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

Abstract

Elucidating the structures of intermediates in the reduction of O 2 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 O 2 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 O 2 evolution in photosynthesis.