FERRICYTOCHROME-C INDUCES MONOPHASIC KINETICS OF FERROCYTOCHROME-C OXIDATION IN CYTOCHROME-C-OXIDASE

The kinetics of ferrocytochrome c oxidation by reconstituted cytochrom e c oxidase (COX) from bovine heart was followed by a spectrophotometr ic method, using on-line data collection and subsequent calculation of reaction rates from a function fitted to the progress curve. When rea ction rates were calculated at increasing reaction times, the multipha sic kinetics of ferrocytochrome c oxidation gradually changed into mon ophasic Michaelis-Menten kinetics. The same phenomenon was observed wh en ferrocytochrome c oxidation was followed in the presence of increas ing 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 s trength conditions. Furthermore, we show that ferricytochrome c inhibi ts the ''high affinity phase'' of ferrocytochrome c oxidation in an ap parently competitive way, while inhibition of the ''low affinity phase '' is noncompetitive. These findings are consistent with a ''regulator y site model'' where both the catalytic and the regulatory site bind f erro- as well as ferricytochrome c.