LACK OF ELECTRON-TRANSFER FROM CYTOCHROME B(5) IN STIMULATION OF CATALYTIC ACTIVITIES OF CYTOCHROME-P450 3A4 - CHARACTERIZATION OF A RECONSTITUTED CYTOCHROME-P450 3A4 NADPH-CYTOCHROME P450 REDUCTASE SYSTEM ANDSTUDIES WITH APO-CYTOCHROME B(5)

Many catalytic activities of cytochrome P450 (P450) 3A4, the major hum an liver P450 enzyme, require cytochrome b(5) (b(5)) for optimal rates , The stimulatory effect of b(5) on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b(5) to the fe rrous P-450-O-2-substrate complex. We found that apo-b(5), devoid of h eme, could substitute for b(5) in stimulating two prototypic activitie s, testosterone 6 beta hydroxylation and nifedipine oxidation. The sti mulatory effect was not seen with albumin, hemoglobin, catalase, or cy tochrome c. Apo-b(5) could not substitute for b(5) in a testosterone 6 beta hydroxylation system composed of NADH-b(5) reductase and P450 3A 4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min(-1)) to support testosterone 6 beta hydroxy lation (similar to 14 min(-1)) unless b(5) or apo-b(5) was present, wh en rates of similar to 700 min(-1) were measured. The oxidation-reduct ion potential (E(m,7)) of the ferric/ferrous couple of P450 3A4 was un changed (similar to-310 mV) under different conditions in which the ki netics of reduction vp ere altered by the addition of substrate and/or apo-b(5). Rapid reduction of P450 3A4 required substrate and a prefor med complex of P450 3A4, NADPH-P450 reductase, and b(5); the rates of binding of the proteins to each other were 2-3 orders of magnitude les s than reduction rates. We conclude that b(5) can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing it s reduction by NADPH-P450 reductase, without directly transferrin,b el ectrons to P450.