Identification of the Fe−O−O Bending Mode in Oxycytochrome P450cam by Resonance Raman Spectroscopy

An oxygen-sensitive mode in oxygenated wild-type cytochrome P450cam (oxyP450cam) is observed at 401 cm - 1 and assigned to the δ(Fe−O−O) bending mode, based upon 16 O 2, 18 O 2 isotopic shifts (19 cm - 1 ) and comparison with Co− and Fe−oxyporphyrin complexes. The detection of this Fe−O−O bending mode has structural implications for enzyme function since its frequency reflects the energies associated with Fe−O−O distortion in oxyhemeprotein active sites. Three body normal coordinate calculations adequately fit the experimental data set with a 125−130° bond angle for the Fe−O−O linkage in oxyP450cam. Observation of low-frequency isotope-sensitive vibrational patterns, some of which are hypothesized to be associated with out-of-plane porphyrin motions are also reported. These patterns, in conjunction with the high frequency of this bending mode and the abnormal isotopic shift of the Fe−O 2 stretching mode, suggest a “strained” Fe−O−O moiety in oxyP450cam, with comparable mobility to HbO 2 and MbO 2 . Possible sources of this “strain” and implications for catalytic dioxygen activation in P450cam are discussed.