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 obtain ed, including sperm whale native, pig wild-type, and the mutants H64L, H64A, V68T, V68N, H64V/V68T, F43W, F46V, and L29F. This series was ch osen in order to study the effect of ligand binding pocket polarity on the positions of the nu(Fe-CO) and delta(Fe-C-O) bands. Spectra of bo th (CO)-C-12 and (CO)-C-13 isotopic forms have been obtained and a det ailed analysis has facilitated the identification of both the ligand-s pecific bands and six underlying polphyrin bands which are insensitive to this isotopic substitution. Along with a band-fitting analysis of infrared spectra, these resonance Raman data provide a comprehensive e valuation of the vibrations of the FeCO unit. The band positions of th e ligand-specific modes are found to depend on the structure of the li gand binding pocket, arising from the strength of back-bonding within the FeCO unit, and clear correlations exist between the nu(Fe-CO), del ta(Fe-C-O), and nu(C-O) band positions which characterize this synergi c bonding. The results are consistent with the proposal that the vibra tional band positions are determined primarily by the electrostatic po tential at the ligand. Five discrete band sets are observed for this s et of mutants, suggesting that 5 discrete conformations occur.