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.     

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.     

Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase

Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-μs electron injection into Cu(A), close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H(+)/COX and 1 H(+)/COX, respectively). The subsequent 10-μs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ～0.5 H(+)/COX at this side to electrostatically compensate the charge of the electron. With ～200 μs, all but 0.4 H(+) at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called "pump site." Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to Cu(A). These results support the idea of a proton-collecting antenna, switched on by electron injection.     

Redox loops and proton pumps

In a Review-Hypothesis, Mitchell [(1987) FEBS Lett. 222,235-245] has recently suggested possible molecular mechanisms for proton translocation by cytochrome oxidase. In describing these mechanisms, he extended his own concept of a redox loop in a manner expected to lead to confusion. He also stated that the term redox-linked proton pump implies an indirect coupling between electron transfer and proton translocation, and that this type of coupling is very difficult to test experimentally. Here it is argued that the original meaning of a redox loop should be maintained, and proper definitions of the terms redox-linked proton pump and direct or indirect coupling are formulated. In addition, it is reasoned that both types of coupling are amenable to experimental tests.     

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.     

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.     

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.     

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.     

The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism

Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.     

Cytochrome oxidase as a proton pump

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H⁺/e ^– stoichiometry of the pump is discussed and results [Wikström (1989),Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. Direct and indirect mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the Cu_A site.     

Cytochrome oxidase: pathways for electron tunneling and proton transfer

 Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by Cu_A in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a ₃–Cu_B. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a ₃–Cu_B, whereas the other mediates transfer of the pumped protons. Received: 23 March 1998 / Accepted: 11 May 1998     

The effect of specific antibodies on oxygen uptake and H+ pumping by cytochrome c oxidase vesicles

Antibodies to solubilized cytochrome c oxidase and to subunit III were incubated with liposomal oxidase. In oxygen uptake experiments, the inhibiting effects on RCI of anti-oxidase (primarily anti- subunits II and IV) and anti-III were by different mechanisms: the former, by inhibiting the uncoupled rate; the later, by stimulating the coupled rate. In experiments with H+ translocation, anti-oxidase was without effect, while anti-III was a potent inhibitor of proton pumping. These results are conclusive evidence for redox-linked proton extrusion from the vesicles by the oxidase (and its subunit III).     

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].     

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.     

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.     

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.     

Electron and proton transport by NADPH oxidases

The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside-out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.     

Proton translocation by cytochrome c oxidase in different phases of the catalytic cycle

Since mitochondrial cytochrome c oxidase was found to be a redox-linked proton pump, most enzymes of the haem-copper oxidase family have been shown to share this function. Here, the most recent knowledge of how the individual reactions of the enzyme's catalytic cycle are coupled to proton translocation is reviewed. Two protons each are pumped during the oxidative and reductive halves of the cycle, respectively. An apparent controversy that concerns proton translocation during the reductive half is resolved. If the oxidised enzyme is allowed to relax in the absence of reductant, the binuclear haem-copper centre attains a state that lies outside the main catalytic cycle. Reduction of this form of the enzyme is not linked to proton translocation, but is necessary for a return to the main cycle. This phenomenon might be related to the previously described "pulsed" vs. "resting" and "fast" vs."slow" forms of haem-copper oxidases.