Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b 5

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydro­epiandrosterone from pregnenolone. The second reaction is the C17–C20 bond scission, which is strongly dependent on the presence of cytochrome b 5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b 5 on C–C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b 5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b 5 (Mn-Cytb 5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe–O mode is observed in the presence of Mn-b 5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe–O–O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb 5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.