Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN

Abstract

Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6β-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded.

Keywords: cytochrome P450; enzyme catalysis; enzyme mechanism; mass spectrometry (MS); nuclear magnetic resonance (NMR); oxygenase; steroid; steroidogenesis.

FIGURE 1.

Steroid 17α-hydroxylation and 17α,20-lyase reactions catalyzed by P450 17A1.

FIGURE 2.

Classic catalytic cycle of P450 enzymes (4). Paths for oxygen surrogates (PhI=O, H₂O₂) are also included. Note the FeO₂⁻ (ferric peroxide) and FeO³⁺ (compound I) forms discussed in the text. In the literature there exists different nomenclature for the same iron intermediates in this P450 catalytic cycle (i.e. Fe^IIIO₂⁻, Fe^IIIO₂H, Fe^IVO^+., and Fe^IVOH) (8, 9). For clarity throughout the text, compound I is referred to interchangeably with FeO³⁺, and ferric peroxide is referred to interchangeably with FeO₂⁻. The electron transfers from the reductase are simplifications in that the course of electron flow is probably from FMNH₂/FADH^• to FMNH^•/FADH^• in the first reduction (step 2) and (assuming that the reductase contributes the second electron to the P450) from FMNH^•/FAD^• to FMNH^•/FAD in the second reduction step 4.

FIGURE 3.

Possible mechanisms of P450 17A1-catalyzed 17α,20-lyase reaction and expected ¹⁸O labeling (7). The course of ¹⁸O (from ¹⁸O₂) and deuterium (D) labels are indicated with an asterisk. A, ferric peroxide mechanism (27–31); B, compound I mechanism with hydrogen atom abstraction from the 17α alcohol followed by C17-C20 bond scission to yield an acetyl radical; C, compound I mechanism with hydrogen atom abstraction from the C16 carbon; D, compound I mechanism with hydrogen atom abstraction from the 17α alcohol followed by C17-C20 bond scission to yield a hydrated acetyl radical (gem-diol); E, compound I mechanism with hydrogen atom abstraction from the C21 methyl group followed by C17-C20 bond scission to yield a C17 radical; F, addition of the 17α-hydroxyl group to compound I to yield an iron peroxide-C17 complex, which can decompose via either (a) a C20 gem-diol or (b) a C17-C20 dioxetane. See text for discussion and also Fig. 19. Mechanisms B–D result in an acetyl radical that undergoes oxygen rebound with Fe-*OH (compound II), with an oxygen atom from molecular oxygen (*O₂) into the acetic acid product.

FIGURE 4.

P450 17A1 incubation with [21,21,21-²H₃]17α-hydroxypregnenolone (1) in the presence of ¹⁸O₂ followed by derivatization and analysis by HRMS. A, scheme showing the incubation of deuterated lyase substrate (1) with P450 17A1 and cytochrome b₅ in the presence of ¹⁸O₂. The acetic acid product (3) was derivatized with the diazoethylpyridine reagent (4) and analyzed by liquid chromatography-HRMS. B, ion chromatograms monitoring the various isotopically labeled acetate products that were derivatized to the pyridylethyl esters (5a–h), with 4 ppm mass tolerance parameter. a, m/z 171 window (d₃, ¹⁸O); b, m/z 169 window (d₃, ¹⁶O); c, m/z 170 window (d₂, ¹⁸O); d, m/z 168 window (d₂, ¹⁶O); e, m/z 169 window (d₁, ¹⁸O); f, m/z 167 window (d₁, ¹⁶O); g, m/z 168 window (d₀, ¹⁸O); h, m/z 166 window (d₀, ¹⁶O). C, mass spectrum of the m/z 166.5–171.3 range by selecting the t_R 3.01–3.12-min time interval in the ion chromatogram corresponding to the pyridine ester retention time. Shown at m/z 167.0901 is the peak corresponding to the acetate from background acetic acid from the natural abundance of ¹³C isotope (5i, expected mass, m/z 167.0896, Δ 3.0 ppm). The peak at m/z 171.1099 corresponds to the acetate derived from the enzymatic product (5a, expected mass, m/z 171.1093, Δ 3.5 ppm). D, expansion of the mass spectrum (m/z 168.95–169.22) from C showing the absence of d₃-labeled acetate with no ¹⁸O incorporation (5b, expected mass, m/z 169.1051). E, expansion of the mass spectrum (m/z 170.95–171.22) from C showing the presence of d₃-labeled acetate with ¹⁸O incorporation (5a, expected mass, m/z 171.1093). p, profile (peaks are shown in profile mode and not “centroid”). ESI, electrospray ionization; RT, retention time; NL, normalized level. More information about the meaning of the settings can be obtained from the Xcalibur Qual Browser User Guide (Thermo Scientific).

FIGURE 5.

