Studies on the mechanism of stimulation of microsomal H2O2 formation and benzo(a)pyrene hydroxylation by substrates and flavone.

The addition of activators like flavone and hexobarbital to hepatic microsomes markedly stimulates H2O2 formation. The similar increase observed with flavone of microsomal hydroxylation of benzo(a)pyrene and its inhibition by catalase and methanol suggests but does not prove a necessary interaction of microsomal H2O2 production with benzo(a)pyrene hydroxylation. Hexobarbital and flavone-stimulated H2O2 formation is optimal at a stoichiometric relationship of these activators and NADPH. This implies either their direct participation as electron donors or their indirect involvement in electron transport by facilitation of stoichiometric substrate cytochrome P-450/NADPH flavoprotein interactions. Steady state kinetics data are consistent with a scheme in which the formation in microsomes of a complex of 1 mole of NADPH with NADPH-cytochrome P-450 reductase and 1 mole hexobarbital with cytochrome P-450 regulates H2O2 formation.