Similar Rates of Second Electron Transfer and Single-Turnover Dehydroepiandrosterone Formation for Oxyferrous Human Cytochrome P450 17A1 (Steroid 17-Hydroxylase/17,20-lyase)-17-hydroxypregnenolone Complex with Either Human Cytochrome P450-Oxidoreductase or Human Cytochrome b5
Year: 2025
Type: article
Abstract: The 17-hydroxylase and 17,20-lyase activities of cytochrome P450 17A1 are required for androgen biosynthesis, which is the target of the prostate-cancer drug abiraterone acetate. Cytochrome b 5 (b5) stimulates the 17,20-lyase activity 8-fold in reconstituted systems containing P450-oxidoreductase (POR); however, the mechanism of the b5 effect and the rate-limiting step(s) of these catalytic cycles are not known. Using stopped flow spectroscopy and rapid chemical quench under single-turnover conditions, we determined the effects of b5 on rates of individual steps of the 17-hydroxylase and 17,20-lyase reactions. Steps prior to and including oxygen binding were rapid for both reactions (>9 s –1 ), and rates of dehydroepiandrosterone release (4–5 s –1 ) were also fast and not increased by b5. Starting with 17-hydroxypregnenolone-bound oxyferrous P450 17A1, the electron transfer rate was slower from b5 than from POR (2.9 ± 0.2 versus 7.4 ± 0.1 s –1 ), whereas return to ferric P450 17A1 was faster with b5 than from POR (1.7 ± 0.3 versus 1.3 ± 0.1 s –1 ). Using the same conditions as electron transfer experiments for rapid chemical quench, rates of dehydroepiandrosterone formation were equivalent with reduced POR or b5 (2.4 ± 0.4 versus 2.3 ± 0.3 s –1, respectively); b5 reduced hydrogen peroxide formation under multiple turnover conditions. We conclude that rates of electron transfer and product formation for the 17,20-lyase reaction starting with reduced oxyferrous P450 17A1 are similar and partially rate-limiting to either POR or b5. These data suggest that the b5 effect on the 17,20-lyase reaction manifests only during multiple turnover conditions rather than enhancing single-turnover kinetics.
Source: Biochemistry
Institutions: University of Michigan, Ann Arbor VA Medical Center
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W4410242866