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

Abstract

Heme-copper oxidases catalyze the four-electron reduction of O₂ to H₂O 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 (<b>P_R</b> ; Fe⁴⁺=O^2-, Cu_B²⁺-OH^-). We show that with the <i>Thermus thermophilus</i> ba₃ 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 <b>P_R</b> ferryl (τ ≅110 μs), which indicates that O₂ 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 [Fe³⁺-O^--O^-(H⁺)], which is formed before the <b>P_R</b> ferryl. The rates of heme b oxidation and <b>P_R</b> 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 O₂ reduction by heme-copper oxidases.