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

Abstract

A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the CuA 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 CuA as the initial electron entry site in cytochrome c oxidase. Partial rapid reduction (ca. 20%) of the modified CuA site suggests that the mononuclear copper ion has a redox potential ca. 100 mV lower than the wild type, and that internal electron transfer to cytochrome a is > or = 10(3)-fold slower than with the wild type enzyme.