Electron entry in a CuA mutant of cytochrome c oxidase from Paracoccus denitrificans.: Conclusive evidence on the initial electron entry metal center

A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the Cu-A site was changed by site-directed mutagenesis to a mononuclear copper site [Zickermann, V., Wittershagen, A., Kolbesen, B.O. and Ludwig, B. Biochemistry 36 (1997) 3232-3236] was investigated by stopped-flow spectroscopy, Contrary to the behavior of the wild type enzyme, in this mutant cytochrome a cannot be reduced by excess cytochrome c in the millisecond time scale in which cytochrome c oxidation is observed. The results conclusively identify and establish Cu-A as the initial electron entry site in cytochrome c oxidase, Partial rapid reduction (ca, 20%) of the modified Cu-A site suggests that the mononuclear copper ion has a redox potential ca, 100 mV lower than the mild type, and that internal electron transfer to cytochrome a is greater than or equal to 10(3)-fold slower than with the mild type enzyme. (C) 1998 Federation of European Biochemical Societies.