P450 17A1 incubation with 17α-hydroxy-[2,2,4,6,6,21,21,21-²H₈]progesterone (1b) in the presence of ¹⁸O₂ followed by derivatization and analysis by HRMS. A, scheme showing the incubation of deuterated lyase substrate (1b) with P450 17A1 and cytochrome b₅ in the presence of ¹⁸O₂. The acetic acid product (3) was derivatized with the diazoethylpyridine reagent (4) and analyzed by liquid chromatography-HRMS. B, ion chromatograms monitoring the various isotopically labeled acetate products that were derivatized to the pyridylethyl esters (5a–h), with 6 ppm mass tolerance parameter. a, m/z 171 window (d₃, ¹⁸O); b, m/z 169 window (d₃, ¹⁶O); c, m/z 170 window (d₂, ¹⁸O); d, m/z 168 window (d₂, ¹⁶O); e, m/z 169 window (d₁, ¹⁸O); f, m/z 167 window (d₁, ¹⁶O); g, m/z 168 window (d₀, ¹⁸O); h, m/z 166 window (d₀, ¹⁶O). C, mass spectrum of the m/z 166.5–171.3 range by selecting the t_R 3.10–3.19-min time interval in the ion chromatogram corresponding to the pyridine ester retention time. Shown at m/z 167.0890 is the peak corresponding to the acetate from background acetic acid from the natural abundance of ¹³C isotope (5i, expected mass, m/z 167.0896, Δ 3.6 ppm). The peak at m/z 171.1099 corresponds to the acetate derived from the enzymatic product (5a, expected mass, m/z 171.1093, Δ 4.1 ppm). D, expansion of the mass spectrum (m/z 168.95–169.22) (from C) showing the detection of d₃-labeled acetate with no ¹⁸O incorporation (5b, expected mass, m/z 169.1051, Δ 5.3 ppm). E, expansion of the mass spectrum (m/z 170.95–171.22) from C showing the presence of d₃-labeled acetate with ¹⁸O incorporation (5a, expected mass, m/z 171.1093). p, profile (peaks are shown in profile mode and not “centroid”). ESI, electrospray ionization. RT, retention time. NL, normalized level. More information about the meaning of the settings can be obtained from the Xcalibur Qual Browser User Guide (Thermo Scientific).

FIGURE 6.

Formation of 16,17α-dihydroxyprogesterone and androstenedione from 17α-hydroxyprogesterone by P450 supported by the oxygen surrogate iodosylbenzene. Retention times (t_R) and integration units are indicated on the chromatograms. A, standard compounds. B, reaction (0.5 μm P450 17A1) supported by NADPH-P450 reductase (2.0 μm), cytochrome b₅ (0.5 μm), and NADPH (30-s incubation). C, reaction (0.5 μm P450 17A1 and cytochrome b₅ (0.5 μm)) with 0.30 mm iodosylbenzene (30-s incubation). In control experiments with only cytochrome b₅ and iodosylbenzene (2 mm) mixed with the 17α-hydroxysteroids, the amounts of androstenedione detected were <15% of the amounts observed in this and similar studies with both 17α-hydroxysteroids.

FIGURE 7.

Identification of 16,17α-dihydroxyprogesterone as a product of 17α-hydroxyprogesterone. HRMS spectrum of 16,17-dihydroxyprogesterone formed in a reaction with NADPH-P450 reductase, cytochrome b₅, and NADPH. Exact mass 346.2217 (protonated species): observed for MH⁺, m/z 347.2184 (Δ 9.5 ppm).

FIGURE 8.

Time course and effect of cytochrome b₅ on 19-carbon steroid formation in the presence of iodosylbenzene (PhIO) or the typical NADPH-supported reaction. A, oxidation of 17α-hydroxyprogesterone. B, oxidation of 17α-hydroxypregnenolone. The insets show the NADPH-supported reactions in the presence of cytochrome b₅. The points are means of duplicate assays, shown as means ± range.

FIGURE 9.

Time course and effect of cytochrome b₅ on steroid 16-hydroxylation in the presence of iodosylbenzene (PhIO) or the typical NADPH-supported reaction. A, oxidation of 17α-hydroxyprogesterone. B, oxidation of 17α-hydroxypregnenolone. The points are means of duplicate assays, shown as means ± range.

FIGURE 10.

Reaction products formed from 17α-hydroxyprogesterone and 17α-hydroxypregnenolone in P450 17A1 reactions supported by various factors. Retention times (t_R) and integration units are indicated on the chromatograms. A–C, 17α-hydroxyprogesterone; D–F, 17α-hydroxypregnenolone. A and D, NADPH-P450 reductase, cytochrome b₅, and NADPH; B and E, H₂O₂ (10 mm) (with cytochrome b₅); C and F, iodosylbenzene (PhI=O, 300 μm) (with cytochrome b₅). In these studies the Δ⁵ products (formed from 17α-hydroxypregnenolone) were oxidized to Δ⁴ products to facilitate LC-UV analysis.

FIGURE 11.

Identification of 16-hydroxy steroids as reaction products formed from DHEA and androstenedione. A, authentic steroid standards: 16α-hydroxyandrostenedione, algestone (16α,17α-dihydroxyprogesterone), androstenedione, and 17α-hydroxyprogesterone; B, 10-min DHEA incubation (with products treated with cholesterol oxidase); C, 10-min androstenedione incubation; D, mass spectrum of peak identified as 16-hydroxyandrostenedione (formed from androstenedione); E, MS/MS analysis of m/z 303.2 peak of D.

