Compound I is the reactive intermediate in the first monooxygenation step during conversion of cholesterol to pregnenolone by cytochrome P450scc (CYP11A1): EPR / ENDOR/cryoreduction-annealing studies

摘要

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 the C20-C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77K 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 22R-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77K 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 145K. 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 185K 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 sKIE, 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 agent that hydroxylates CH, rather than the hydroperoxy-ferric heme.