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 was measured as a function of temperature between 20 and 300 K. The data were analyzed by using an earlier model relating each transition into the vibronic 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 facilitated by comparison with vibronic coupling parameters derived from the line intensities in the polarized Raman spectra taken with Sorer excitation. From the depolarization ratios of several Raman lines, the existence of asymmetric heme macrocycle distortions was inferred, which lift the degeneracy of the excited B state. Raman intensities and depolarization ratios were then analyzed by a theory that formulates the polarizability tensor in terms of a time-independent perturbation theory. The vibronic coupling parameters thus obtained are linearly related to normal coordinate deformations of the heme macrocycle. The results obtained from this analysis of the Raman data 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 slightly lower than the glass temperature of the glycerol/water solvent. This clearly indicates the onset of anharmonic motions within the protein environment, 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 deoxymyoglobin. This indicates that oxidation and the spin state of the central iron atom have a significant impact on its dynamic properties. From the analysis of the depolarization ratio dispersion and the resonance excitation profiles of the oxidation marker band, we infer a rhombic distortion of the heme group that gives rise to nonequivalent Fe-N distances. Finally, the appearance of polarized Raman lines arising from A(2u) type vibrations indicates that the heme group is somewhat domed despite its hexacoordinated state.