FIGURE 12.

Time course of 16-hydroxylation of androstenedione and DHEA by P450 17A1.

FIGURE 13.

Rate of conversion of 16α,17α-dihydroxyprogesterone to 6β,16α,17α-trihydroxyprogesterone (Fig. 15) by P450 17A1. The points are means of duplicate assays, shown as means ± range.

FIGURE 14.

Characterization of 16α-hydroxy-DHEA. The product was formed in an incubation of an NADPH-reconstituted P450 17A1 system with 16α,17α-dihydroxypregnenolone and isolated by preparative HPLC. A, HRMS spectrum of [DHEA + 16]⁺ peak, 16α-hydroxy-DHEA (theoretical m/z for MH⁺ 305.2111, found m/z 305.2094). B, NMR spectra of 16α,17α-dihydroxypregnenolone (a) and product (b) in CDCl₃ (600 MHz). See text for discussion.

FIGURE 15.

Characterization of 6β,16α,17α-trihydroxyprogesterone. The product was formed in an incubation of an NADPH-reconstituted P450 17A1 system with 16α,17α-dihydroxyprogesterone and isolated by preparative HPLC. A, HRMS spectrum (theoretical m/z for MH⁺ 363.2166, found m/z 363.2160). B, UV spectra of product (b) compared with 16α,17α-dihydroxyprogesterone (a). C, ¹H NMR spectra of product (b) and 16α,17α-dihydroxyprogesterone (a) in CDCl₃ (600 MHz). Note that the C-18, C-19, and C-21 methyl signals are intact and the chemical shifts of the H-7 protons appear to be moved upfield, as predicted (Table 1). See text and Ref. for discussion, and see supplemental Figs. S-1–S-4 for two-dimensional NMR spectra.

FIGURE 16.

Kinetic solvent isotope effects on 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions catalyzed by P450 17A1 (in the presence of NADPH-P450 reductase, NADPH, and cytochrome b₅). Results are shown as means of four individual experiments ± S.D.

FIGURE 17.

Solvent kinetic deuterium isotope effects on 17α-hydroxyprogesterone and 17α-hydroxypregnenolone reactions catalyzed by P450 17A1 (in the presence of NADPH-P450 reductase and cytochrome b₅). Retention times (t_R) and integration units are indicated on the chromatograms. The substrate concentration was 30 μm in all cases, and the reactions were done in either H₂O or 95% D₂O (v/v) at pH or pD 7.4. A and B, 17α-hydroxyprogesterone; C and D, 17α-hydroxypregnenolone. A and C, H₂O; B and D, 95% D₂O (v/v).

FIGURE 18.

Possible oxidizing alternative to compound I in the iodosylbenzene (PhI=O)-supported reactions (46).

FIGURE 19.

Mechanisms of P450 17A1-catalyzed 17α,20-lyase reaction consistent with ¹⁸O labeling (7), oxygen surrogate results, and solvent kinetic isotope results. The course of an ¹⁸O label (from ¹⁸O₂) is indicated with an asterisk (7, 40). A, compound I mechanism with hydrogen atom abstraction from the 17α alcohol followed by C17-C20 bond scission to yield an acetyl radical; B, addition of the 17α hydroxyl group to compound I to yield an iron peroxide-C17 complex, followed by decomposition via a C17-C20 dioxetane; C, compound I mechanism with hydrogen atom abstraction from the C16 carbon. See text for discussion and also Fig. 3.

FIGURE 20.

Sites of hydroxylation of progesterone by P450 17A1. A, chair configuration of progesterone, with the four sites of attack indicated by arrows. B, wire diagram of 17α-hydroxyprogesterone (17OHP, red) and 16α,17α-dihydroxyprogesterone (16,17OHP, blue) overlaid, with the latter in an alternative configuration to show the proximity of the C-6 atom of 16α,17α-hydroxyprogesterone with the 17-hydroxy group of 17α-hydroxyprogesterone. The model was made using Chem3D, with a minimum root mean square error of 0.1 and minimum root mean square gradient of 0.01. The C14, C10, and O3 atoms of 17α-hydroxyprogesterone were aligned with the C10, C14, and O16 atoms of 16α,17α-dihydroxyprogesterone, respectively, by displaying the distance measurements of each pair of atoms and then running an overlay minimization calculation. The green lines indicate the pair of atoms that were aligned (after overlay minimization, the distances between C14 of 17-OHP and C10 of 16,17-OHP; C10 of 17-OHP and C14 of 16,17-OHP; and O3 of 17-OHP and O17 of 16,17-OHP were 1.2, 1.3, and 1.4 Å, respectively, and are shown as green lines). The distance between the O17 atom of 17α-hydroxyprogesterone and C6 atom of 16α,17α-dihydroxyprogesterone was 1.4 Å.

FIGURE 21.

Summary of current known reactions of human P450 17A1. See also Refs. , , . Rates determined at high substrate concentrations (approximating k_cat conditions) in this study are indicated, in units of nanomoles of product formed/min/nmol P450 17A1, when available. A, products formed from progesterone; B, products formed from pregnenolone.