Interaction of apo-cytochrome b(5) with cytochromes P4503A4 and P45017A: Relevance of heme transfer reactions

Maximal activity of CYP3A4 is obtained using a reconstitution system consis ting of NADPH-P450 reductase (CPR), dioleoylphosphatidylcholine (DOPC), an ionic detergent, and cytochrome b(5) (b(5)). The mechanism by which bs stim ulates the catalytic activity of CYP3A4 is controversial. Recent data repor t that apo-cytochrome-b(5) (apo-b(5)) can substitute for holo-b(5) by servi ng as an allosteric effector. These authors concluded that b(5) is not dire ctly involved in electron transfer reactions to CYP3A4. We have studied the effect of apo-bs on the ability of purified CYP3A4 to catalyze the 6 beta -hydroxylation of testosterone and horse CYP17A to catalyze the 17,20-lyase reaction. The high molecular weight form of holo-b(5) (HMW-holo-b(5)) stim ulates the GP-hydroxylation of testosterone while the low molecular weight (truncated) form of holo-b(5) (LMW-holo-b(5)) does not. When added to the r econstituted system, HMW-apo-b(5) stimulates the activity of CYP3A4 to a le vel 50-60% of that obtained with HMW-holo-b(5). A similar stimulation of 17 alpha -hydroxyprogesterone metabolism is seen when studying the CYP17A-cat alyzed reaction. Neither LMW-holo-b(5) nor LMW-apo-b(5) stimulates the acti vity of CYP3A4 or CYP17A. CYP3A4 forms a complex during affinity chromatogr aphy with immobilized HMW-holo-b(5) but not with immobilized HMW-apo-b(5). incubation of apo-b(5) with CYP3A4, using conditions required for reconstit ution of enzymatic activities, results in the transfer of heme from the CYP 3A4 preparation to apo-bs, thereby forming holo-b(5) The separation of heme proteins by thiol-disulfide exchange chromatography confirms the formation of holo-b(5). A His67Ala mutant of HMW-b(5) as well as the Zn-substituted protoporphyrin derivative of HMW-b(5) do not stimulate the activity of eith er CYP3A4 or CYP17A. These data show that the mechanism of stimulation of C YP3A4 and CYP17A activities by apo-b(5) results from the formation of holo- b(5) by a heme transfer reaction.