title: Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.
abstract: Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (<10 s(-1)). Mixing BsFd(red) with BsFNR(ox) induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs.
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: dseo@se.kanazawa-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Seo
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Soeta
authors.affiliation: Research Institute for Photobiological Hydrogen Production, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Sakurai
authors.affiliation: CEA, iBiTecS, 91191 Gif sur Yvette, France; CNRS/Université Paris-Sud/CEA, I2BC, 91190 Gif sur Yvette, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Sétif
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
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D009249:NADP; D005287:Ferredoxin-NADP Reductase
keywords: Adrenodoxin; Bacillus; Ferredoxin; Flavodoxin; Putidaredoxin; yumC