Relationship between the interconversion of cytochrome P-450 and P-420 and its activities in hydroxylations and demethylations by P-450 oxidase systems

Abstract

The relationship between the interconversion of microsomal cytochrome P-450 and P-420 and the hydroxylation and demethylation activities of the P-450 oxidase system was studied. The hydroxylation and demethylation activities could only be partly restored by complete reconversion of P-420 to P-450 by addition of polyols by dilution or by washing. The P-420 produced by detergents, monohydric alcohols or anilines did not retain hydroxylation and demethylation activities. Cytochrome P-450 is not rate limiting in the overall reaction for hydroxylation and demethylation of compounds such as anilines, nitroanisoles and aminopyrine. The substrate specificities of the hydroxylations and demethylations by the P-450 oxidase systems of different tissues differ. The rate of hydroxylation of anilines at the p-position was influenced by not only the hydrophobic character, but also by electronic and steric effects of the substrate. The magnitude of the spectral changes of the microsomes induced by the substrate corresponded to the ESR signal height at g m = 2.25 of microsomal Fe x rather than to the absorption peak of the CO complex of P-450 at 450 mμ. The hydroxylation and demethylation activities were observed with either NADPH or NADH. Evidence is presented that microsomal NADPH-cytochrome c reductase is reduced by NADH and the K m for the reductase with NADH is greater than the K m for NADPH.