The proton pump of cytochrome c oxidase and its stoichiometry.

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikstr?m, M. F. K. (1977) Nature (Lond.) 266, 271--273].     

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.     

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.     

Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase

Mitochondrial oxidative phosphorylation provides most cellular energy. As part of this process, cytochrome c oxidase (CcO) pumps protons across the inner mitochondrial membrane, contributing to the generation of the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. During acute inflammation, as in sepsis, aerobic metabolism appears to malfunction and switches to glycolytic energy production. The pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been shown to play a central role in inflammation. We hypothesized that TNFalpha-triggered cell signaling targets CcO, which is a central enzyme of the aerobic energy metabolism and can be regulated through phosphorylation. Using total bovine and murine hepatocyte homogenates TNFalpha treatment led to an approximately 60% reduction in CcO activity. In contrast, there was no direct effect of TNFalpha on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFalpha-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFalpha treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was approximately 50 and 70% inhibited at high cytochrome c concentrations in the presence of allosteric activator ADP and inhibitor ATP, respectively. CcO phosphorylation occurs on tyrosine 304 as demonstrated with a phosphoepitope-specific antibody. Furthermore, the mitochondrial membrane potential was decreased in H2.35 cells in response to TNFalpha. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFalpha-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients.     

Biosynthesis of cytochrome c oxidase by isolated rat liver mitochondria.

When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo.     

The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system

The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6. With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium. When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed. This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin. Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c. We thus conclude that cytochrome c oxidase of P. denitrificans is a proton pump. Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers.     

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.     

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.     

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

The mechanism of transmembrane delta muH+ generation in mitochondria by cytochrome c oxidase.

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.     

Purification and characterization of cytochrome c oxidase from rat liver mitochondria.

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.     

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.     

Prevention of leak in the proton pump of cytochrome c oxidase

The cytochrome c oxidases (CcO), which are responsible for most O(2) consumption in biology, are also redox-linked proton pumps that effectively convert the free energy of O(2) reduction to an electrochemical proton gradient across mitochondrial and bacterial membranes. Recently, time-resolved measurements have elucidated the sequence of events in proton translocation, and shed light on the underlying molecular mechanisms. One crucial property of the proton pump mechanism has received less attention, viz. how proton leaks are avoided. Here, we will analyse this topic and demonstrate how the key proton-carrying residue Glu-242 (numbering according to the sequence of subunit I of bovine heart CcO) functions as a valve that has the effect of minimising back-leakage of the pumped proton.     

A near-infrared investigation of cytochrome c oxidase in higher plant mitochondria

Optical features of cytochrome c oxidase in potato mitochondria have been characterized in the near-ir region. In order to discriminate the respective properties of the various redox centers, the redox state was monitored from free and inhibited, bound species. Appropriate comparisons singled out difference spectra which can be attributed specifically to CuA and CuB. The CuA difference spectrum (red-ox) exhibits a negative band centered at 812 nm and, analogous to its mammalian counterpart, the so-called 830-nm band (delta epsilon red/ox = -2.0 mM-1 cm-1). The unusual difference spectrum (red-ox) assigned to CuB is characterized by a broad positive band also centered at 812 nm with an extinction coefficient of delta epsilon red/ox = 4.3 mM-1 cm-1.     

Quantum molecular dynamics simulation of proton transfer in cytochrome c oxidase

Proton transfer/translocation is studied in cytochrome c oxidase (CcO) by a combination of quantum mechanics (QM) for the transferring protons and classical molecular dynamics (MD) for the protein and solvent. The possibility of a glutamate, Glu286 in the Rhodobacter sphaeroides numbering scheme, acting as a rely point for proton translocation is investigated. The MD finds a hydrogen-bonded cycle of two waters and the carboxylate oxygens of Glu286. The possibility of protonating Glu286 to form neutral GluH is studied and we find that, as experimentally inferred, this glutamate can spend most of its time as GluH. Since translocation relies on the presence of water chains within CcO channels, MD is used to assess their formation. Glu286 and Mg(2+) can be connected by continuous hydrogen-bonded chains that are robust, though transient, and the protein appears spongy above (toward the outer membrane) the Mg(2+). In contrast, the D-channel spanning Asp132, close to the inner membrane surface, to Glu286, forms water chains that are much sparser and do not continuously connect these residues. Rather, there are chains spanning Glu286 to the vicinity of Asn140, and other more robust and ramified water structures that connect Asp132 with waters close to Asn140.