title: 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.
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 20α,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). (1)H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe(III) is coordinated to the 22R-O(1)H of 22-HC, where the (1)H 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.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Gilep
authors.forename: Natallia V
authors.initials: NV
authors.lastname: Strushkevich
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Usanov
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011284:Pregnenolone / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D011284:Pregnenolone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D010100:Oxygen