Anharmonic protein motions and heme deformations in myoglobin cyanide probed by absorption and resonance Raman spectroscopy

The Soret absorption of myoglobin cyanide in a 65% glycerol/water mixture w as measured as a function of temperature between 20 and 300 K. The data wer e analyzed by using an earlier model relating each transition into the vibr onic manifold of the electronic B-state to a Voigtian band profile (Cupane et al. Eur. Biophys. J. 1995, 23, 385, 1995). Its Gaussian part contains a temperature-dependent component due to the coupling of low-frequency modes to the Soret transition. The analysis of the vibronic substructure was faci litated by comparison with vibronic coupling parameters derived from the li ne intensities in the polarized Raman spectra taken with Sorer excitation. From the depolarization ratios of several Raman lines, the existence of asy mmetric heme macrocycle distortions was inferred, which lift the degeneracy of the excited B state. Raman intensities and depolarization ratios were t hen analyzed by a theory that formulates the polarizability tensor in terms of a time-independent perturbation theory. The vibronic coupling parameter s thus obtained are linearly related to normal coordinate deformations of t he heme macrocycle. The results obtained from this analysis of the Raman da ta suggest a Sorer band splitting of ca. 130 cm(-1). This finding was then explicitly taken into account in the analysis of the Sorer band absorption. The temperature dependence of the Gaussian broadening was found to deviate from the predictions of a harmonic model above a temperature that is sligh tly lower than the glass temperature of the glycerol/water solvent. This cl early indicates the onset of anharmonic motions within the protein environm ent, which are coupled to out-of-plane vibrations of the central iron atom. At room temperature, the degree of anharmonicity is much larger than that observed for myoglobin carbonmonoxide and is comparable with that of deoxym yoglobin. This indicates that oxidation and the spin state of the central i ron atom have a significant impact on its dynamic properties. From the anal ysis of the depolarization ratio dispersion and the resonance excitation pr ofiles of the oxidation marker band, we infer a rhombic distortion of the h eme group that gives rise to nonequivalent Fe-N distances. Finally, the app earance of polarized Raman lines arising from A(2u) type vibrations indicat es that the heme group is somewhat domed despite its hexacoordinated state.