Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography–mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process. This work utilizes GC/MS analysis to document product formation and show that, upon annealing, the Cytochrome P450 17A1 samples generate expected lyase products Dehydroepiandrosterone (DHEA) and Androstenedione (AD). [Display omitted] •Cytochrome P450 17A1 catalyzes the production of androgens.•The precise mechanism for the lyase reaction has been debated for decades.•Cryoradiolysis generates expected lyse products DHEA and Androstenedione.•Careful extraction of cryoreduced samples allows isolation of lyase products.•GC/MS reveals expected lyase products consistent with peroxo-mediated reaction.