The Inhibition Mechanism of the Cytochrome Oxidase ReactionI. Inhibition by Hydroxylamine*

Abstract

1. Unusual phenomena in the time course of the oxidation of ferrocytochrome c in the presence of cytochrome a [cytochrome oxidase, EC 1.9.3.1] and hydroxylamine were studied by examining the preincubation effect of cytochrome a with hydroxylamine alone or with the mixture of hydroxylamine and cytochrome c under various conditions. The results indicated that the formation of the hydroxylamine-cyto-chrome a complex was not instantaneous and that the enzyme-inhibitor complex was not formed unless the enzyme reacted with the substrate in the presence of the inhibitor. It was also found that the cytochrome oxidase reaction was reversibly inhibited by hydroxylamine. 2. Based on the kinetic examination of the effect of hydroxylamine on the cytochrome oxidase activity and its dependence on the substrate concentration, a simple scheme which is consistent with the present results was proposed as follows; cytochrome a contains at least two groups, each of which is capable of combining with hydroxylamine, and the enzyme species combined with one molecule of hydroxylamine has a residual activity while the one combined with two molecules of hydroxylamine is completely inactive.