title: A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid.
abstract: We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P, were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)(2)phenA (1) and Ru(phen)(2)phenA (2) (bpy=bipyridine, phen=1,10-phenanthroline, and phenA=5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding.
authors.affiliation: San José State University, Department of Chemistry, One Washington Square, San José, CA 95192-0101, USA.
authors.forename: Ngoc-Han
authors.initials: NH
authors.lastname: Tran
authors.forename: Ngoc
authors.initials: N
authors.lastname: Huynh
authors.forename: Garrett
authors.initials: G
authors.lastname: Chavez
authors.forename: Angelina
authors.initials: A
authors.lastname: Nguyen
authors.forename: Sudharsan
authors.initials: S
authors.lastname: Dwaraknath
authors.forename: Thien-Anh
authors.initials: TA
authors.lastname: Nguyen
authors.forename: Maxine
authors.initials: M
authors.lastname: Nguyen
authors.forename: Lionel
authors.initials: L
authors.lastname: Cheruzel
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D007850:Lauric Acids / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055668:Photochemical Processes; D011485:Protein Binding / Q000235:genetics; D012428:Ruthenium / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D007850:Lauric Acids; C030358:lauric acid; D012428:Ruthenium; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases