Spectrophotometric Examination of Exogenous-Ligand Complexes of Ferric Cytochrome P-450.1 Characterization of the Axial Ligand trans to Thiolate in the Native Ferric Low-Spin Form

The alpha band of the native ferric low-spin form of cytochrome P-450 is more intense than the β band and this is characteristic of the cytochrome. When the native sixth ligand of P-4501, a low-spin cytochrome P-450 of rabbit liver micro-somes, was replaced with an exogenous ligand, the α band diminished and the cytochrome lost the above-mentioned spectral characteristics. This indicated that the intense a band of the cytochrome was related to the nature of its sixth ligand. P-4481, a rabbit liver microsomal cytochrome P-450 which is essentially in a high-spin state, was converted to low-spin complexes upon binding with various exogenous ligands. The intensity of the α band of low-spin P-4481 derivatives increased in the order of 1-methylimidazole and pyridine complexes, 2-methylim-idazole and 2-methylpyridine complexes, and lower n-alkanol complexes. This order was coincident with the decreasing order of field strength of the exogenous ligands, indicating that the intensity of the α band is inversely proportional to the field strength of the sixth ligand. The absorption spectrum of the 1-propanol complex of P-4481 was essentially identical with that of native low-spin cytochrome P-450, whereas spectra of other P-4481 derivatives were superimposable on those of the corresponding P-4501 derivatives. P-4501 and metmyoglobin showed an orderly red-shift of the Soret band upon binding with pyridine, 1-methylimidazole and cyanide. The order and the degree of the red-shift of the Soret band of P-4501 were essentially the same as those of metmyoglobin, indicating that the native sixth ligand of P-4501 may resemble that of metmyoglobin, a water molecule. From these observations, we propose that the native sixth ligand of ferric low-spin cytochrome P-450 is an oxygenous one such as a water molecule or an oxy-amino acid side chain from the apoprotein.