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An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe–4S] clusters

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Abstract

The biosynthesis of the organometallic H cluster of [Fe–Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe–S protein with GTPase activity. The [4Fe–4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe–4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe–4S]2+/+ clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe–4S] cluster and the non-imidazole-bound [4Fe–4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe–4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.

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Acknowledgments

G.B. gratefully acknowledges support from the Bengt Lundqvist Minnesfond, FORMAS (contract number 213-2010-563), and the Swedish Royal Academy of Sciences. EPR and resonance Raman studies were supported by a grant from the National Institutes of Health (GM62524 to M.K.J.).

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Author notes

  1. Gustav Berggren

    Present address: Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 16, 106 91, Stockholm, Sweden

Authors and Affiliations

  1. Laboratoire de Chimie et Biologie des Métaux, Équipe «Biocatalyse», Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV-LCBM/Biocat, UMR 5249 CEA/CNRS/UJF, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France

    Gustav Berggren, Xavier Brazzolotto, Mohamed Atta & Marc Fontecave

  2. Laboratoire de Chimie et Biologie des Métaux, Équipe “Physicochimie des Métaux en Biologie”, Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV-LCBM/pmb, UMR 5249 CEA/CNRS/UJF, CEA/Grenoble, Grenoble, France

    Ricardo Garcia-Serres, Martin Clemancey & Jean-Marc Latour

  3. Laboratoire “Résonance Magnétique”, Université Joseph Fourier, Grenoble 1/CEA/Institut Nanoscience et Cryogénie/SCIB, UMR-E3, Grenoble, France

    Serge Gambarelli

  4. Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA, 30602, USA

    Heather L. Hernández, Sowmya Subramanian & Michael K. Johnson

  5. Collège de France, 11 place Marcellin-Berthelot, Paris, France

    Marc Fontecave

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  1. Gustav Berggren
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Correspondence to Marc Fontecave.

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Berggren, G., Garcia-Serres, R., Brazzolotto, X. et al. An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe–4S] clusters. J Biol Inorg Chem 19, 75–84 (2014). https://doi.org/10.1007/s00775-013-1062-9

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