cupric hydroxide, and tyrosinate (3, 14). In P_M the tyrosine is almost certainly in the form of a neutral radical (3, 14), so the reaction P_M → P_R is a proton-coupled electron transfer reaction (PCET) that initiates the reactions of the proton pump (3, 12). Note that in the state P_R the proton at the PLS partially neutralizes the electron in the BNC (3) in accordance with the charge-neutralization principle of the BNC (15). However, an important question arises: how can proton transfer from Glu242 to the BNC be prevented, which would short-circuit one step of proton pumping and form the next stable intermediate F? In the O_H → E_H transition of the catalytic cycle this short-circuit is minimized because reduction of the low-spin heme is thought to raise the pK_a of the PLS sufficiently to lead to its protonation before transfer of the electron to the BNC (3, 10, 13, 16–18), and uncompensated proton transfer to the BNC is endergonic in nature (refs. 13,16,17; cf. ref. 19). In contrast, the likelihood of a proton leak in the A → P_R transition increases manifold because the electron transfer from heme a to the BNC is required for loading of the PLS with a proton (3, 12). This facet is analyzed in the current work, and it is proposed that it is the orientation of the water molecules in the nonpolar cavity above Glu242 that effectively gates the pump and minimizes such a short-circuit.     

The proton donor for O-O bond scission by cytochrome c oxidase

Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate P(R) of the binuclear active site composed of heme a(3) and Cu(B). The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu(B). To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the P(R) intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximately 60 micros) appearance of the P(R) species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the P(R) species suggests transfer of a proton over a distance of approximately 4 A perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O(2) reduction in conditions where heme a is oxidized.     

Kinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation.

The kinetics of oxidation of reduced cytochrome c by cytochrome c oxidase reconstituted into unilamellar vesicles (COV) has been followed by stopped-flow method in the time range 3 msec-1 sec. In the presence of valinomycin, the oxidation of cytochrome c is linked to proton ejection in the external medium, with an apparent stoichiometry (H+/e-) of 0.93 +/- 0.22, under conditions in which the enzyme is in the more active "pulsed" state (i.e., having undergone oxidation-reduction cycles). The time course of reaction indicates that the conformational change(s) involved in coupling the redox reaction to proton translocation is fast. Similar experiments carried out with cytochrome c oxidase depleted of subunit III show that proton-pumping is maintained, although with a lower efficiency (H+/e- = 0.5). The number of protons ejected per electron appears to be correlated to the value of the respiratory control ratio; although this result is partly due to an increase in the rate of diffusion back into the vesicles, a relationship between the respiratory control ratio and the efficiency of the proton pump may be inferred, suggesting a control of the H+/e-ratio.     

A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized “as-isolated” form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529–533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-Å resolution by limiting the X-ray dose for each shot and by using many (≈400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O₂ reduction site (Fe³⁺-O⁻-O⁻-Cu²⁺), in contrast to a ferric hydroxide (Fe³⁺-OH⁻) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O₂ reduction site suppresses the proton pumping function.     

Proton pumping by cytochrome oxidase as studied by time-resolved stopped-flow spectrophotometry.

The H+/e- stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination of transient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alkalinization fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/e- ratio under various experimental conditions. The proton-pumping stoichiometry was found to be 1.01 +/- 0.10, independent of the number of turnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse).     

Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.

A single catalytic site model is proposed to account for the multiphasic kinetics of oxidation of ferrocytochrome c by cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). This model involves nonproductive binding of substrate to sites near the catalytic site on cytochrome c oxidase for cytochrome c, decreasing the binding constant for cytochrome c at the catalytic site. This substrate inhibition results in an increase in the first-order rate constant for the dissociation of the ferricytochrome c-cytochrome c oxidase complex, the rate-limiting step in the steady-state turnover of electrons between cytochrome c and cytochrome c oxidase in the spectrophotometric assay, yielding increases in the initial rate as well as the Michaelis constant--namely, multiple kinetic phases.     

Rapid electrostatic evolution at the binding site for cytochrome c on cytochrome c oxidase in anthropoid primates

Cytochrome c (CYC) oxidase (COX), a multisubunit enzyme that functions in mitochondrial aerobic energy production, catalyzes the transfer of electrons from CYC to oxygen and participates in creating the electrochemical gradient used for ATP synthesis. Modeling three-dimensional structural data on COX and CYC reveals that 57 of the >1,500 COX residues can be implicated in binding CYC. Because of the functional importance of the transfer of electrons to oxygen, it might be expected that natural selection would drastically constrain amino acid replacement rates of CYC and COX. Instead, in anthropoid primates, although not in other mammals, CYC and COX show markedly accelerated amino acid replacement rates, with the COX acceleration being much greater at the positions that bind CYC than at those that do not. Specifically, in the anthropoid lineage descending from the last common ancestor of haplorhines (tarsiers and anthropoids) to that of anthropoids (New World monkeys and catarrhines) and that of catarrhines (Old World monkeys and apes, including humans), a minimum of 27 of the 57 COX amino acid residues that bind CYC were replaced, most frequently from electrostatically charged to noncharged residues. Of the COX charge-bearing residues involved in binding CYC, half (11 of 22) have been replaced with uncharged residues. CYC residues that interact with COX residues also frequently changed, but only two of the CYC changes altered charge. We suggest that reducing the electrostatic interaction between COX and CYC was part of the adaptive evolution underlying the emergence of anthropoid primates.     

