title: Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies.
abstract: Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208-3113, United States.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus , Kuprevicha Street 5/2, Minsk 220141, Belarus.
authors.forename: Natallia
authors.initials: N
authors.lastname: Strushkevich
authors.affiliation: Structural Genomics Consortium, University of Toronto , 101 College Street, Toronto, ON, Canada M5G 1L7.
authors.forename: David
authors.initials: D
authors.lastname: Smil
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus , Kuprevicha Street 5/2, Minsk 220141, Belarus.
authors.forename: Aliaksei
authors.initials: A
authors.lastname: Yantsevich
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus , Kuprevicha Street 5/2, Minsk 220141, Belarus.
authors.forename: Andrey
authors.initials: A
authors.lastname: Gilep
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus , Kuprevicha Street 5/2, Minsk 220141, Belarus.
authors.forename: Sergey
authors.initials: S
authors.lastname: Usanov
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208-3113, United States.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D006888:Hydroxycholesterols / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D010084:Oxidation-Reduction; D011284:Pregnenolone / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006888:Hydroxycholesterols; C003585:22-hydroxycholesterol; D011284:Pregnenolone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme