Cytochrome P450BM-3 and P450 11A1 retain Compound I (FeO3+) chemistry with electrophilic substrates poised for Compound 0 (Fe3+O2-) reactions

Abstract

The catalytic cycle of cytochrome P450 (P450) enzymes involves ferric peroxide anion (Fe³⁺O₂^-, Compound 0) and perferryl oxygen (FeO³⁺, Compound I) intermediates. Compound I is generally viewed as responsible for most P450-catalyzed oxidations, but Compound 0 has been implicated in the oxidation of some carbonyl compounds, particularly deformylation reactions. We considered the hypothesis that Compound 0 could also attack other electrophilic carbon atoms and accordingly positioned keto groups at preferred hydroxylation sites of substrates for two P450s with well-defined catalytic reactions, bacterial P450_BM-3 (102A1), and human P450 11A1. The predicted products of Compound I and Compound 0 reactions were analyzed. With the normally preferred ω-1 site blocked, P450_BM-3 oxidized 12-oxotridecanoic acid (12-oxo C13:0) only at the ω-2 position (yielding 11-hydroxy,12-oxotridecanoic acid), indicative of a Compound I oxidation. P450 11A1 is highly selective for catalyzing the 22R-hydroxylation of cholesterol (and some other sterols) in the first step of its overall side-chain cleavage reaction. With 22-oxocholesterol as the substrate, P450 11A1 (slowly) generated only 23-hydroxy,22-oxocholesterol, indicative of Compound I oxidation. Neither P450 generated the products expected from nucleophilic Compound 0 reactions. We conclude that the strategic placement of electrophilic oxo substituents at sites of substrate hydroxylation failed to divert the oxidation mechanism to a Compound 0 pathway with either enzyme. Instead, the Compound I mechanism-blocked at the preferred reaction site-was redirected to neighboring carbons, suggesting that the basis for Compound 0-mediated reactions lies in chemical properties of the enzyme rather than those of the substrate.

Keywords: 11A1; BM-3; CYP; Compound 0; Compound I; cytochrome P450; enzyme mechanism; fatty acid oxidation; steroid oxidation.

Figure 1

Classical mechanism for P450-catalyzedoxidations.

Figure 2

Generalized mechanism for P450-catalyzedoxidations.

Figure 3

Three-step oxidation of cholesterol topregnenolone by P450 11A1.

Figure 4

Potential P450_BM-3Compound 0 reactions with12-oxotridecanoicacid.

Figure 5

Potential P450 11A1 Compound 0 reactions with22-oxocholesterol.

Figure 6

Potential P450_BM-3Compound I reactions with 12-oxotridecanoic acid.

Figure 7

Stimulation of NADPH oxidation by P450_BM-3 and fatty acids. P450_BM-3 (0.25 μM) and NADPH (150 μM) were incubated with: A, lauric acid (C12:0), B, 12-oxotridecanoic acid (12-oxo C13:0), C, myristic acid (C14:0), and D, palmitic acid (C16:0) at fatty acid concentrations of 100 μM (red) or 1000 μM (blue). E, plot of NADPH oxidation rate vs. concentration of 12-oxotridecanoic acid (0–1200 μM). F, comparison of NADPH oxidation rates (n = 1) stimulated by each fatty acid (100 μM). Black lines (Parts A–D): no fatty acid added.

