Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b 5 and epoxide hydrase in reconstituted phospholipid vesicles

Abstract

The roles of rabbit liver cytochrome b 5, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450 LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450 LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450 LM3. Cytochrome b 5 enhanced cytochrome P-450 LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450 LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b 5 was present in the membrane.