Cytochrome c oxidase: the mechanistic significance of structural H+ in energy transduction

Changes in the bulk-phase concentration of O₂ and H⁺ associated with the reduction of O₂ to water are simultaneously determined in reactions catalyzed by fully reduced cytochrome c oxidase both isolated and embedded in liposomes. Consistent with the polyphasic kinetics of electron transfer through the oxidase, the time course of O₂ consumption and H⁺ translocation exhibit the following novel characteristics: (1) The uptake of scalar protons (H_m⁺), the ejection of vectorial protons (H⁺_v), and the consumption of O₂, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O₂ uptake and H⁺ transfer are extremely fast and compatible with the rates of electron flow through the oxidase. (3) The K_m of the oxidase for O₂ is close to 75 M, the same for O₂ consumption and scalar H⁺ uptake. The V_max of O₂ reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 × 10⁴ s^–1. (4) The extent of vectorial H⁺ ejection by cytochrome c oxidase embedded in liposomes is an exponential function dependent on both enzyme concentration and extent of O₂ consumption. (5) The H⁺/O stoichiometry of H⁺ ejection is a variable that may reach a maximum value of 4.0 only when the enzyme undergoes net oxidation at extremely high enzyme/O₂ molar ratios. It is postulated that the generation of useful energy at the level of cytochrome c oxidase depends not only on the number of molecules of O₂ reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.