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Characterization of the [FeFe]-Hydrogenase Maturation Protein HydF by EPR Techniques: Insights into the Catalytic Mechanism

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Abstract

The catalytic site of [FeFe]-hydrogenase, the “H-cluster”, composed by a [4Fe–4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN− and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE and HydF. In this contribution we review our studies on HydF, a protein which acts as scaffold and carrier for the [2Fe] unit of the H-cluster. HydF is a complex enzyme which contains one [4Fe–4S] cluster binding site, with three conserved cysteine residues and a non-Cys ligand. We have exploited EPR, HYSCORE and PELDOR spectroscopies to get insight into the structure and chemical role of HydF. On the basis of the results we discuss the possibility that the non-Cys ligated Fe atom of the [4Fe–4S] cluster, is the site where the [2Fe] subcluster precursor is anchored and finally processed to be delivered to the hydrogenase (HydA). Our PELDOR experiments on the isolated GTPase domain of HydF, have also suggested that interactions with HydG and HydE proteins may be regulated by the binding of the nucleotide.

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Acknowledgments

This work has been supported by the CARIPARO Foundation (M3PC Project) by the MIUR (PRIN2010-2011 Project 2010FM38P_004).

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Authors and Affiliations

  1. Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padua, Italy

    Marco Albertini, Laura Galazzo & Marilena Di Valentin

  2. Department of Biology, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy

    Lorenzo Maso, Edith De Rosa & Paola Costantini

  3. Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35131, Padua, Italy

    Francesca Vallese, Paola Berto & Donatella Carbonera

Authors
  1. Marco Albertini
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  2. Laura Galazzo
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  3. Lorenzo Maso
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  4. Francesca Vallese
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  5. Paola Berto
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  6. Edith De Rosa
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  7. Marilena Di Valentin
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  8. Paola Costantini
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  9. Donatella Carbonera
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Corresponding authors

Correspondence to Paola Costantini or Donatella Carbonera.

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Albertini, M., Galazzo, L., Maso, L. et al. Characterization of the [FeFe]-Hydrogenase Maturation Protein HydF by EPR Techniques: Insights into the Catalytic Mechanism. Top Catal 58, 708–718 (2015). https://doi.org/10.1007/s11244-015-0413-x

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  • DOI: https://doi.org/10.1007/s11244-015-0413-x

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