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Structural and functional characterization of the hydrogenase-maturation HydF protein

Nature Chemical Biology volume 13, pages 779–784 (2017)Cite this article

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Abstract

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H+ and H2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN− and azapropanedithiolate (adt2−) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe2(adt)(CO)4(CN)2]2− is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.

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Figure 1: Structures of the H cluster, chemical analogs of the 2Fe subcluster, and the 6Fe units in HydF hybrids described in this study.
Figure 2: EPR and structural characterization of FeS–HydF from T. melanesiensis.
Figure 3: Hyperfine sublevel correlation (HYSCORE) spectra of reduced FeS–TmeHydF.
Figure 4: FTIR spectra of 1–TmeHydF and 2–TmeHydF.
Figure 5: Hydrogen evolution activity of 2–HydF and 2–HydA.

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References

  1. Lubitz, W., Ogata, H., Rüdiger, O. & Reijerse, E. Hydrogenases. Chem. Rev. 114, 4081–4148 (2014).

    CAS  PubMed  Google Scholar 

  2. Armstrong, F.A. et al. Dynamic electrochemical investigations of hydrogen oxidation and production by enzymes and implications for future technology. Chem. Soc. Rev. 38, 36–51 (2009).

    CAS  PubMed  Google Scholar 

  3. Cracknell, J.A., Vincent, K.A. & Armstrong, F.A. Enzymes as working or inspirational electrocatalysts for fuel cells and electrolysis. Chem. Rev. 108, 2439–2461 (2008).

    CAS  PubMed  Google Scholar 

  4. Woolerton, T.W., Sheard, S., Chaudhary, Y.S. & Armstrong, F.A. Enzymes and bio-inspired electrocatalysts in solar fuel devices. Energy Environ. Sci. 5, 7470–7490 (2012).

    CAS  Google Scholar 

  5. Peters, J.W., Lanzilotta, W.N., Lemon, B.J. & Seefeldt, L.C. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853–1858 (1998).

    CAS  PubMed  Google Scholar 

  6. Pandey, A.S., Harris, T.V., Giles, L.J., Peters, J.W. & Szilagyi, R.K. Dithiomethylether as a ligand in the hydrogenase h-cluster. J. Am. Chem. Soc. 130, 4533–4540 (2008).

    CAS  PubMed  Google Scholar 

  7. Nicolet, Y., Piras, C., Legrand, P., Hatchikian, C.E. & Fontecilla-Camps, J.C. Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Structure 7, 13–23 (1999).

    CAS  PubMed  Google Scholar 

  8. Nicolet, Y. et al. Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans. J. Am. Chem. Soc. 123, 1596–1601 (2001).

    CAS  PubMed  Google Scholar 

  9. Berggren, G. et al. Biomimetic assembly and activation of [FeFe]-hydrogenases. Nature 499, 66–69 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Roche, B. et al. Reprint of: Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity. Biochim. Biophys. Acta 1827, 923–937 (2013).

    CAS  PubMed  Google Scholar 

  11. Suess, D.L.M. et al. Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster. Proc. Natl. Acad. Sci. USA 112, 11455–11460 (2015).

    CAS  PubMed  Google Scholar 

  12. Dinis, P. et al. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly. Proc. Natl. Acad. Sci. USA 112, 1362–1367 (2015).

    CAS  PubMed  Google Scholar 

  13. Kuchenreuther, J.M. et al. The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster. Science 343, 424–427 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Nicolet, Y. et al. X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima. J. Biol. Chem. 283, 18861–18872 (2008).

    CAS  PubMed  Google Scholar 

  15. Nicolet, Y., Rohac, R., Martin, L. & Fontecilla-Camps, J.C. X-ray snapshots of possible intermediates in the time course of synthesis and degradation of protein-bound Fe4S4 clusters. Proc. Natl. Acad. Sci. USA 110, 7188–7192 (2013).

    CAS  PubMed  Google Scholar 

  16. Betz, J.N. et al. [FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis. Biochemistry 54, 1807–1818 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Rohac, R. et al. Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE. Nat. Chem. 8, 491–500 (2016).