Figure 8

Fatty acid binding and oxidation kinetics of P450_BM-3. A, binding spectra of P450_BM-3 (1 μM) and 12-oxotridecanoic acid (200 μM (blue) and 1000 μM (magenta) (bound vs free P450 difference spectra). B, binding isotherm of 12-oxotridecanoic acid (12-oxo C13:0, 0–1200 μM) and P450_BM-3 (1 μM). Substrate binding is plotted as ΔA₃₉₀-A₄₂₀. C, selected ion (SIR) chromatograms of proposed P450 Compound 0 products (black) and the [M-H+16]⁻ product of the P450_BM-3 reaction with 12-oxo C13:0. Di-acid, dodecanedioic acid; 11-OH, 11-hydroxy undecanoic acid; Sub, substrate (12-oxotridecanoic acid); BV, Baeyer-Villiger product; 10-und, 10-undecanoic acid (Fig. 4). D, chromatogram of P450_BM-3 incubations (10 min (gray), 30 min (blue), and 90 min (red)) with 12-oxo C13:0. The [M-H+16]⁻ product ion (m/z 246.3) channel is shown. E, peak area integration (n = 2) of the m/z 246.3 product ion. F, fatty acid hydroxylation rate of P450_BM-3 with lauric (C12:0), 12-oxotridecanoic (12-oxo C13:0), myristic (C14:0), and palmitic (C16:0) acids (n = 2).

Figure 9

Characterization of P450_BM-3 fatty acid hydroxylation product. A, scheme depicting the formation of both possible hydroxylation products (at carbons 11 (C₁₁, red) and 13 (C₁₃, blue) (R = C₁₀H₁₈O₂^-). The products of the P450_BM-3 reaction were reduced (with NaBH₄) and the product (a diol) was cleaved (with HIO₄) to yield the possible products displayed. A dashed arrow and red cross indicate a molecule that was not detected. B, D, F, and H, chromatograms of a P450_BM-3 incubation with 12-oxotridecanoic acid with (red) and without (black) initiation by NADPH (A, m/z channel 243.1601) that was subsequently treated with D, NaBH₄ (m/z channel 245.1758) and then F and H, HIO₄ (m/z channels of 199.1339 and m/z 213.1496, respectively). C, E, and G, m/z spectra collected at the retention times (t_R) of the products identified in B, D, and F, respectively. All chromatograms are extracted with a 10 ppm m/z window from the theoretical m/z of the product. The corresponding chemical structures of each product are displayed.

Figure 10

Sterol binding and oxidation kinetics of P450 11A1. A, binding spectra of P450 11A1 (1 μM) and 22-oxocholesterol (5 μM, red) that was then treated with cholesterol (5 μM, blue). B, binding isotherm of 22-oxocholesterol (0–100 μM) and P450 11A1 (1 μM). Substrate binding is plotted as ΔA₄₁₀-A₄₂₈. C, chromatogram of the oxidation product ([M+H-H₂O]⁺ ion, m/z 399.3258 ± 5 ppm) of the P450 11A1 reaction (5, 15, and 60 min) with 22-oxocholesterol. D, comparison of 22-oxocholesterol hydroxylation rate (estimated from substrate consumption of three independent 60 min reactions with 25 μM substrate), to that of cholesterol, 22R-hydroxy cholesterol (22R-OH), and 20R,22R-dihydroxy cholesterol (from two replicates of 5 min reactions of 20 μM substrate).

Figure 11

Characterization of P450 11A1 sterol oxidation product.A, scheme depicting the formation of both possible hydroxylation products (at carbons 23 (C₂₃, red) and 20 (C₂₀, blue) (R = C₁₉H₂₈⁺). The products of the P450 11A1 hydroxylation reaction were subsequently reduced (with NBH₄) and the product (a diol) was cleaved (with HIO₄) to yield the possible products displayed. The resulting aldehyde was oxidized (with NaClO₂) to an acid, the identity of which was confirmed with an authentic commercial standard. A dashed arrow and red cross indicate a molecule that was not detected. B, D, F, and H, APCI-MS chromatograms (of the [M+H-H₂O]⁺ ions) of a P450 11A1 incubation with 22-oxocholesterol with (red) and without (black) initiation by NADPH (B, m/z channel 399.3258 ± 3 ppm) that was subsequently treated with NaBH₄ (D, m/z channel 401.3414 ± 10 ppm), cleaved with HIO₄ (F, m/z channel 313.2526) and oxidized with NaClO₂ (H, m/z channel 329.2475) (33, 51). C, E, G, and I, m/z spectra collected at the retention time (t_R) of the product identified in Parts B, D, F, and H, respectively.