Two Km values for cytochrome c of aa3-type two-subunit cytochrome c oxidase from Nitrobacter agilis

Abstract

Oxidation of ferrocytochrome c by aa 3-type two-subunit cytochrome c oxidase from Nitrobacter agilis was measured polarographically and the results obtained were analyzed by means of Eadie-Hofstee plots. Two apparent K m values of ∼3.0 × 10 −8 and 2.0 × 10 −6 M were obtained for horse cytochrome c in 25 mM phosphate buffer, pH 6.5. Also when N. agilis ferrocytochrome c was oxidized by the enzyme in 25 mM phosphate buffer (pH 6.5), two apparent K m values of ∼2.0 × 10 −8 and 4.0 × 10 −6 M were obtained although the break point in the Eadie-Hofstee plot was as clear as in the case of horse cytochrome c. The results show that two reactive sites for cytochrome c also occur in the bacterial cytochrome c oxidase composed of two subunits and will give further support to the idea that only the larger two subunits of eukaryotic cytochrome c oxidase are responsible for oxidation of ferrocytochrome c.