    CAS  PubMed  Google Scholar 

  18. Cendron, L. et al. Crystal structure of HydF scaffold protein provides insights into [FeFe]-hydrogenase maturation. J. Biol. Chem. 286, 43944–43950 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Shepard, E.M. et al. [FeFe]-hydrogenase maturation. Biochemistry 53, 4090–4104 (2014).

    CAS  PubMed  Google Scholar 

  20. Shepard, E.M. et al. Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold. Proc. Natl. Acad. Sci. USA 107, 10448–10453 (2010).

    CAS  PubMed  Google Scholar 

  21. Vallese, F. et al. Biochemical analysis of the interactions between the proteins involved in the [FeFe]-hydrogenase maturation process. J. Biol. Chem. 287, 36544–36555 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. McGlynn, S.E. et al. HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis. FEBS Lett. 582, 2183–2187 (2008).

    CAS  PubMed  Google Scholar 

  23. Czech, I., Silakov, A., Lubitz, W. & Happe, T. The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN- ligated iron cofactor. FEBS Lett. 584, 638–642 (2010).

    CAS  PubMed  Google Scholar 

  24. Artero, V. et al. From enzyme maturation to synthetic chemistry: the case of hydrogenases. Acc. Chem. Res. 48, 2380–2387 (2015).

    CAS  PubMed  Google Scholar 

  25. Esselborn, J. et al. A structural view of synthetic cofactor integration into [FeFe]-hydrogenases. Chem. Sci. 7, 959–968 (2016).

    CAS  PubMed  Google Scholar 

  26. Schweins, T. & Wittinghofer, A. GTP-binding proteins. Structures, interactions and relationships. Curr. Biol. 4, 547–550 (1994).

    CAS  PubMed  Google Scholar 

  27. Albertini, M. et al. Characterization of the [FeFe]-hydrogenase maturation protein HydF by EPR techniques: insights into the catalytic mechanism. Top. Catal. 58, 708–718 (2015).

    CAS  Google Scholar 

  28. Berggren, G. 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).

    CAS  PubMed  Google Scholar 

  29. Esselborn, J. et al. Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic. Nat. Chem. Biol. 9, 607–609 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Caserta, G. et al. Chemical assembly of multiple metal cofactors: the heterologously expressed multidomain [FeFe]-hydrogenase from Megasphaera elsdenii. Biochim. Biophys. Acta 1857, 1734–1740 (2016).

    CAS  PubMed  Google Scholar 

  31. Berto, P. et al. The [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies. Biochim. Biophys. Acta 1817, 2149–2157 (2012).

    CAS  PubMed  Google Scholar 

  32. Kuchenreuther, J.M., Britt, R.D. & Swartz, J.R. New insights into [FeFe] hydrogenase activation and maturase function. PLoS One 7, e45850 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Czech, I. et al. The [FeFe]-hydrogenase maturation protein HydF contains a H-cluster like [4Fe4S]-2Fe site. FEBS Lett. 585, 225–230 (2011).

    CAS  PubMed  Google Scholar 

  34. Joshi, N. et al. Iron-sulfur cluster coordination in the [FeFe]-hydrogenase H cluster biosynthetic factor HydF. FEBS Lett. 586, 3939–3943 (2012).

    CAS  PubMed  Google Scholar 

  35. Albertini, M. et al. Probing the solvent accessibility of the [4Fe-4S] cluster of the hydrogenase maturation protein HydF from Thermotoga neapolitana by HYSCORE and 3p-ESEEM. J. Phys. Chem. B 119, 13680–13689 (2015).

    CAS  PubMed  Google Scholar 

  36. Shepard, E.M., Byer, A.S., Betz, J.N., Peters, J.W. & Broderick, J.B. A redox active [2Fe-2S] cluster on the hydrogenase maturase HydF. Biochemistry 55, 3514–3527 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jiang, H. et al. Asp97 is a crucial residue involved in the ligation of the [Fe4S4] cluster of IscA from Acidithiobacillus ferrooxidans. J. Microbiol. Biotechnol. 18, 1070–1075 (2008).

