Resonance Raman detection of the hydroperoxo intermediate in the cytochrome P450 enzymatic cycle.

[Figure: see text] The Resonance Raman spectra of the hydroperoxo complex of camphor-bound CYP101 have been obtained by cryoradiolytic reduction of the oxygenated ferrous form that had been rapidly frozen in water-glycerol frozen solution; EPR spectroscopy was employed to confirm the identity of the trapped intermediate. The ν(O-O) mode, appearing at 799cm, is observed for the first time in a peroxo-heme adduct. It is assigned unambiguously by employing isotopomeric mixtures of oxygen gas containing 50% OO, confirming the presence of an intact O-O fragment. The ν(Fe-O) mode is observed at 559 cm (HO). Furthermore, both modes shift down by 3 cm, documenting the formulation as a hydroperoxo-complex, in agreement with EPR data.