Investigations of the resonance Raman excitation profiles of cytochrome P450cam

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Abstract
We have measured the resonance Raman excitation profiles (REP’s) of several vibrational modes associated with the heme active site of cytochrome P450cam. The important Fe–S axial ligand mode (351 cm−1) of the substrate bound (high‐spin ferric) complex is found to have structure in its blue shifted REP. Inverse transform techniques allow the line shape of the resonant charge transfer absorption to be reconstructed directly from the REP data. The observed splitting (3200 cm−1) is associated with an inequivalence in the resonant S→Fe charge transfer excitations. The position of the high‐energy component (∼323 nm) is found to be in excellent agreement with z‐polarized single crystal measurements, while the low‐energy component (∼360 nm) is not clearly observed in the single crystal analysis. The ‘‘spin‐marker’’ band ν3 is found to have a REP that is significantly red shifted with respect to the theoretical predictions. A variety of perturbations including non‐Condon and multiple state coupling, as well as state dependent damping, are unable to account for the observed red shift. Studies of other ferric heme protein systems, including substrate free cytochrome P450cam (low‐spin) and aquomet myoglobin (high‐spin) reveal ‘‘normal’’ behavior of their REP’s. The electron–nuclear coupling strengths are directly extracted from the measured absolute cross sections in these cases.