title: Probing the interactions of putidaredoxin with redox partners in camphor P450 5-monooxygenase by mutagenesis of surface residues.
abstract: The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.
authors.affiliation: Biotechnology Division, Chemical Sciences and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001, USA. marcia.holden@nist.gov
authors.forename: M
authors.initials: M
authors.lastname: Holden
authors.forename: M
authors.initials: M
authors.lastname: Mayhew
authors.forename: D
authors.initials: D
authors.lastname: Bunk
authors.forename: A
authors.initials: A
authors.lastname: Roitberg
authors.forename: V
authors.initials: V
authors.lastname: Vilker
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase / Q000378:metabolism*; D004926:Escherichia coli; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013054:Spectrophotometry, Atomic
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D009243:NAD; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase