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

Changes in the bulk-phase concentration of O-2 and H+ associated with the reduction of O-2 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 O-2 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 O-2, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O-2 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 O-2 is close to 75 muM, the same for O-2 consumption and scalar H+ uptake. The V-max of O-2 reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 x 10(4) 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 O-2 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/O-2 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 O-2 reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.