title: Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: implications for substrate binding.
abstract: A previous study [Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850] revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to that of CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In our study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant, and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme's ability to regio- and stereospecifically metabolize some ligands while allosterically activating others.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA. a1roberts@ucsd.edu
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cheesman
authors.forename: Andrew
authors.initials: A
authors.lastname: Primak
authors.forename: Michael K
authors.initials: MK
authors.lastname: Bowman
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002850:Chromatography, Gel; D065729:Cytochrome P-450 CYP2C9; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D006418:Heme; D006639:Histidine; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases
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