Interaction of the substrate analogue of cytochrome P-450 and mixed function oxidases.

ABSTRACT

The interaction of a spin labeled compound carrying an alkylating group 4-(3-iodo-2-oxopropylidene)-2,2,3,5,5-pentamethylimidozolydene-1-oxyl (RJ) and capable of binding covalently to mixed function oxidases (MFO) was studied. Measurements of the difference spectrum of cytochrome P-450 demonstrated that RJ induces spectral changes characteristic of type I substrates (lambda max = 403 nm; lambda min = 418 nm). The spectral binding constant (Ks) was 66 microM as determined from the difference spectrum. RJ inhibited the microsomal oxidation of substrates of cytochrome P-450 (aniline, aminopyrine and benzo [a]pyrene). This inhibition was shown not to be associated with the conversion of cytochrome P-450 to cytochrome P-420, or with the suppression of the activities of NADPh-cytochrome c reductase and NADPH-cytochrome P-450 reductase. Thus, evidence was obtained for the possible interaction of RJ with cytochrome P-450. RJ injected to rats (5 mg/100 g body wt, i.p.), inhibited the hydroxylation of benzo[a]pyrene, a type I substrate, (21%) and aniline, a type II substrate, (40%) in the microsomes from their livers. The presence of a paramagnetic center in RJ made it possible to study its interaction with microsomes. The electron paramagnetic resonance (EPR) spectrum of RJ was recorded in the rat liver microsomal fraction after in vivo administration of RJ. In rats treated with RJ (5 mg/100 g), hexobarbital sleeping time was prolonged 1.5-fold. Alkylating analogs of substrates of cytochrome P-450 are suggested as agents for structural studies of the active center of cytochrome P-450 and the development of efficient inhibitors of reactions catalyzed by this enzyme.