pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase.: The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O-2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R -> O transition) decreased from approximate to 2.4 at pH 6.5 to approximate to 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O -> R transition), the H+/COX ratio for proton release from COV increased from, approximate to 0.3 at pH 6.5 to approximate to 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O-2 versus ferricyanide oxidation) approaching 1 H+/COX at pH <= 6.5 is associated with the reduction of O-2 by the reduced metal centers. At pH >= 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O-2 of the fully reduced oxidase directly followed by re-reduction (R -> O -> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e(-) cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O-2 to H2O is presented.