Resonance Raman spectroscopy is used to document, for the first time, a 6 cm−1 decrease of the Fe-S stretch by introducing an H-bond donor into the proximal pocket of a cytochrome P450, which interacts with the key cysteine thiolate axial ligand. The anticipated trans-effect on bound exogenous ligands is also confirmed and evidence is obtained supporting intimate interaction of the new histidyl-imidazole fragment with the heme periphery.