Photosynthesis Research

, Volume 125, Issue 1–2, pp 321–328 | Cite as

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

  • Daisuke Seo
  • Hiroshi Naito
  • Erika Nishimura
  • Takeshi Sakurai
Regular Paper


Ferredoxin-NAD(P)+ oxidoreductases ([EC], [EC], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.


Ferredoxin Flavin adenine dinucleotide NADPH Tyrosine 





Flavin mononucleotide


Flavin adenine dinucleotide




Ferredoxin-NAD(P)+ oxidoreductase




Glucose-6-phosphate dehydrogenase


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


Dissociation constant


Michaelis constant


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Bacterial NADPH-thioredoxin reductase


Wild type



We are grateful to Profs. Hitoshi Tamiaki and Tadashi Mizoguchi from Ritsumeikan University for their help with the measurements of fluorescence spectra.


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Daisuke Seo
    • 1
  • Hiroshi Naito
    • 1
  • Erika Nishimura
    • 1
  • Takeshi Sakurai
    • 1
  1. 1.Division of Material Science, Graduate School of Natural Science and TechnologyKanazawa UniversityKanazawaJapan

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