A cytochrome c oxidase proton pumping mechanism that excludes the O2 reduction site

A redox-coupled conformational change in Asp51 of subunit I and a hydrogen-bond network connecting Asp51 with the matrix surface have been deduced from X-ray structures of bovine heart cytochrome c oxidase. This has provided evidence that Asp51 may play a role in the proton pumping process. However, the lack of complete conservation of a residue analogous to Asp51, the inclusion of a peptide bond in the hydrogen-bonding network and the lack of apparent involvement of the O2 reduction site have been used as arguments against the involvement of Asp51 in the mechanism of proton pumping. This minireview re-examines these arguments.     

Proton pumping mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.     

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a(3) as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK(a) values during the pumping process are derived for comparison to experiments.     

Reaction mechanism of bovine heart cytochrome c oxidase

The 1.9 A resolution X-ray structure of the O2 reduction site of bovine heart cytochrome c oxidase in the fully reduced state indicates trigonal planar coordination of CuB by three histidine residues. One of the three histidine residues has a covalent link to a tyrosine residue to ensure retention of the tyrosine at the O2 reduction site. These moieties facilitate a four electron reduction of O2, and prevent formation of active oxygen species. The combination of a redox-coupled conformational change of an aspartate residue (Asp51) located near the intermembrane surface of the enzyme molecule and the existence of a hydrogen bond network connecting Asp51 to the matrix surface suggest that the proton-pumping process is mediated at Asp51. Mutation analyses using a gene expression system of the Asp51-containing enzyme subunit yield results in support of the proposal that Asp51 plays a critical role in the proton pumping process.     

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.     

Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's CuB center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model-the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from delta-nitrogen of His291 ligand of CuB center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pKa of His291 for an extended model in which the whole Fe(a3)-CuB center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pKa units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.     

The mechanism for proton pumping in cytochrome c oxidase from an electrostatic and quantum chemical perspective

The mechanism for proton pumping in cytochrome c oxidase in the respiratory chain, has for decades been one of the main unsolved problems in biochemistry. However, even though several different suggested mechanisms exist, many of the steps in these mechanisms are quite similar and constitute a general consensus framework for discussing proton pumping. When these steps are analyzed, at least three critical gating situations are found, and these points are where the suggested mechanisms in general differ. The requirements for gating are reviewed and analyzed in detail, and a mechanism is suggested, where solutions for all the gating situations are formulated. This mechanism is based on an electrostatic analysis of a kinetic experiment fior the O to E transition. The key component of the mechanism is a positively charged transition state. An electron on heme a opens the gate for proton transfer from the N-side to a pump loading site (PLS). When the negative charge of the electron is compensated by a chemical proton, the positive transition state prevents backflow from the PLS to the N-side at the most critical stage of the pumping process. The mechanism has now been tested by large model DFT calculations, and these calculations give strong support for the suggested mechanism.     

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.     

Deuterium isotope effect of proton pumping in cytochrome c oxidase

In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron-proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3-->F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3-->O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.     

X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase

X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state shows a peroxide bridging between Fe2+ and Cu2+ in the O2 reduction site. The bond distances for Fe-O and Cu-O are 2.52 and 2.16 A, respectively. The structure is consistent with antiferromagnetic coupling between the two metals, which has long been known and to recent redox titration results [J. Biol. Chem. 274 (1999) 33403]. The trigonal planer coordination of Cu1+ in the O2 reduction site is consistent with the very weak interaction between Cu1+ and O2 bound at Fe2+ revealed by time-resolved resonance Raman investigations. One of the three histidine imidazoles coordinated to the Cu ion in the O2 reduction site fixes a tyrosine phenol group near the O2 reduction site with the direct covalent link between the two groups. The structure suggests that the phenol group is the site for donating protons to the bound O2. Redox-coupled conformational change in an extramembrane loop indicates that an aspartate (Asp51) in the loop apart from the O2 reduction site is the site for 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.     

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 proton/electron coupling ratio at heme a and Cu(A) in bovine heart cytochrome c oxidase.

