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

摘要

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 ( P_R ; Fe4+=O2?, Cu_B2+–OH?). We show that with the Thermus thermophilus 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 P_R 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 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 O₂ reduction by heme-copper oxidases.