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. 2024 Aug 12;63(33):e202406542.
doi: 10.1002/anie.202406542. Epub 2024 Jul 17.

Roles of Ferric Peroxide Anion Intermediates (Fe3+O2 -, Compound 0) in Cytochrome P450 19A1 Steroid Aromatization and a Cytochrome P450 2B4 Secosteroid Oxidation Model

Yasuhiro Tateishi  1 , Kevin D McCarty  1 , Martha V Martin  1 , Francis K Yoshimoto  2 , F Peter Guengerich  1
Affiliations

Roles of Ferric Peroxide Anion Intermediates (Fe3+O2 -, Compound 0) in Cytochrome P450 19A1 Steroid Aromatization and a Cytochrome P450 2B4 Secosteroid Oxidation Model

Yasuhiro Tateishi et al. Angew Chem Int Ed Engl. .

Abstract

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3-step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C-C bond scission (removing the 19-oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2 -, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen-18 labeling, i.e., from 18O2 and H2 18O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19-oxo-androstenedione by human P450 19A1 and of a model secosteroid, 3-oxodecaline-4-ene-10-carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non-enzymatic acid-catalyzed deformylation, yielding 19-norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe3+O2 -) in both P450 19A1 oxidation of 19-oxo-androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016-16025), attributed to several technical modifications.

Keywords: cytochrome P450; enzyme mechanisms; mass spectrometry; oxidation mechanisms; steroid biosynthesis.

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Figures

Figure 1.
Figure 1.
Acid-catalyzed degradation of ODEC and 19-oxo-androstenedione.
Figure 2.
Figure 2.
UPLC-HRMS traces of incorporation of 18O2 into formic acid in 19-oxo-androstenedione oxidation. A–C) Representative mass traces of derivatized DCOOH (panel A, m/z 167.0925) and DC18OOH (panel B, m/z 169.0968), and 17α-OH progesterone (panel C, m/z 331.2268 for 16O product and m/z 333.2310 for 18O product) with 5 ppm mass tolerance. Panels A and B shows peaks with (red line) or without (black line) the NADPH-generating system at the time of incubation, and panel C is from the (+) NADPH sample. D) Summary of 18O incorporation assay. Values were normalized by the percentage of 18O incorporation calculated with progesterone 17α-hydroxylation by P450 17A1. Data represent the means ± standard deviation from triplicate assays. Separate trials were run on different days. See Figure S5 for raw data.
Figure 3.
Figure 3.
UPLC-HRMS traces of incorporation of H218O into formic acid in 19-oxo-androstenedione oxidation. A–C) Representative mass traces of derivatized DCOOH (panel A, m/z 167.0925), DC18OOH (panel B, m/z 169.0968), and DC18O18OH (panel C, m/z 171.1010) with 5 ppm mass tolerance. Each trace shows peaks with (red line) or without (black line) the NADPH-generating system at the time of incubation. D) Representative mass traces of 19-oxo-androstenedione-d in (–) NADPH samples with 5 ppm mass tolerance (m/z 302.1861 for 16O product and m/z 304.1903 for 18O product). See Figure S6 for raw data.
Figure 4.
Figure 4.
UPLC-HRMS traces of incorporation of 18O2 into formic acid in ODEC oxidation. A and B) Representative mass traces of derivatized DCOOH (panel A, m/z 167.0925) and DC18OOH (panel B, m/z 169.0968) with 5 ppm mass tolerance. Each trace shows peaks with (red line) or without (black line) the NADPH-generating system at the time of incubation. The peak of interest in panel A is at tR=4.55 min. The peaks at tR= 5.32 and 5.56 min are not NADPH dependent. C) Representative mass trace (with 5 ppm mass tolerance) of derivatized ODEC-COOH (16O product, m/z 314.1751; 18O product, m/z 316.1793) from (+) NADPH sample. D) Normalized 18O incorporation into formic acid from the P450 2B4 assay. Data represent the mean ± standard deviation from triplicate samples. See Figure S8 for raw data.
Scheme 1.
Scheme 1.
P450-catalyzed reactions focused on in this study. A) P450 19A1 reaction sequence beginning with androstenedione. A similar reaction series begins with testosterone and yields 17β-estradiol.; B) P450 2B4-catalyzed model steroid aromatization of ODEC.
Scheme 2.
Scheme 2.
Two major proposed mechanisms for the third step of P450 19A1 (10-deformylation).[11] Steroid C and D rings are removed for clarity (i.e., mechanism for ODEC). The colored oxygen atoms show the course of oxygen atoms from O2 (orange) and H2O (blue) oxygen incorporation into formic acid. A) Compound 0 mechanism. B) Compound I mechanism.
Scheme 3.
Scheme 3.
General scheme for concomitant Compound 0 and Compound I reactions by P450 enzymes.
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