Monte Carlo simulations of proton pumps: on the working principles of the biological valve that controls proton pumping in cytochrome c oxidase

Gaining a detailed understanding of the proton-pumping process in cytochrome c oxidase (COX) is one of the challenges of modern biophysics. Recent mutation experiments have highlighted this challenge by showing that a single mutation (the N139D mutation) blocks the overall pumping while continuing to channel protons to the binuclear center without inhibiting the oxidase activity. Rationalizing this result has been a major problem because the mutation is quite far from E286, which is believed to serve as the branching point for the proton transport in the pumping process. In the absence of a reasonable explanation for this important observation, we have developed a Monte Carlo simulation method that can convert mutation and structural information to pathways for proton translocation and simulate the pumping process in COX on a millisecond and even subsecond time scale. This tool allows us to reproduce and propose a possible explanation to the effect of the N139D mutation and to offer a consistent model for the origin of the "valve effect" in COX, which is crucial for maintaining uphill proton pumping. Furthermore, obtaining the first structure-based simulation of proton pumping in COX, or in any other protein, indicates that our approach should provide a powerful tool for verification of mechanistic hypotheses about the action of proton transport proteins.     

Glutamic acid 242 is a valve in the proton pump of cytochrome c oxidase

Aerobic life is based on a molecular machinery that utilizes oxygen as a terminal electron sink. The membrane-bound cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water in mitochondria and many bacteria. The energy released in this reaction is conserved by pumping protons across the mitochondrial or bacterial membrane, creating an electrochemical proton gradient that drives production of ATP. A crucial question is how the protons pumped by CcO are prevented from flowing backwards during the process. Here, we show by molecular dynamics simulations that the conserved glutamic acid 242 near the active site of CcO undergoes a protonation state-dependent conformational change, which provides a valve in the pumping mechanism. The valve ensures that at any point in time, the proton pathway across the membrane is effectively discontinuous, thereby preventing thermodynamically favorable proton back-leakage while maintaining an overall high efficiency of proton translocation. Suppression of proton leakage is particularly important in mitochondria under physiological conditions, where production of ATP takes place in the presence of a high electrochemical proton gradient.     

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Despite promising mechanistic proposals, most works have not related the activation barriers of the different assumed steps to the protein structure, and there has not been a physically consistent model that reproduced the barriers needed to create a working pump. This work reevaluates the activation barriers for the primary proton transfer (PT) steps by calculations that reflect all relevant free energy contributions, including the electrostatic energies of the generated charges, the energies of water insertion, and large structural rearrangements of the donor and acceptor. The calculations have reproduced barriers that account for the directionality and sequence of events in the primary PT in CcO. It has also been found that the PT from Glu-286 (E) to the propionate of heme a(3) (Prd) provides a gate for an initial back leakage from the high pH side of the membrane. Interestingly, the rotation of E that brings it closer to Prd appears to provide a way for blocking competing pathways in the primary PT. Our study elucidates and quantifies the nature of the control of the directionality in the primary PT in CcO and provides instructive insight into the role of the water molecules in biological PT, showing that "bridges" of several water molecules in hydrophobic regions present a problem (rather than a solution) that is minimized in the primary PT.     

Gating of proton and water transfer in the respiratory enzyme cytochrome c oxidase

The membrane-bound enzyme cytochrome c oxidase is responsible for cell respiration in aerobic organisms and conserves free energy from O2 reduction into an electrochemical proton gradient by coupling the redox reaction to proton-pumping across the membrane. O2 reduction produces water at the bimetallic heme a3/CuB active site next to a hydrophobic cavity deep within the membrane. Water molecules in this cavity have been suggested to play an important role in the proton-pumping mechanism. Here, we show by molecular dynamics simulations that the conserved arginine/heme a3 delta-propionate ion pair provides a gate, which exhibits reversible thermal opening that is governed by the redox state and the water molecules in the cavity. An important role of this gate in the proton-pumping mechanism is supported by site-directed mutagenesis experiments. Transport of the product water out of the enzyme must be rigidly controlled to prevent water-mediated proton leaks that could compromise the proton-pumping function. Exit of product water is observed through the same arginine/propionate gate, which provides an explanation for the observed extraordinary spatial specificity of water expulsion from the enzyme.     

A plausible two-state model for cytochrome c oxidase.

The catalytic properties of pulsed and resting cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1), expressed in terms of a minimal kinetic scheme and simulated by numerical computations, were successfully described. A two-state model, in which the relative amounts of the enzyme present in each conformation are regulated by the rates of electron flux and O2 binding on one side and the interconversion rates on the other, accounts for the activation of cytochrome c oxidase during turnover.     

Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼10⁴ s⁻¹) can be restored in the Asp-132–Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139–Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn²⁺ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn²⁺ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139–Thr/Asp-132–Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.     

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by (1)H-(15)N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field (1)H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by (1)H- and (1)H, (15)N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.     

Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2, 2'-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (tau(1) approximately 20 micros and tau(2) approximately 175 micros) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, tau(2) is increased to 900 micros. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of approximately 1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of tau(3) approximately 2 ms and tau(4) approximately 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.