P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo‐Mediated Lyase Efficiency

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17−C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17‐OH PREG, not only reveals an initially trapped peroxo‐iron intermediate experiencing an H‐bond interaction of the 17‐OH group with the proximal oxygen of the Fe‐Op‐Ot fragment, facilitating peroxo‐ attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction. Resonance Raman spectroscopy studies confirm that the CYP17A1 T306A mutant, with its disordered proton delivery network, exhibits only residual hydroxylase activity, but retains significant lyase efficiency. In the lyase substrate (17OH‐PREG) bound enzyme, the peroxo‐ferric complex was trapped at 77 K and further converts to the hemiketal intermediate upon annealing to 165 K, providing evidence for the peroxo‐mediated C17−C20 scission mechanism.