title: Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES.
abstract: Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin pi-cation radical (Fe(IV)=O/Por(+))), as well as compound ES (Fe(IV)=O/Tyr()) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-9]. Compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin pi-cation radical of Compound I, and has been characterized by rapid-freeze-quench-Mössbauer/EPR spectroscopy; the tyrosyl radical was assigned to Tyr96 for wild type or to Tyr75 for the Tyr96Phe variant [V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.L. Barra, S.G. Sligar, A.X. Trautwein, J. Biol. Chem. 279 (2004) 10919-10930]. Here we report rapid-scanning stopped-flow studies of the reactions of peracids with substrate-free ferric Y75F, Y96F, and Y96F/Y75F P450cam variants, showing how these active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates. Curiously, rates of generation of Compounds I and ES for both single mutants were not very different from wild type. Contrasting with the earlier EPR results, the Y96F/Y75F variant was also shown to form an ES-like species, but more slowly. When substrate is not present, or is improperly bound, compound I rapidly converts to compound ES, which can be reduced to form H(2)O and ferric P450, thus avoiding the modification of nearby protein groups or release of reactive oxygen species.
authors.affiliation: Department of Biological Chemistry, University of Michigan, 1150 Medical Center Dr, Ann Arbor, MI 48109-0606, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Spolitak
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D005290:Ferric Compounds / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction
chemical_list: D017931:DNA Primers; D005290:Ferric Compounds; D019475:Camphor 5-Monooxygenase