Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Authors

  • Emeline Aubert-Jousset,

    1.  CEA, DSV, IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries & Microalgues, Saint Paul Lez Durance, France
    2.  CNRS, UMR Biologie Végétale & Microbiologie Environnementales, Saint Paul lez Durance, France
    3.  Aix-Marseille Université, Saint Paul lez Durance, France
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  • Melissa Cano,

    1.  CEA, DSV, IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries & Microalgues, Saint Paul Lez Durance, France
    2.  CNRS, UMR Biologie Végétale & Microbiologie Environnementales, Saint Paul lez Durance, France
    3.  Aix-Marseille Université, Saint Paul lez Durance, France
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  • Geneviève Guedeney,

    1.  CEA, DSV, IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries & Microalgues, Saint Paul Lez Durance, France
    2.  CNRS, UMR Biologie Végétale & Microbiologie Environnementales, Saint Paul lez Durance, France
    3.  Aix-Marseille Université, Saint Paul lez Durance, France
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  • Pierre Richaud,

    1.  CEA, DSV, IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries & Microalgues, Saint Paul Lez Durance, France
    2.  CNRS, UMR Biologie Végétale & Microbiologie Environnementales, Saint Paul lez Durance, France
    3.  Aix-Marseille Université, Saint Paul lez Durance, France
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  • Laurent Cournac

    1.  CEA, DSV, IBEB, Laboratoire de Bioénergétique et Biotechnologie des Bactéries & Microalgues, Saint Paul Lez Durance, France
    2.  CNRS, UMR Biologie Végétale & Microbiologie Environnementales, Saint Paul lez Durance, France
    3.  Aix-Marseille Université, Saint Paul lez Durance, France
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L. Cournac, CEA Cadarache, IBEB Bat 161, 13108 Saint Paul Lez Durance, France
Fax: +33 4 42 25 62 65
Tel: +33 4 42 25 43 66
E-mail: laurent.cournac@cea.fr

Abstract

Cyanobacterial NAD(P)+-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe–2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H2 production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H2 oxidation, for which NAD+ was a much better electron acceptor than NADP+. Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H2 production or uptake with NADH/NAD+. However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.

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