Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. II. Spectral properties.

The spectral properties of multiple forms of cytochrome P-450 purified or partially purified from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rabbits have been studied. Both optical absorption and EPR studies have shown that the oxidized forms of P-450(1), P-450(2) (from PB-treated animals), and P-450(3) (from MC-treated animals) are in the low spin state, having a Soret absorption peak at 417-418 nm. Oxidized P-448(1) (from both PB- and MC-treated animals), on the other hand, shows a Soret peak at 393 nm and a weak band at 646 nm. This and EPR evidence indicate that P-448(1) contains heme which is predominantly in the high spin state, though EPR studies at low temperature indicate the presence of a small amount of low spin ferric heme. The presence of tightly bound MC in P-448(1) purified from MM-treated animals is reflected by characteristic absorption peaks in the ultraviolet region, but this does not affect the absorption spectra in the Soret and visible regions. Emulgen 913, a nonionic detergent, causes the conversion of oxidized P-448(1) from the high to the low spin state, as evidenced by optical absorption and EPR results; bound MC inhibits this conversion in a noncompetitive way. Binding of ethyl isocyanide to reduced P-450(1) and P-448(1) results in the appearance of two Soret peaks in the 430 and 455 nm regions, the relative intensities of which are dependent on pH. At any pH the 455 nm peak of P-448(1) is always higher than that of P-450(1). Benzphetamine and aniline, added to oxidized P-450(1), cause Type I and Type II spectral changes, respectively, but the magnitudes of the changes are small in both cases. The Soret peak of oxidized P-448(1) at 393 nm is completely shifted to 420 nm on addition of aniline, resulting in a reverse Type I spectral change; acetanilide causes the conversion of the Soret peak to the low spin state to only a slight extent. The conversions caused by aniline and acetanilide are both inhibited by the presence of tightly bound MC. On the basis of these and other observations, the spin state of these P-450's are discussed.