Splitting of the O-O bond at the heme-copper catalytic site of respiratory oxidases.

Heme-copper oxidases catalyze the four-electron reduction of O to HO at a catalytic site that is composed of a heme group, a copper ion (Cu), and a tyrosine residue. Results from earlier experimental studies have shown that the O-O bond is cleaved simultaneously with electron transfer from a low-spin heme (heme a/b), forming a ferryl state ( ; Fe=O, Cu-OH). We show that with the ba oxidase, at low temperature (10°C, pH 7), electron transfer from the low-spin heme b to the catalytic site is faster by a factor of ~10 (τ ≅ 11 μs) than the formation of the ferryl (τ ≅110 μs), which indicates that O is reduced before the splitting of the O-O bond. Application of density functional theory indicates that the electron acceptor at the catalytic site is a high-energy peroxy state [Fe-O-O(H)], which is formed before the ferryl. The rates of heme b oxidation and ferryl formation were more similar at pH 10, indicating that the formation of the high-energy peroxy state involves proton transfer within the catalytic site, consistent with theory. The combined experimental and theoretical data suggest a general mechanism for O reduction by heme-copper oxidases.