Maximal activity of CYP3A4 is obtained using a reconstitution system consisting of NADPH−P450 reductase (CPR), dioleoylphosphatidylcholine (DOPC), an ionic detergent, and cytochrome b₅ (b₅). The mechanism by which b₅ stimulates the catalytic activity of CYP3A4 is controversial. Recent data report that apo-cytochrome b₅ (apo-b₅) can substitute for holo-b₅ by serving as an allosteric effector. These authors concluded that b₅ is not directly involved in electron transfer reactions to CYP3A4. We have studied the effect of apo-b₅ on the ability of purified CYP3A4 to catalyze the 6β-hydroxylation of testosterone and horse CYP17A to catalyze the 17,20-lyase reaction. The high molecular weight form of holo-b₅ (HMW-holo-b₅) stimulates the 6β-hydroxylation of testosterone while the low molecular weight (truncated) form of holo-b₅ (LMW-holo-b₅) does not. When added to the reconstituted system, HMW-apo-b₅ stimulates the activity of CYP3A4 to a level 50−60% of that obtained with HMW-holo-b₅. A similar stimulation of 17α-hydroxyprogesterone metabolism is seen when studying the CYP17A-catalyzed reaction. Neither LMW-holo-b₅ nor LMW-apo-b₅ stimulates the activity of CYP3A4 or CYP17A. CYP3A4 forms a complex during affinity chromatography with immobilized HMW-holo-b₅ but not with immobilized HMW-apo-b₅. Incubation of apo-b₅ with CYP3A4, using conditions required for reconstitution of enzymatic activities, results in the transfer of heme from the CYP3A4 preparation to apo-b₅, thereby forming holo-b₅. The separation of heme proteins by thiol−disulfide exchange chromatography confirms the formation of holo-b₅. A His67Ala mutant of HMW-b₅ as well as the Zn-substituted protoporphyrin derivative of HMW-b₅ do not stimulate the activity of either CYP3A4 or CYP17A. These data show that the mechanism of stimulation of CYP3A4 and CYP17A activities by apo-b₅ results from the formation of holo-b₅ by a heme transfer reaction.

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