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

Reduced 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 Cu(B) and the rate of the reaction from Oxy to P (P(R)) 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 Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O 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 H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.