Time-resolved resonance Raman study of dioxygen reduction by cytochrome C oxidase

Six oxygen-associated vibrations were observed for reaction intermediates of bovine cytochrome c oxidase with O-2 using time-resolved resonance Raman spectroscopy at room temperature. The isotope frequency shifts for (OO)-O-16-O-18, have established that the primary intermediate is an end-on type dioxygen adduct of Fe-a3, which is followed by two oxoheme intermediates, and that the final intermediate appearing around 3 ms is the Fe-OH heme. The reaction rate between the two oxoheme intermediates was significantly slower in D2O than in H2O, suggesting tight coupling with proton translocation at this step. It is noted that the reaction intermediates of oxidized enzyme with hydrogen peroxide yields the same three sets of oxygen isotope-sensitive bands as those seen for oxoheme intermediates of the dioxygen reduction, indicating the identity of intermediates.