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

Abstract

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.

A cooperative model for protonmotive heme‐copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

“…The results of the investigations, based on measurement of the rates of electron £ow and proton ejection at level £ow, showed that the H /e 3 ratio of proton pumping by the oxidase varies, both in intact mitochondria and in the isolated-reconstituted oxidase, from around zero, at extremes of low and high rates, to about one at intermediate rates (see also [14]). It was also found that the H /e 3 ratios for proton ejection driven by ferrocytochrome c oxidation in COV increase with pH, up to about one at a pH around 8 and then decline at higher pHs [21]. When compared with the pH dependence of redox Bohr effects in the soluble oxidase, the pH dependence of the H /e 3 ratio for proton pumping is similar to that observed for the H /e 3 ratio of the Bohr e¡ect linked to oxido-reduction of heme a (Fig.…”

“…The observed non-linearity of the steady-state relationship between respiratory rate and vp is taken by some authors as evidence of slip in proton pumps [19], by others as due to non-ohmic increases of membrane proton conductance at high vp (leak) [20]. Papa et al have carried out an extensive study of the in£u-ence of kinetic and thermodynamic factors on the H /e 3 stoichiometry of cytochrome c oxidase in mitochondria and in the isolated-reconstituted state (COV) [17,18,21]. The results of the investigations, based on measurement of the rates of electron £ow and proton ejection at level £ow, showed that the H /e 3 ratio of proton pumping by the oxidase varies, both in intact mitochondria and in the isolated-reconstituted oxidase, from around zero, at extremes of low and high rates, to about one at intermediate rates (see also [14]).…”

The stoichiometry of charge translocation by cytochrome oxidase and the cytochrome bc1 complex of mitochondria at high membrane potential

“…The measured q+/2e stoichiometry of cytochrome oxidase at low Ay approaches the most widely accepted value of 4 at level flow [22][23][24]26, 271. As we have indicated [l], this method may slightly underestimate the stoichiometry; therefore a reasonable interpretation of the observed drop in the q+/2e stoichiometry of cytochrome oxidase in these experiments is that it changes from 4 at low values of Ap to 2 at high values of Ap, although other absolute values of 4+/2e are not rigorously excluded by these results.…”

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

“…that (for example) values considerably less than unity have been observed (3,(13)(14)(15), depending on the total number of turnovers, the absolute turnover rate, and the initial state of the enzyme, either pulsed or resting (for definition, see refs. 16 and 17).…”