Limited-turnover studies on proton translocation in reconstituted cytochrome c oxidase-containing vesicles

Abstract

We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.

Functional reassembly of membrane proteins in planar lipid bilayers

“…The fast proton efflux was fully abolished by proton ionophores or by omitting valinomycin from the medium (Krab & Wikstrom, 1978). Casey, Chappell & Azzi (1979) confirmed these results with cytochrome oxidase vesicles. Furthermore, by careful titration with the proton ionophore FCCP they showed that proton ejection was indeed the result of proton translocation and reported a (H+/e~) ratio of 0-9.…”

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

“…Ferrocytochrome c, prepared as in [30], was measured using a A~5 5 0 (reduced-oxidized) = 21 mM-' cm-' [34]. Ferrocytochrome c was oxidized by adding a few grains of ferricyanide.…”

“…Reconstitution of the cytochrome oxidase vesicles was performed by the cholate dialysis method as described in [30]. The percentage of total cytochrome oxidase incorporated 'right-side out' was calculated as in [31] or after reduction by impermeant and permeant reducing agents in anaerobiosis in the absence of cyanide.…”

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

“…Oxygen uptake assays used conditions similar to [20]; uncoupling for respiratory control measurements was as in [7]. Proton translocation was at 3°C using phenol red [6] as modified by [7] except that the reduced cytochrome c was chromatographed and stored in 50 mM K$SOJl mM MOPS (pH 7.2). Protein determination was as in [21], compensating for phospholipid interference.…”