Resonance Raman Spectroscopy Reveals That Substrate Structure Selectively Impacts the Heme-Bound Diatomic Ligands of CYP17

Abstract

An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the cortico-steroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17 α -hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17 α -hydroxyprogesterone (17-OH PROG). The hydroxylated products then are either used for production of corticoids or undergo a second CYP17 catalyzed-transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions.

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis

“…As was reported in an earlier work [29], binding of pregnenolone to substrate-free CYP17A1 causes a spin state conversion from almost pure low spin (LS) to largely high spin (HS) form, while binding of 17α-hydroxypregnenolone generates a lower HS fraction. The persistence of more LS component being attributed to the tendency of the 17α-hydroxy fragment of the substrate to directly interact with the heme iron or promote retention of distal pocket water molecules.…”

Spectral Characterization of a Novel NO Sensing Protein in Bacteria: NosP

“…All three samples exhibit spectra corresponding to a 6-coordinated low spin species, with the ν 4 , ν 3 , and ν 2 modes occurring at 1373, 1498 and 1590 cm −1 , respectively, consistent with previously published data for other heme proteins. 74, 75 No evidence of a band at 1358 cm −1 is detected, which is a marker band of unligated ferrous heme, demonstrating that the spectra are not contaminated with modes of photo-dissociated species. All three samples exhibit quite similar spectral patterns, with the ν (Fe-C) frequency observed at the range of 496 to 497 cm −1 , with its correlated C-O stretching mode at 1955~1959 cm −1 .…”

“…These assignments are within the typical range of well-documented frequencies of other heme proteins, which have generally been verified by 13 CO isotope substitution. 75–77 …”

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

“…Second, resonance Raman spectroscopy supports differential hydrogen bonding of the efficient and poor lyase substrates to the proximal or distal oxygens, respectively, for the ferrous dioxygen state immediately preceding the peroxy state in the P450 catalytic cycle (24). Third, resonance Raman studies with CYP17A1 bound to CO demonstrate that two distinct ironcarbon-oxygen vibrational modes exist for 17␣-hydroxypregnenolone, and only a single mode exists in the presence of hydroxylase substrates and the poor lyase substrate, 17␣-hydroxyprogesterone (49). Thus the recent functional and current structural evidence converges to strongly support two binding modes for 17␣-hydroxypregnenolone, one of which is oriented appropriately to stabilize the peroxo intermediate and undergo the lyase reaction.…”