Investigations of the resonance Raman excitation profiles of cytochrome P450cam

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.

The Interaction of Covalently Bound Heme with the Cytochrome c Maturation Protein CcmE

“…6, spectrum a) that was absent from the spectrum of the Y134F variant (spectrum b). Although it is known that 363.8-nm excitation gives an Fe-Cys stretching mode around 350 cm Ϫ1 for Cys-bound ferric 5cHS hemes (28,29), the observed frequency (600 cm Ϫ1 ) is too high to assign it to the Fe-S stretching mode, and the soluble domain of CcmE studied in this work contains no Cys residues. Considering that the 600 cm Ϫ1 band was not observed for the holo-Y134F variant and is in good agreement with those observed for proximal Tyr variants of Hb and Mb (578 -603 cm Ϫ1 ), it is likely that this band arises from the Fe-Tyr stretching mode.…”

Ligand?protein interactions in nitric oxide synthase

“…Similar superoxide character of the heme-bound dioxygen is found in the oxy-derivatives of P450 and CPO. In P450s, as well as their model complexes, the O-O stretching mode is found in the 1139-1147 cm À1 region as summarized in Table 2 [88][89][90][91][92]. Similarly, CPO has a split O-O stretching mode with components at 1137 and 1124 cm À1 (Dr. Ryu Makino of Rikko University, private communication).…”

Polarized resonance Raman dispersion spectroscopy on metalporphyrins

“…By using the transform theory the asymmetric band shape is already produced when only FC-coupling is taken into consideration S We have referred to the earlier phase of work on the transform analysis of heme protein Raman lines because we wanted to focus on the basic principles of the method. It should be noted, however, that the above analysis of ferrocytochrome c lines has been extended to other heme proteins such as myoglobin [47] and cytochrome P450 [48]. The cytochrome data have been reanalyzed in a subsequent study by Schomaker et al [26] which combines the transform method with time correlator calculations to obtain the influence of inhomogeneous and thermal broadening.…”