title: Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.
abstract: Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa, 920-1192, Japan, dseo@se.kanazawa-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Seo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Naito
authors.forename: Erika
authors.initials: E
authors.lastname: Nishimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakurai
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009249:NADP / Q000378:metabolism*; D016415:Sequence Alignment; D014443:Tyrosine
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; C008173:isoalloxazine; D009249:NADP; D005287:Ferredoxin-NADP Reductase