Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1

Abstract

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 17␣-hydroxylated products to yield 16,17␣-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6␤-hydroxylation of 16␣,17␣-dihydroxyprogesterone and the oxidation of both 16␣,17␣-dihydroxyprogesterone and 16␣,17␣-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 17␣,20-lyase reaction cannot be excluded. Cytochrome P450 (P450)3 enzymes catalyze oxidations of more chemicals than any other group of proteins (1). The list of reactions includes aliphatic and aromatic hydroxylations, heteroatom oxidations, epoxidations, and reactions involving both ring formation and cleavage (2-4). Many P450 reactions are important in the biosynthesis and degradation of steroids and sterols (4, 5), including several critical C-C bond cleavage reactions, i.e. those catalyzed by P450s 11A1, 17A1 (Fig. 1), 19A1, and 51A1 (6, 7).The mechanisms of the C-C cleavage reactions have been the subject of considerable interest and debate. One of the questions with P450s 17A1, 19A1, and 51A1 has been whether the active oxidant is a ferric peroxide (FeO 2 Ϫ ), which is an early intermediate following oxygen addition to the iron (Fig. 2, step 4) or the FeO 3ϩ species (Fig. 2, step 6), often referred to as compound I (4, 10, 11). With P450s 17A1 and 19A1, a variety of approaches has been applied, including theoretical calculations, biomimetic models, spectroscopy, substrate atom labeling, and kinetics (12-32).These C-C bond cleavage reactions are complex, and many of the results are ambiguous; also, a "mixed" mechanism would not be discerned in many of these experiments. One powerful approach originally used by Akhtar and co-workers (27-31) analyzes the actual reaction and can provide discrimination between the nucleophilic FeO 2 Ϫ and electrophilic FeO 3ϩ reactions (Fig. 2), based on the incorporation of 18 O label from O 2 into the carboxylic acid products (Fig. 3) (7). However, these experiments are complicated due to the ubiquitous presence of formic acid (P450 19A1 and 51A1 reactions) and acetic acid (P450 17A1) in laboratory settings. Thus, the data from such experiments are interpreted with the most confidence when the steroid substrates are labeled with 2 H or 13 C isotopes to facilitate analysis (15,33). Even then, the mass spectrometry results can be problematic, particularly if a shift of only one atomic mass unit is introduced and isotopologues derived from 18 O incorporation are not discriminated from molecules containing natural abundance 13 C atoms (33). The incorporation of one atom of 18 O label from O 2 into formic acid (F...

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO3+, for the Carbon–Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1

“…We have shown that P450 17A1 incorporates a single oxygen ( 18 O) atom from molecular oxygen ( 18 O 2 ) into the acetic acid product in the lyase step (ii) when 17 α -hydroxy-[21,21,21- 2 H 3 ]-pregnenolone is used as the substrate. 15 Therefore, in order to determine 18 O-incorporation into pregnenolone by P450 11A1, the derivatized acetic acid product was analyzed for incorporation of one or two 18 O atoms. If the derivatized acetate product contained two 18 O atoms, then the mechanism shown in Scheme 2Ba would be supported.…”

“…The detection of the acetate with a single 18 O atom indicates the possibility of some exchange of the C20-oxygen in the 21-carbon steroid products (Scheme 4, 4a to 4b or 16a to 16b ) during the enzyme-coupled assay. However, the LC-MS analysis of the synthetic pyridine acetate standard with no 18 O label (Supporting Information), which was prepared by treating acetic anhydride with the corresponding alcohol, 15 did show the presence of some acetate containing one 18 O atom (0.003% 18 O). Therefore, the source of the mono- 18 O-containing acetate detected in the positive control experiment (Figures 3B and 3C, 18d-1 ) may be from the acetate derived from background acetic acid.…”

“…The ethylpyridine acetate standard (compound 8d-0 ) was synthesized according to our previous study. 15 …”

“…15,32 The two layers were mixed with a vortex device and the diethyl ether layer was extracted and dried with anhydrous MgSO 4 (~50 mg). The organic layer was filtered through a Pasteur pipet with a cotton plug and was used immediately to treat the incubation extracts prepared above.…”

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

“…In addition, the H 2 O 2 ‐dependent CYPs have been deployed to oxidize non‐native substrates in the presence of decoy molecules . Further, single‐oxygen donors (e.g., periodate and iodosobenzene) are also found to promote CYP‐catalyzed monooxygenation reactions, resulting in a highly reactive ferryl heme π‐cation radical known as compound I . However, few CYPs are known to display such characteristics in the presence of specific chemical compounds.…”