title: Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy.
abstract: EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.forename: Roshan
authors.initials: R
authors.lastname: Perera
authors.forename: Shengxi
authors.initials: S
authors.lastname: Jin
authors.forename: Tran-Chin
authors.initials: TC
authors.lastname: Yang
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Bryson
authors.forename: Masanori
authors.initials: M
authors.lastname: Sono
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002164:Camphor / Q000031:analogs & derivatives / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D000218:Adamantane; C001862:adamantanone