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

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 mu s 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 Cue 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,, Fel, M, J,, Libeu, C, P,, Mizushima, T,, et nl. 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.