Mechanistic and spectroscopic characterization of human CYP17A1 in Nanodiscs

The human cytochrome P450 CYP17A1 plays a critical role in the production of steroid hormones, converting pregnenolone to dehydroepiandrosterone and progesterone to androstenedione. Sequential reactions catalyzed by CYP17A1 are hydroxylation at C17 position, followed by C17 - C20 carbon‑carbon bond scission. The mechanism of the lyase reaction is still debated, with two proposed reaction pathways favoring either a peroxo- (Compond 0) or iron-oxo (Compound 1) driven catalysis. In this review we summarize the results obtained through collaboration between the Sligar laboratory at University of Illinois and the Kincaid laboratory at Marquette University over the last 15 years. We used a combination of spectroscopic and functional studies of human CYP17A1 incorporated in lipid Nanodiscs, mimicking the native membrane environment, to dissect the elementary steps of P450 reaction cycle and characterize the iron‑oxygen intermediates in the presence of substrates for both reactions catalyzed by CYP17A1. In addition, we used the mutations E305G and T306A to probe the effect of perturbing the proton delivery required for the formation of Compound 1, but not for Compound 0, and the mutation N202S involved in substrate positioning at the active site. Resonance Raman spectra, in combination with cryo-radiolytic reduction of the oxy-complex of CYP17A1, provided a detailed picture of hydrogen bonding and protonation of peroxo- and hydroperoxo- intermediates and identified a new transient hemiketal complex on the peroxo-driven pathway of lyase reaction. These results consistently demonstrated the predominant role of the peroxo-driven catalysis for the lyase reaction in CYP17A1 incorporated in lipid Nanodiscs.