Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O(2) reduction at the haem a(3)-Cu(B) centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikstr?m, Nature 266 (1977) 271; M. Wikstr?m, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 ). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 ). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Delta-propionate of haem a(3) (pumping), or to haem a(3)-Cu(B) (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a(3)-Cu(B), which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O(2) reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O₂ reduction at the haem a₃-Cu_B centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271 [1]; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 [2]). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 [3]). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Δ-propionate of haem a₃ (pumping), or to haem a₃-Cu_B (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a₃-Cu_B, which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O₂ reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.     

Electrostatic control of proton pumping in cytochrome c oxidase

As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O2 to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.     

Solvent proton relaxation studies of cytochrome c oxidase solutions

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.     

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.     

A quantitative characterisation of H+ translocation by cytochrome c oxidase vesicles

A quantitative analysis of H+ extrusion by reconstituted cytochrome c oxidase vesicles is presented with particular regard to the decay kinetics of the extruded proton pulse and to the structural heterogeneity of the vesicle preparation. The decay of the extruded H+ pulse under conditions typical of those used for its measurement is much slower than expected from the passive proton permeability of the vesicle membranes. It is shown that this apparent anomaly results from insufficient transmembrane charge equilibration via valinomycin and K+ during oxidase turnover. This situation can be remedied by increasing the valinomycin concentration or by replacing this counterion system with 1 mM tetraphenylphosphonium. Under these latter conditions, the decay kinetics can be described as the sum of two exponential terms. To facilitate interpretation of the proton pump decay kinetics, a structural analysis of the oxidase vesicle preparation is presented. The bulk of the reconstituted vesicles (i.e., those representing approx. 80% of the total oxidase and lipid) are 30-62 nm in diameter. At least 70% of the reconstituted oxidase molecules are contained individually in separate vesicles, indicating that the enzyme monomer is competent in H+ translocation.     

Alternative hypotheses of proton ejection in cytochrome oxidase vesicles. Transmembrane proton pumping or redox-linked deprotonation of phospholipid-cytochrome c complex(es)

A review of published experimental and interpretative knowledge concerning proton ejection associated with cytochrome c oxidation by artificial phospholipid vesicles inlaid with cytochrome c oxidase indicates that the detailed characteristics of the redox-linked proton ejection cannot be simply explained by proton pumping. We propose an alternative hypothesis according to which proton ejection is due to the redox-linked deprotonation of a complex involving phospholipid and cytochrome c at the surface of the vesicles. The postulates upon which this hypothesis depends are explicitly outlined, and some methods of testing the hypothesis are suggested.     

The membrane modulates internal proton transfer in cytochrome c oxidase

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.     

Palmitate decreases proton pumping of liver-type cytochrome c oxidase.

The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.     

Gating and regulation of the cytochrome c oxidase proton pump

As a consumer of 95% of the oxygen we breathe, cytochrome c oxidase plays a major role in the energy balance of the cell. Regulation of its oxygen reduction and proton pumping activity is therefore critical to physiological function in health and disease. The location and structure of pathways for protons that are required to support cytochrome c oxidase activity are still under debate, with respect to their requirements for key residues and fixed waters, and how they are gated to prevent (or allow) proton backflow. Recent high resolution structures of bacterial and mammalian forms reveal conserved lipid and steroid binding sites as well as redox-linked conformational changes that provide new insights into potential regulatory ligands and gating modes. Mechanistic interpretation of these findings and their significance for understanding energy regulation is discussed.     

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.     

Proton translocation by cytochrome oxidase vesicles catalyzing the peroxidatic oxidation of ferrocytochrome c

Coupled with the peroxidatic oxidation of ferrocytochrome c under anaerobic conditions, proteoliposomes reconstituted with a purified preparation of bovine heart cytochrome oxidase ejected protons into the external medium with an apparent H+/e- ratio of 0.9. At the same time, protons in the intravesicular space were consumed. Dicyclohexylcarbodiimide significantly inhibited the proton translocation. Cyanide (0.14 mM) completely inhibited both the peroxidase and proton translocating activities. On the contrary, in the presence of 1 mM CO the proton ejection was abolished almost completely, but 50% of the peroxidase activity persisted. This result suggests the operation of multiple mechanisms in the peroxidase reaction and that the CO-sensitive one is coupled to the proton translocation.