An Anomaly in the Resonance Raman Spectra of Cytochrome P-450_cam in the Ferrous High-spin State

Abstract

Resonance Raman spectra of cytochrome P-450_cam (P-450_cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450_cam was unusual in the sense that the “oxidation-state marker” appeared at an unexpectedly lower frequency (1346 cm^-1) in comparison with those of other reduced hemoproteins (_??_1355-1365 cm^-1), whereas that of oxidized P-450_cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450_cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding π orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm^-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450_cam to P-420. The Raman spec-trum of reduced P-450_cam•metyrapone complex was very similar to that of ferrous cyto-chrome b₅.