THE EFFECTS OF CYTOCHROME B(5), NADPH-P450 REDUCTASE, AND LIPID ON THE RATE OF 6-BETA-HYDROXYLATION OF TESTOSTERONE AS CATALYZED BY A HUMANP450 3A4 FUSION PROTEIN

The recombinant fusion protein containing the heme domain of human P45 0 3A4 and the flavin domains of rat NADPH-cytochrome P450 (P450) reduc tase (rF450[mHum3A4/mRat0R]L1) requires both phospholipid and detergen t as well as cytochrome bs (b(5)) for the NADPH-dependent catalysis of the 6 beta-hydroxylation of testosterone. NADPH oxidation results in the formation of hydrogen peroxide in the presence or absence of phosp holipid and detergent. NADPH oxidation and hydrogen peroxide formation are inhibited by the addition of b(5) and stimulated greater than 3-f old by the addition of testosterone. Marked differences in the ability of various phospholipids to support the P450-dependent 6 beta-hydroxy lation of testosterone by the fusion protein were seen. Addition of a 4-fold excess of purified NADPH-P450 reductase, in the presence of pho spholipid, detergent, and b(5), stimulates the rate of testosterone 6 beta-hydroxylation approximately 10-fold, providing turnover rates as high as 80 min(-1) for P450 3A4. Approximately 30% of the rate bf hydr ogen peroxide formation is not sensitive to inhibition by the P450 inh ibitor ketoconazole, suggesting hydrogen peroxide (or superoxide anion ) formation/directly from the reduced flavin domains of the fusion pro tein. it is proposed that the stimulation of NADPH oxidation observed following the addition of testosterone to the fusion protein may serve as a useful means of monitoring the interaction of other substrates w ith this P450 and thereby permit the rapid screening of chemicals to e valuate their potential metabolism by a human P450. (C) 1995 Academic Press, Inc.