title: Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.
abstract: Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.
authors.affiliation: The Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Takeda
authors.forename: Junichi
authors.initials: J
authors.lastname: Sato
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Goto
authors.forename: Takanori
authors.initials: T
authors.lastname: Fujita
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Abo
authors.forename: Etsuro
authors.initials: E
authors.lastname: Yoshimura
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakagawa
authors.forename: Akira
authors.initials: A
authors.lastname: Abe
authors.forename: Shinji
authors.initials: S
authors.lastname: Kawasaki
authors.forename: Youichi
authors.initials: Y
authors.lastname: Niimura
mesh_terms: D004492:Edetic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038181:FMN Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007502:Iron Chelating Agents / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D007502:Iron Chelating Agents; D016877:Oxidants; D011994:Recombinant Proteins; D009243:NAD; C077565:fpr protein, E coli; D005182:Flavin-Adenine Dinucleotide; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D005287:Ferredoxin-NADP Reductase; D038181:FMN Reductase; C023903:ferric citrate iron reductase; C116054:NfsB protein, E coli; D009601:Nitroreductases; C019179:Fe(III)-EDTA