A chemometric analysis of the resonance Raman spectra of mutant carbonmonoxy-myoglobins reveals the effects of polarity.

Resonance Raman spectra of 10 carbonmonoxy-myoglobins have been obtained, including sperm whale native, pig wild-type, and the mutants H64L, H64A, V68T, V68N, H64V/V68T, F43W, F46V, and L29F. This series was chosen in order to study the effect of ligand binding pocket polarity on the positions of the v(Fe-CO) and delta (Fe-C-O) bands. Spectra of both 12CO and 13CO isotopic forms have been obtained and a detailed analysis has facilitated the identification of both the ligand-specific bands and six underlying porphyrin bands which are insensitive to this isotopic substitution. Along with a band-fitting analysis of infrared spectra, these resonance Raman data provide a comprehensive evaluation of the vibrations of the FeCO unit. The band positions of the ligand-specific modes are found to depend on the structure of the ligand binding pocket, arising from the strength of back-bonding within the FeCO unit, and clear correlations exist between the v(Fe-CO), delta (Fe-C-O), and v(C-O) band positions which characterize this synergic bonding. The results are consistent with the proposal that the vibration band positions are determined primarily by the electrostatic potential at the ligand. Five discrete band sets are observed for this set of mutants, suggesting that 5 discrete conformations occur.