Active site proton delivery and the lyase activity of human CYP17A1

Cytochrome P450 CYP17A1 plays a central role in steroid hormone metabolism by catalyzing key steps at the junction of corticoid and androgen biosynthesis. This bifunctional enzyme exhibits 17α-hydroxylase activity, converting the Δ⁴- and Δ⁵-steroids progesterone and pregnenolone into their corresponding 17α-hydroxy derivatives, and 17,20-lyase activity, which cleaves the C17–C20 bond of these intermediates to generate the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While 17α-hydroxylation proceeds via the canonical “Compound I” rebound mechanism, the lyase reaction is thought to involve an iron–peroxy intermediate.
We investigated the functional role of Thr306, a conserved residue within the acid/alcohol pair critical for proton delivery required to generate Compound I in P450 enzymes. Using Nanodisc-reconstituted wild-type 17-OH PREG and T306A mutant CYP17A1 with its redox partner cytochrome P450 oxidoreductase (POR), we assessed turnover and coupling efficiencies for both Δ⁴- and Δ⁵-substrates. In the T306A mutant, NADPH coupling during hydroxylation of pregnenolone and progesterone dropped sharply to 0.9% and 0.7%, respectively, compared to 97% and 22% in the wild-type. Despite increased NADPH oxidation, hydroxylase activity was severely compromised in the mutant, indicating extensive uncoupling where electrons and protons were diverted into non-productive pathways—consistent with prior findings in other P450 hydroxylation systems.
Interestingly, disruption of the acid-alcohol pair had minimal impact on the lyase activity. This suggests that carbon-carbon bond cleavage in CYP17A1 operates through a mechanism independent of proton delivery, supporting a model in which a nucleophilic peroxo-anion, rather than Compound I, serves as the active intermediate.