title: EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding.
abstract: The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.
authors.affiliation: Department of Biological Chemistry, Medical School, Ann Arbor, Michigan 48109-0606, USA. jel@umich.edu
authors.forename: J E
authors.initials: JE
authors.lastname: LeLean
authors.forename: N
authors.initials: N
authors.lastname: Moon
authors.forename: W R
authors.initials: WR
authors.lastname: Dunham
authors.forename: M J
authors.initials: MJ
authors.lastname: Coon
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006418:Heme / Q000737:chemistry; D009154:Mutation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D017382:Reactive Oxygen Species; D013113:Spin Labels; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase