Reduction and activity of cytochrome c in the cytochrome c-cytochrome aa3 complex

Uncharged reductants, such as NNN'N'-tetramethyl-p-phenylenediamine and diaminodurene, reduce cytochrome c at both high and low ionic strength, unlike ascorbate, which is effective only at low ionic strength. The 'tightly bound' cytochrome c-cytochrome c oxidase complex, with 1 equiv. of cytochrome c per cytochrome aa3, can be prepared by simple mixing of the two component species. Its properties are not affected by co-sonication of the mixture. Bound cytochrome c is more rapidly reduced by NNN'N'-tetramethyl-p-phenylenediamine and diaminodurene than is free cytochrome c. At high ionic strength, when the complex is largely dissociated, addition of reductant under aerobic conditions in the presence of cyanide, or under anaerobic conditions, induces a rapid reduction of cytochrome c followed by the reduction of cytochrome a. At low ionic strength, addition of reductant induces a rapid reduction of cytochrome a while cytochrome c remains largely oxidized, the rate-limiting step now being the reduction of cytochrome c. The results are interpreted in terms of direct reduction of cytochrome c in its tight complex with the oxidase, followed by rapid intramolecular electron transfer to both cytochrome a and the associated e.p.r.-detectable Cu atom.