title: Comparisons of hydroperoxide isomerase and monooxygenase activities of cytochrome P450 for conversions of allylic hydroperoxides and alcohols to epoxyalcohols and diols: probing substrate reorientation in the active site.
abstract: Ferric cytochrome P450 (P450) catalyzes intramolecular rearrangements of hydroperoxides to diols by heterolytic scission of the O-O bond and insertion of the terminal oxygen atom into the resulting alcohol. The goals of this work were to further characterize the regio- and stereochemistry of P450 isomerase activity using allylic hydroperoxides and to compare these reactions with NADPH-supported monooxygenations of the corresponding alcohols. Microsomes from phenobarbital-treated rats or purified P450 2B1 catalyzed the conversions of several peroxyquinols, including 2-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,5-dien-1-one (BMPOOH) and its 2,6-dimethyl derivative (TMPOOH), to diols and to alpha,beta-epoxyquinols by predominant (>93%) cis addition of oxygen to the least hindered double bond. Monooxygenation of the 4-hydroxy analogues (quinols) yielded identical cis-epoxyquinols, and both isomerization and monooxygenation reactions exhibited similar enantioselectivities (32-74% enantiomer excess). Regioselectivities were similar for BMPOOH and BMPOH (epoxyquinol:diol ratios of 5.8-7.6), but quite different for TMPOOH and TMPOH (ratios of 0.6 and 6.4, respectively). Bulky peroxyquinols and quinols derived from the A-rings of 17beta-estradiol and estrone were utilized to further compare these reactions. Both estradiol derivatives underwent approximately equal amounts of 6beta-hydroxylation and 1,2-epoxidation. The estrone derivatives also underwent 6beta-hydroxylation, but only estrone quinol yielded a second product consistent with hydroxylation at position 16. The results support several conclusions. (i) Allylic hydroperoxides may be isomerized to alpha,beta-epoxyalcohols by a heterolytic O-O cleavage mechanism with high stereoselectivity. (ii) Hydroperoxide isomerization is an efficient process relative to monooxygenation. (iii) Isomerase substrates remain in proximity to the P450 oxoferryl intermediate and are rapidly captured by the oxidant. Monooxygenase substrates, on the other hand, may bind to ferric P450 in multiple orientations and undergo more extensive substrate reorientation prior to oxidative attack.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kupfer
authors.forename: S Y
authors.initials: SY
authors.lastname: Liu
authors.forename: A J
authors.initials: AJ
authors.lastname: Allentoff
authors.forename: J A
authors.initials: JA
authors.lastname: Thompson
mesh_terms: D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism; D006873:Hydroquinones / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008956:Models, Chemical; D010545:Peroxides / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D004852:Epoxy Compounds; D006873:Hydroquinones; D010545:Peroxides; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase