Correlation of type I, type II, and reverse type I difference spectra with absolute changes in spin state of hepatic microsomal cytochrome P-450 iron from five mammalian species

The spin state of cytochrome P-450 ferric iron in ui uo and in oitro was examined in freshly prepared liver microsomes from rabbit, guinea pig, hamster, mouse, and rat by low temperature electron paramagnetic resonance CEPR) spectroscopy and compared with room temperature difference spectral changes.Simple liquids of known relatively "pure" type I, reverse type I, and type II tcyclohexane, butanol-1, and octylamine-1, respectively) were used to produce changes in the in vitro spin state of P-450 iron.The in uiuo spin state in intact freshly prepared microsomes reflects the environment of the P-450 iron itself in the vicinity of the heme protein, which may include bound endogenous compounds, whereas the in vitro spin state reflects binding of added exogenous compounds to the iron (or binding in the vicinity of the heme iron).A method is proposed for determining the percentage of high spin P-450 iron in uiuo per total P-450 content, by knowing the g, = &O/g, = 2.24 ratio of EPR signal heights.Because of broadening, g value shifts, and splittings of the EPR derivative spectra during these in uiuo or in vitro manipulations, it is demonstrated why this method cannot be more precise than just an approximation.Nevertheless, this method is shown to give a good approximation among all five mammalian species examined and during various in uiuo treatments with inducers of certain forms of P-450 and during in vitro additions of chemicals.In order of decreasing amounts of high spin P-450 iron in uiuo, the species may be ranked: rabbit > guinea pig = hamster -mouse > rat.The EPR data with a series of compounds of increasing ligand field strengths added in vitro are consistent with the hypothesis that the sixth coordination site for in uiuo low spin P-450 ferric iron is an oxygen atom from an adjacent amino acid residue or a water molecule, rather than a stronger ligand such as a nitrogen atom.It also is shown