Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer

In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17 alpha-hydroxylation of al-carbon steroids and the 17,20-lyase activity that cleaves the C-17,-C-20,, bond to produce C-19, sex steroids. Cytochrome b(5), augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b(5) in Saccharomyces cerevisiae and analyzed the 17 alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17 alpha-hydroxylase and trace 17,20 lyase activities toward both Delta(4) and Delta(5) steroids. Coexpression of human OR with P450c17 increases the V-max,,, of both the 17 alpha-hydroxylase and 17,20-lyase reactions B-fold; coexpression of human b(5), with P450c17 also increases the V-max,, of the 17,20-lyase reactions but not of the 17 alpha-hydroxylase reactions. Simultaneous expression of human b(5), with P450c17 and OR, or addition of purified human b(5), to microsomes from yeast coexpressing human P450c17 and OR, further increases the V-max,, of the 17,20-lyase reaction without altering 17 alpha-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b(5),, apo-b(5),, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b(5), does not require electron transfer from b(5), to P450c17. These data suggest that human b(5), acts principally as an allosteric effector that interacts primarily with the P450c17 OR complex to stimulate 17,20-lyase activity.