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(2) to H(2)O at a catalytic site that is composed of a heme group, a copper ion (Cu(B)), 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 (P(R); Fe(4+)=O(2−), Cu(B)(2+)–OH(−)). We show that with the Thermus thermophilus ba(3) 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 P(R) ferryl (τ ≅110 μs), which indicates that O(2) 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(3+)–O(−)–O(−)(H(+))], which is formed before the P(R) ferryl. The rates of heme b oxidation and P(R) 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(2) reduction by heme-copper oxidases.