Active Site Structures of CYP11A1 in the Presence of Its Physiological Substrates and Alterations upon Binding of Adrenodoxin

Abstract

The rate-limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at positions 22 and 20, generating 20(R),22(R)-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. This work provides detailed information about the active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high-quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered during the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups, and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O fragments, as revealed by telltale shifts of the observed vibrational modes. Specifically, the vibrational mode patterns observed for the Fe-O-O fragments with the first and third substrates are consistent with H-bonding interactions with the terminal oxygen, a structural feature that tends to promote O-O bond cleavage to form the Compound I intermediate. Furthermore, such spectral data are acquired for complexes with the natural redox partner, adrenodoxin (Adx), revealing protein-protein-induced active site structural perturbations. While this work shows that Adx has an only weak effect on ferric and ferrous CO states, it has a relatively stronger impact on the Fe-O-O fragments of the functionally relevant oxy complexes.

Quantum-Mechanical/Molecular-Mechanical Studies of CYP11A1-Catalyzed Biosynthesis of Pregnenolone from Cholesterol Reveal a C–C Bond Cleavage Reaction That Occurs by a Compound I-Mediated Electron Transfer

“…Indeed, this is in accord with the recent resonance Raman spectra experiments. 9 A substrate-assisted proton-relay mechanism was suggested for Cpd 0→Cpd I conversion in CYP107A1, 43 but through QM/MM studies Thiel et al 44 found the alternative water channel was involved in the proton transfer during Cpd I formation in CYP107A1. Thus, it is interesting to investigate here whether the substrate's hydroxyl group can indeed be directly involved in Cpd I formation in CYP11A1 or other water channels exist.…”

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

“…[10,27,28] Resonance Raman (rR) spectroscopy proved to be am ost valuable tool to investigate the active site structure and hydrogen bonding network in CYP17A1a nd many other heme proteins, [29][30][31] with recent studies of cytochromesP 450 effectively demonstrating its uniquep otential for revealing differential interactions of active site H-bond donors, including substrates, with the keyF e-O-O fragment of enzymatic intermediates. [27,[32][33][34][35] Thus, in rR studies of WT CYP17A1, [10,13] it was clearly shown that the 17OH-PROG substrate, with its relativelyl ow lyase efficiency,i ncludesd onation of an H-bond to the terminalo xygen (O t )o ft he Fe-O p -O t fragment, whereas the 17-OH PREG substrate, more efficiently undergoing the CÀCb ond cleavage reaction, adopts ap osition with its OH-fragment oriented toward the proximal oxygen (Op). This impressive level of structurald efinition provides crucial insight into functional variability,a sf ollows.…”

A requirement for an active proton delivery network supports a compound I-mediated C–C bond cleavage in CYP51 catalysis

“…In more recent studies, a ferric peroxide mechanism has been supported by a kinetic solvent isotope effect (12) and resonance Raman spectroscopy (13,14) for the substrate-induced lyase reaction of CYP17A1, although evidence for a compound I pathway has also been presented (15,16). Compound I was identified as the active oxidant for the lyase step of CYP19A1 (steroid aromatase) using resonance Raman spectroscopy (17), kinetic solvent isotope effects (18), and 18 O isotope labeling (11) and for CYP11A1 (P450scc) using electron paramagnetic resonance/electron nuclear double resonance spectroscopy (19), 18 O isotope labeling (20), and resonance Raman spectroscopy (21). For the third step of the CYP51 reaction, theoretical studies have proposed the operation of a ferric peroxo anion (compound 0) (22), but definitive experimental evidence so far has been lacking.…”