title: The use of deuterated camphor as a substrate in (1)H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound I.
abstract: Electron paramagnetic resonance and (1)H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1R)-camphor (H(2)-camphor) and (1R)-5,5-dideuterocamphor (D(2)-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam-O(2)-substrate complex. Hydroxylation of H(2)-camphor produced a primary product state in which 5-exo-hydroxycamphor is coordinated with Fe(III). ENDOR spectra contained signals derived from two protons [Fe(III)-bound C5-OH(exo) and C5-H(endo)] from camphor. When D(2)-camphor was hydroxylated under the same condition in H(2)O or D(2)O buffer, both ENDOR H(exo) and H(endo) signals are absent. For D(2)-camphor in H(2)O buffer, H/D exchange causes the C5-OH(exo) signal to reappear during relaxation upon annealing to 230 K; for H(2)-camphor in D(2)O, the magnitude of the C5-OH(exo) signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.forename: Roshan
authors.initials: R
authors.lastname: Perera
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D002164:Camphor / Q000737:chemistry*; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D003080:Cold Temperature; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006900:Hydroxylation; D008958:Models, Molecular; D010084:Oxidation-Reduction; D055550:Protein Stability
chemical_list: D006418:Heme; D002164:Camphor; D003903:Deuterium; D019475:Camphor 5-Monooxygenase