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Chloroperoxidase (CPO) and cytochrome P450cam have been shown by several techniques to have similar active site properties. Recent resonance Raman investigations using isotopically enriched 34S-labeled samples have demonstrated thiolate ligation in the P450cam system. We report here on a number of parallel studies involving CPO. On the basis of isotopic labeling (34S, 13CO), we assign the Fe-S and Fe-CO stretching frequencies of CPO at 347 (-vFe-S) and 488 cm-1 (-vFe-CO). The differences of the -vFe-S and -vFe-CO in CPO and P450cam may suggest subtle differences in the thiolate binding in the two systems.

We present the first Stokes and anti-Stokes Raman intensity measurements of a molecular system using laser excitation throughout a resonant electronic transition. Experiments on oxidized and reduced cytochrome c at both room and cryogenic temperatures are reported. We employ a Kramers–Kronig transform method in our analysis and use the absorption and Raman cross sections on an absolute scale in order to find the linear Franck–Condon coupling strengths of the observed low frequency modes. A general expression for the Boltzmann factor is also derived in terms of the resonant Stokes and anti-Stokes scattering intensities. This expression is appropriate for the determination of temperatures in absorbing media. The traditional Stokes/anti-Stokes ratio used for this purpose is shown to fail both experimentally and theoretically when the laser excitation is in resonance with an electronic transition of the material under study. Finally, we discuss the frequency domain approximations necessary for application of the transform method at finite temperature and quantitatively treat the dispersion of the coupling strength across the Raman spectrum of cytochrome c. This latter phenomenon leads naturally to the concept of a coupling strength ‘‘spectrum.’’ This spectrum is calculated and subsequently used to estimate a parameter of interest (the Stokes shift, Δ) in recent theories of vibrationaly assisted electron transport.

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.