Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5

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, 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.

Automated summary

This study used resonance Raman spectroscopy to compare the effect of Mn-substituted cytochrome b(5) on the oxy complex of CYP17A1, resulting in a specific allosteric interpretation for the more pronounced acceleration seen with the 17OH-PROG substrate. The presence of Mn-b led to a reorientation of a hydrogen bond between the 17OH group of the substrate and the proximal oxygen atom, favoring lyase catalysis, but this effect was not observed for 17OH-pregnenolone.