Low frequency spectral density of ferrous heme: perturbations induced by axial ligation and protein insertion

Femtosecond coherence spectroscopy is used to probe low frequency (20-400 cm(-1)) modes of the ferrous heme group in solution, with and without 2-methyl imidazole (2MeIm) as an axial ligand. The results are compared to heme proteins (CPO, P450(cam), HRP, Mb) where insertion of the heme into the protein results in redistribution of the low frequency spectral density and in (approximately 60%) longer damping times for the coherent signals. The major effect of imidazole ligation to the ferrous heme is the "softening" of the low frequency force constants by a factor of approximately 0.6 +/- 0.1. The functional consequences of imidazole ligation are assessed and it is found that the enthalpic CO rebinding barrier is increased significantly when imidazole is bound. The force constant softening analysis, combined with the kinetics results, indicates that the iron is displaced by only approximately 0.2 A from the heme plane in the absence of the imidazole ligand, whereas it is displaced by approximately 0.4 A when imidazole (histidine) is present. This suggests that binding of imidazole (histidine) as an axial ligand, and the concomitant softening of the force constants, leads to an anharmonic distortion of the heme group that has significant functional consequences.