Compound i is the reactive intermediate in the first monooxygenation step during conversion of cholesterol to pregnenolone by cytochrome P450scc: EPR/ENDOR/cryoreduction/annealing studies

Roman Davydov, Andrey A. Gilep, Natallia V. Strushkevich, Sergey A. Usanov*, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22R-hydroxycholesterol (22-HC) and then 20±,22R-dihydroxycholesterol (20,22-DHC), followed by C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-ferriheme P450scc intermediate, along with a minor fraction of peroxo-ferriheme intermediate that converts into a new hydroperoxo-ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). 1H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O1H of 22-HC, where the 1H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.

Original languageEnglish (US)
Pages (from-to)17149-17156
Number of pages8
JournalJournal of the American Chemical Society
Volume134
Issue number41
DOIs
StatePublished - Oct 17 2012

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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