Measurements of the H(+)/heme a, Cu(A) ratios for proton-electron coupling at these centers (redox Bohr effect) in CO-inhibited cytochrome c oxidase purified from bovine heart mitochondria, both in the soluble state and reconstituted in liposomes, are presented. In the soluble oxidase, the H(+)/heme a, Cu(A) ratios were experimentally determined upon oxidation by ferricyanide of these centers as well as upon their reduction by hexammineruthenium(II). These measurements showed that in order to obtain H(+)/heme a, Cu(A) ratios approaching 1, one-step full oxidation of both metal centers by ferricyanide had to be induced by a stoicheiometric amount of the oxidant. Partial stepwise oxidation or reduction of heme a and Cu(A) did produce H(+)/heme a, Cu(A) ratios significantly lower or higher than 1, respectively. The experimental H(+)/heme a, Cu(A) ratios measured upon stepwise reduction/oxidation of the metals were reproduced by mathematical simulation based on the coupling of oxido-reduction of both heme a and Cu(A) to pK shifts of common acid-base groups. The vectorial nature of the proton-electron coupling at heme a/Cu(A) was analyzed by measuring pH changes in the external bulk phase associated with oxido-reduction of these redox centers in the CO-inhibited oxidase reconstituted in liposomes. The results show that the proton release associated with the oxidation of heme a and Cu(A) takes place in the external aqueous phase. Protons taken up by the oxidase upon rereduction of the centers derive, on the other hand, from the inner space. These results provide evidence supporting the view that cooperative proton-electron coupling at heme a/Cu(A) is involved in the proton pump of the oxidase.     

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.     

Preparation and properties of bovine heart cytochrome c oxidase

Cytochrome c oxidase is isolated from bovine heart by a procedure that involves differential precipitation, fractionation with ammonium sulfate in 0.5% cholate, and removal of residual cholate by molecular sieve chromatography. The oxidase is highly active and is unusually soluble in phosphate buffer without added detergent; solutions with several millimolar concentrations, yet low viscosities, are readily prepared. The preparation contains ca. 20% lipid with a Cu to Fe ratio of 1:1. Intensities of visible and Soret bands in oxidized and reduced states are ca. 25% lower than in the presence of detergent (0.75% Tween 20). Oxidized cytochrome c inhibits and binds more tightly than does the reduced species (K_I, 18 μM; K_M, 25 μM) as noted in mitochondria.     

Structural studies on bovine heart cytochrome c oxidase

Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8?. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (Cu(A), heme a (Fe(a)), Cu(B), heme a(3) (Fe(a3))) and 3 redox-inactive metal sites (Mg(2+), Zn(2+) and Na(+)). The effects of various O(2) analogs on the X-ray structure suggest that O(2) molecules are transiently trapped at the Cu(B) site before binding to Fe(a3)(2+) to provide O(2)(-). This provides three possible electron transfer pathways from Cu(B), Fe(a3) and Tyr244 via a water molecule. These pathways facilitate non-sequential 3 electron reduction of the bound O(2)(-) to break the OO bond without releasing active oxygen species. Bovine heart CcO has a proton conducting pathway that includes a hydrogen-bond network and a water-channel which, in tandem, connect the positive side phase with the negative side phase. The hydrogen-bond network forms two additional hydrogen-bonds with the formyl and propionate groups of heme a. Thus, upon oxidation of heme a, the positive charge created on Fe(a) is readily delocalized to the heme peripheral groups to drive proton-transport through the hydrogen-bond network. A peptide bond in the hydrogen-bond network and a redox-coupled conformational change in the water channel are expected to effectively block reverse proton transfer through the H-pathway. These functions of the pathway have been confirmed by site-directed mutagenesis of bovine CcO expressed in HeLa cells.     

The mechanism of cytochrome oxidase and other reaction centres for electron/proton pumping

The functional significance of the metal centres of cytochrome oxidase is deduced from the ways in which the centres are bound into its peptides. To this end use is made of structural knowledge of other metalloproteins for dioxygen binding, haemocyanin and haemoglobin, and for electron transfer, cytochromes b and azurin. The order and manner in which the motions of helical sections of the oxidase are linked to proton pumping are suggested and a comparison is made with other proton pumps, for example that of ATP synthetases.