Interaction of Apo-cytochrome b5 with Cytochromes P4503A4 and P45017A: Relevance of Heme Transfer Reactions

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Abstract
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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