title: Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.
abstract: The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Caroline
authors.initials: C
authors.lastname: Aldag
authors.forename: Igor A
authors.initials: IA
authors.lastname: Gromov
authors.forename: Inés
authors.initials: I
authors.lastname: García-Rubio
authors.forename: Konstanze
authors.initials: K
authors.lastname: von Koenig
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
authors.forename: Bernhard
authors.initials: B
authors.lastname: Jaun
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D019943:Amino Acid Substitution; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D015336:Molecular Probe Techniques; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017279:Selenocysteine / Q000235:genetics*
chemical_list: D008024:Ligands; D017279:Selenocysteine; D006418:Heme; D019475:Camphor 5-Monooxygenase