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Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

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Abstract

Ferredoxin:NADP+ oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP+ is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

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Abbreviations

ACN:

Acetonitrile

DBMIB:

Dibromothymoquinone

Cyt b6f:

Cytochrome b6f complex

FAD:

Flavin adenine dinucleotide

FA:

Formic acid

Fd:

Ferredoxin

FNR:

Ferredoxin:NADP+ oxidoreductase

NADP:

Nicotinamide adenine dinucleotide phosphate

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Acknowledgements

The authors are grateful to Dr. Maciej Kotlinski from Department of Plant Molecular Biology, Warsaw University, Miecznikowa 1, PL-02-096 Warsaw, Poland and Dr. Adam Jagielski for help in MS analysis. The authors also would like to thank Dr. Andrzej Waloszek for his critical reading of the manuscript. This work was financed from the budget of the Polish Ministry of Science for the years 2005–2008 under project No: 2P04A06328.

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  1. Joanna Grzyb

    Present address: Weizmann Institute of Science, Rehovot, Israel

Authors and Affiliations

  1. Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387, Kraków, Poland

    Joanna Grzyb, Przemysław Malec & Kazimierz Strzałka

  2. Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland

    Izabela Rumak & Maciej Garstka

  3. Department of Plant Anatomy and Cytology, Institute of Plant Experimental Biology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland

    Izabela Rumak

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Correspondence to Kazimierz Strzałka.

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Grzyb, J., Malec, P., Rumak, I. et al. Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization. Photosynth Res 96, 99–112 (2008). https://doi.org/10.1007/s11120-008-9289-y

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