Cytochrome b₅ enhances androgen synthesis by rapidly reducing the CYP17A1 oxy-complex in the lyase step

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two-step reaction process: allylic hydroxylation and carbo-carbon bond scission. Cytochrome b₅ (Cyt-b₅) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt-b₅. To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt-b₅, we measured the reduction rates of oxy-ferrous substrate-bound CYP17A1 by Cyt-b₅ and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt-b₅ reduces oxy-ferrous CYP17A1 10-fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins.