Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa,) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046-7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa(3) are higher than those found in the native aa(3) enzyme. However, (3) K-m and V-max values obtained from the non-linear Eadie-Hofstee plots are very similar in both caa(3) and aa(3). The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.