Ferredoxin-NADP+ reductase from Pseudomonas putida functions as a ferric reductase.
Pseudomonas putida harbors two ferredoxin-NADP(+) reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k(cat)/K(m) value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.
Yeom
Jinki
J
Korea University, Seoul, Republic of Korea.
Jeon
Che Ok
CO
Madsen
Eugene L
EL
Park
Woojun
W
D003470:Culture Media; D038181:FMN Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology
D003470:Culture Media; D007501:Iron; D005287:Ferredoxin-NADP Reductase; D038181:FMN Reductase; C023903:ferric citrate iron reductase