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Kinetics of NADP+/NADPH reduction–oxidation catalyzed by the ferredoxin-NAD(P)+ reductase from the green sulfur bacterium Chlorobaculum tepidum

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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 CtFNRox with 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 CtFNRred was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRox with 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.

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Abbreviations

A :

Absorbance

Ad:

Adrenodoxin

AdR:

Adrenodoxin reductase

Bs :

Bacillus subtilis

Ct :

Chlorobaculum tepidum

CT:

Charge transfer

CTC:

Charge transfer complex

Ec :

Escherichia coli

FAD:

Flavin adenine dinucleotide

Fd:

Ferredoxin

FNR:

Ferredoxin-NAD(P)+ oxidoreductase

GR:

Glutathione reductase

HEPES:

2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid

NADPD:

(4S-2H)-reduced nicotinamide adenine dinucleotide phosphate

Pd:

Putidaredoxin

PdR:

Putidaredoxin reductase

TrxR:

Bacterial NADPH-thioredoxin reductase

Ox:

Oxidized

Red:

Reduced

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Acknowledgments

We thank Dr. Hidehiro Sakurai for stimulating discussions. This work was supported in part by a grant-in-aid for Scientific Research on Innovative Areas (No. 24107004) and the Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to KI).

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Authors and Affiliations

  1. Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa, 920-1192, Japan

    Daisuke Seo & Takeshi Sakurai

  2. Department of Biological Sciences, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan

    Masaharu Kitashima & Kazuhito Inoue

  3. Research Institute for Integrated Science, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa, 259-1293, Japan

    Masaharu Kitashima & Kazuhito Inoue

Authors
  1. Daisuke Seo
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  2. Masaharu Kitashima
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  3. Takeshi Sakurai
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  4. Kazuhito Inoue
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Correspondence to Daisuke Seo.

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Seo, D., Kitashima, M., Sakurai, T. et al. Kinetics of NADP+/NADPH reduction–oxidation catalyzed by the ferredoxin-NAD(P)+ reductase from the green sulfur bacterium Chlorobaculum tepidum . Photosynth Res 130, 479–489 (2016). https://doi.org/10.1007/s11120-016-0285-3

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