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

Abstract

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.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1

“…Clearly seen are two positive bands at 797 and 779 cm −1 in the mid-frequency region, both shifting as expected (38 and 40 cm −1 ) upon 18 O 2 substitution. Following our earlier work, [16][17][18]43 these features are most reasonably assigned to the ν(O−O) modes of the peroxo-and hydroperoxo-intermediates, respectively. The corresponding ν(Fe−O) modes are seen at 560 cm −1 (peroxo) and 570 cm −1 (hydroperoxo).…”

“…Based on the crystallographic studies of CYP17 by Scott and coworkers, 21 the most reasonable candidates for such donors are the C 17 −OH groups of the two lyase substrates, as depicted in Scheme 1. Figure 3 shows the 16 [15][16][17][18]43 Comparison of these results for the E305G variant with those previously obtained for the wild-type enzyme supports the conclusion that the hydrogen-bonding patterns observed for these two lyase substrates are shifted. That is, with wild type, the bound 17OH-PREG provides an H-bond to the proximal oxygen, exhibiting a ν(Fe− 16 O) at 526 cm −1 , whereas for the E305G variant, this same substrate is oriented to generate an H-bonding interaction primarily with the terminal oxygen, exhibiting a ν(Fe− 16 O) at 540 cm −1 .…”

“…The rR difference spectrum obtained with the lyase substrate 17OH-PREG is shown in Figure 4A. Clearly seen are two positive bands at 797 and 779 cm −1 in the mid-frequency region, both shifting as expected (38 and 40 cm −1 ) upon 18 O 2 substitution. Following our earlier work, [16][17][18]43 these features are most reasonably assigned to the ν(O−O) modes of the peroxo-and hydroperoxo-intermediates, respectively.…”

“…Previous work in our laboratory discovered a key intermediate in the nucleophilic mechanism of carbon−carbon 1). 16,18,22 The rR spectra for E305G mutant bound with 17OH-PREG was obtained after annealing of the cryoreduced sample at 165 K for 1 min then returning to 77 K for data collection (Figure 5A). Excitation of the Raman bands with 442 nm radiation, close to the 440 nm Soret peak of the peroxo states, 16,18,22 enhances Fe−O−O modes and reveals that the peroxo anion intermediate has disappeared, leaving only weak difference features observed at 779/740 cm −1 which are associated with the ν(O−O) mode of the hydroperoxo-species, whose ν(Fe−O) mode is seen at 570/545 cm −1 .…”

Nanodiscs: A toolkit for membrane protein science

“…Kinetic solvent isotope effects proved to be a robust probe for the proton transfer events that control the population of iron‐bound oxidants 164,165 . Cryotrapping and annealing at increasing temperatures monitors the protonation events and led to the identification of a new hemiacetal intermediate, characterized by resonance Raman spectroscopy, in human P450 CYP17A1 163,166,167 …”