Ferricytochrome c induces monophasic kinetics of ferrocytochrome c oxidation in cytochrome c oxidase.

The kinetics of ferrocytochrome c oxidation by reconstituted cytochrome c oxidase (COX) from bovine heart was followed by a spectrophotometric method, using on-line data collection and subsequent calculation of reaction rates from a function fitted to the progress curve. When reaction rates were calculated at increasing reaction times, the multiphasic kinetics of ferrocytochrome c oxidation gradually changed into monophasic Michaelis-Menten kinetics. The same phenomenon was observed when ferrocytochrome c oxidation was followed in the presence of increasing amounts of ferricytochrome c. From these results we conclude that ferricytochrome c shifts the multiphasic kinetics of ferrocytochrome c oxidation by COX into monophasic kinetics, comparable to high ionic strength conditions. Furthermore, we show that ferricytochrome c inhibits the "high affinity phase" of ferrocytochrome c oxidation in an apparently competitive way, while inhibition of the "low affinity phase" is noncompetitive. These findings are consistent with a "regulatory site model" where both the catalytic and the regulatory site bind ferro- as well as ferricytochrome c.