Time-resolved resonance Raman investigation of oxygen reduction mechanism of bovine cytochrome c oxidase.

Six oxygen-associated resonance Raman bands were identified for intermediates in the reaction of bovine cytochrome c oxidase with O2 at room temperature. The primary intermediate, corresponding to Compound A of cryogenic measurements, is an O2 adduct of heme a3 and its isotope frequency shifts for 16O18O have established that the binding is of an end-on type. This is followed by two oxoheme intermediates, and the final intermediate appearing around 3 ms is the Fe-OH heme. The reaction rate between the two oxoheme intermediates is significantly slower in D2O than in H2O, suggesting that the electron transfer is regulated by proton translocations at this step. It is noted that the reaction intermediates of oxidized enzyme with hydrogen peroxide yield the same three sets of oxygen isotope-sensitive bands as those of oxoheme intermediates seen for O2 reduction and that the O-O bond has already been cleaved in the so-called peroxy form (or 607 nm form).