Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.