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Resonance Raman Detection of the Hydroperoxo Intermediate in the Cytochrome P450 Enzymatic Cycle

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journal contribution
posted on 23.05.2007, 00:00 by Piotr J. Mak, Ilia G. Denisov, Doreen Victoria, Thomas M. Makris, Tianjing Deng, Stephen G. Sligar, James R. Kincaid
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 799 cm-1, is observed for the first time in a peroxo-heme adduct. It is assigned unambiguously by employing isotopomeric mixtures of oxygen gas containing 50% 16O18O, confirming the presence of an intact O−O fragment. The ν(Fe−O) mode is observed at 559 cm-1 (H2O). Furthermore, both modes shift down by 3 cm-1, documenting the formulation as a hydroperoxo complex, in agreement with EPR data.