Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

AbstractReduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron–oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with CuB and the rate of the reaction from Oxy to P (PR) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fea3–O–O–CuB peroxy bridged structure. The Fea3O stretching (νFeO) frequency of the PR intermediate, 804/764 cm−1 for 16O/18O, is distinctly higher than that of F intermediate, 785/750 cm−1. The rate of reaction from P to F in D2O solution is evidently slower than that in H2O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H2O2 gave the νFeO band at 803/769 cm−1 for H216O2/H218O2 and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O2 provided the P intermediate (PM) giving rise to an absorption peak at 607 nm and the νFeO bands at 804/768 cm−1. Thus, three kinds of P intermediates are considered to have the same oxo-heme a3 structure. The ν4 and ν2 modes of heme a3 of the P intermediate were identified at 1377 and 1591 cm−1, respectively. The Raman excitation profiles of the νFeO bands were different between P and F. These observations may mean the formation of a π cation radical of porphyrin macrocycle in P