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Electron entry in a Cu A mutant of cytochrome c oxidase from Paracoccus denitrificans . Conclusive evidence on the initial electron entry metal center
Author(s) -
Malatesta Francesco,
Nicoletti Flavia,
Zickermann Volker,
Ludwig Bernd,
Brunori Maurizio
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01006-0
Subject(s) - paracoccus denitrificans , cytochrome c oxidase , cytochrome , cytochrome c , chemistry , cytochrome c peroxidase , cytochrome c1 , electron transfer , electron transport chain , cytochrome b , oxidase test , site directed mutagenesis , coenzyme q – cytochrome c reductase , biochemistry , mutant , enzyme , photochemistry , mitochondrion , mitochondrial dna , gene
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 wild type, and that internal electron transfer to cytochrome a is ≥10 3 ‐fold slower than with the wild type enzyme.