Spectral Evidence for Weak Ligand in Sixth Position of Hepatic Microsomal Cytochrome P-450 Low Spin Ferric Iron in vivo

Abstract

The spin state of cytochrome P-450 ferric iron in vivo and in vitro was examined in freshly prepared liver microsomes from rabbit, guinea pig, hamster, mouse and rat by room-temperature difference spectrophotometry. The administration of compounds which induce certain forms of P-450 (phenobarbital, .beta.-naphthoflavone or 3-methylcholanthrene) to each of these species produces consistent detectable changes in the spin state of P-450 iron in vivo. Simple liquids of known relatively pure type I, reverse type I or type II character (cyclohexane, butanol-1 and octylamine-1, respectively) produce detectable changes in the in vitro spin state of P-450 iron. By the method of off-balance spectrophotometry (in which microsomal cytochrome b5 is made equal in both cuvettes), these differences in spin state in vivo and in vitro were shown to vary among the 5 mammalian species and are altered by prior treatment in vivo with the inducers of P-450 and by addition in vitro of such chemicals as cyclohexane, butanol-1 and octylamine-1. The magnitude of the peak-to-trough height in type I, reverse type I and type II difference spectra (or whether any spectrum is detected) is shown to depend largely on the in vivo spin state of the P-450 iron in freshly prepared microsomes at the start of the experiment. The presence of a 410 nm trough in the type II difference spectrum represents a ligand having reverse type I character in the 6th coordination site of in vivo low spin iron. Apparently the 6th ligand for in vivo low spin P-450 ferric iron is a hydroxyl group from an adjacent amino acid residue of the P-450 protein (or moiety of similar ligand field strength, e.g., a water model molecule) rather than a stronger ligand such as one having lone pair electrons from an N atom.