Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Chemistry Department and Laser Laboratory, Michigan State University, East Lansing, Michigan 48824-1322

Communicated by Harry B. Gray, California Institute of Technology, Pasadena, CA (received for review February 22, 1998)

Abstract

Elucidating the structures of intermediates in the reduction of O₂ to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O₂ with the mixed-valence enzyme, in which only heme a ₃ and Cu_B in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O=O bond cleavage occurs within the first 200 μs after reaction initiation; the presence of a uniquely stable Fe—O—O(H) peroxy species is not detected. The product of this rapid reaction is a heme a ₃ oxoferryl (Fe^IV=O) species, which requires that an electron donor in addition to heme a ₃ and Cu_B must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A., Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547–10553] show that one of the Cu_B ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O—O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His—Tyr species to produce the product oxoferryl species, Cu_B ²⁺—OH⁻, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O—O bond forming chemistry that occurs during O₂ evolution in photosynthesis.

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: babcock{at}cemvax.cem.msu.edu.
↵ † Several different approaches have been used to generate a form of CcO at the peroxy (2e⁻) oxidation level (11, 16, 18–21). In the literature, these species have been referred to variously as compound C, peroxy, “607 nm” form, or P. In this paper, these will be referred to collectively as P. Similarly, a form of CcO at the ferryl (3e⁻) oxidation level, which is referred to as ferryl, “580 nm” from, or F, will be designated as F to promote simplicity in comparison with earlier work. The P and F nomenclature is a formal construct and should not be taken as necessarily indicating the physical structure existing at the active site.
ABBREVIATIONS:

CcO,

cytochrome c oxidase;

MV-CcO,

mixed-valence CcO;

P,

spectral form of CcO at the 2e− reduced level;

F,

spectral form of CcO at the 3e− reduced level