title: Kinetics of NADP+/NADPH reduction-oxidation catalyzed by the ferredoxin-NAD(P)+reductase from the green sulfur bacterium Chlorobaculum tepidum.
abstract: Ferredoxin-NAD(P)+oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum (CtFNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. CtFNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of CtFNR with NADP+, NADPH, and (4S-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNRoxwith NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When CtFNRredwas mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRoxwith an apparent rate of ~4 s-1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP+/NADPH is reversible, in accordance with its physiological function.
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.affiliation: Department of Biological Sciences, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan.|Research Institute for Integrated Science, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kitashima
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa, 920-1192, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Department of Biological Sciences, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan.|Research Institute for Integrated Science, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Inoue
mesh_terms: D041883:Chlorobium / Q000201:enzymology* / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods
chemical_list: D009249:NADP; D005287:Ferredoxin-NADP Reductase
keywords: Adrenodoxin; Charge transfer complex; Flavoenzyme; Kinetic isotope effect; Putidaredoxin; Stopped flow