I.The polyphasic reduction of oxygen to water by purified cytochrome c oxidase

The time course of oxygen consumption by purified cytochrome oxidase has been studied in reactions where the fully reduced enzyme was rapidly mixed with molecular oxygen. Similar to intact mitochondria (Reynafarje & Davies, Am. J. Physiol. 258, 1990), the enzyme reduces oxygen to water in a kinetically and well defined polyphasic event. The initial rates of O₂ consumption depended hyperbolically on O₂ concentration, with a bimolecular rate constant of near 10⁷ M-1 s-1. The V_max of O₂ uptake was, however, a complex function of the concentrations of both enzyme and cytochrome c. It is concluded that the reduction of oxygen to water takes place in a cyclic process in which the oxidase undergoes redox changes at rates depending on the relative concentration of the enzyme and its 3 substrates: O₂, electrons and protons. No evidence was found for impairments in the intramolecular flow of electrons per se.