Oxygen "pulsed" cytochrome c oxidase: functional properties and catalytic relevance.

Abstract

The kinetics of the reaction of cytochrome c with solubilized beef heart of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was studied by a stopped-flow technique under 2 different experimental situations: the completely oxidized enzyme (resting oxidase as obtained from the preparation) was mixed with reduced cytochrome c, and the completely reduced enzyme in the presence of reduced cytochrome c was exposed to a pulse of O2 (pulsed oxidase). Both sets of experiments were performed with either limiting or excess O2 (relative to oxidase), in the presence or absence of CO. Both the pre-steady-state events and the steady-state kinetics of cytochrome oxidase were found to be different in the 2 cases. This showed that the product of the reaction of fully reduced oxidase with O2 (pulsed oxidase) was functionally different from the oxidase as prepared (resting oxidase). These differences were interpreted with the assumption of a different rate of intramolecular electron transfer in the pulsed and resting oxidases. Implications of these experimental findings were discussed in the general framework of a tentative model for the catalytic cycle of the oxidase.