    CAS  PubMed  Google Scholar 

  38. Calzolai, L. et al. 1H NMR investigation of the electronic and molecular structure of the four-iron cluster ferredoxin from the hyperthermophile Pyrococcus furiosus. Identification of Asp14 as a cluster ligand in each of the four redox states. Biochemistry 34, 11373–11384 (1995).

    CAS  PubMed  Google Scholar 

  39. Muraki, N. et al. X-ray crystal structure of the light-independent protochlorophyllide reductase. Nature 465, 110–114 (2010).

    CAS  PubMed  Google Scholar 

  40. Lee, M. et al. Biosynthesis of isoprenoids: crystal structure of the [4Fe-4S] cluster protein IspG. J. Mol. Biol. 404, 600–610 (2010).

    CAS  PubMed  Google Scholar 

  41. Brazzolotto, X. et al. The [Fe-Fe]-hydrogenase maturation protein HydF from Thermotoga maritima is a GTPase with an iron-sulfur cluster. J. Biol. Chem. 281, 769–774 (2006).

    CAS  PubMed  Google Scholar 

  42. Loiseau, L. et al. Analysis of the heteromeric CsdA-CsdE cysteine desulfurase, assisting Fe-S cluster biogenesis in Escherichia coli. J. Biol. Chem. 280, 26760–26769 (2005).

    CAS  PubMed  Google Scholar 

  43. Fish, W.W. Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol. 158, 357–364 (1988).

    CAS  PubMed  Google Scholar 

  44. Beinert, H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal. Biochem. 131, 373–378 (1983).

    CAS  PubMed  Google Scholar 

  45. Li, H. & Rauchfuss, T.B. Iron carbonyl sulfides, formaldehyde, and amines condense to give the proposed azadithiolate cofactor of the Fe-only hydrogenases. J. Am. Chem. Soc. 124, 726–727 (2002).

    CAS  PubMed  Google Scholar 

  46. Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).

    CAS  PubMed  Google Scholar 

  47. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  49. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  50. Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 103, 8060–8065 (2006).

    CAS  PubMed  Google Scholar 

  51. Terwilliger, T.C. et al. phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta. J. Struct. Funct. Genomics 13, 81–90 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    CAS  PubMed  Google Scholar 

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Acknowledgements

Diffraction data were collected at the synchrotron SOLEIL, beamlines Proxima 1 and Proxima 2 (Saint-Aubin, France). We are most grateful to the beamline groups for making these experiments possible. V.A., M.A. and S.R. acknowledge support from the French National Research Agency (Labex program ARCANE, ANR-11-LABX-0003-01). G.C., L.P., C.P. and M.F. acknowledge support from the French National Research Agency (Labex program DYNAMO, ANR-11-LABX-0011) and from Fondation de l'Orangerie for Individual Philanthropy and its donors.

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Authors and Affiliations

  1. Laboratoire de Chimie des Processus Biologiques, Collège de France, Université Pierre et Marie Curie, CNRS UMR 8229, Paris, France

    Giorgio Caserta, Ludovic Pecqueur, Cecilia Papini & Marc Fontecave

  2. Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, Germany

    Agnieszka Adamska-Venkatesh, Edward Reijerse & Wolfgang Lubitz

  3. Laboratoire de Chimie et Biologie des Métaux, Université Grenoble Alpes, CEA, CNRS, Grenoble, France

    Souvik Roy, Vincent Artero & Mohamed Atta

Authors
  1. Giorgio Caserta
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Contributions

G.C., L.P. and M.F. designed the study; G.C. performed protein purification, characterization and crystallogenesis, and sample preparation for spectroscopy experiments; C.P. performed mutagenesis and characterization of mutant proteins; G.C. and C.P. assayed hydrogenase activity; L.P. determined the three-dimensional structures; A.A.-V., E.R. and W.L. performed and analyzed the EPR, HYSCORE and FTIR experiments; V.A. and S.R. contributed to synthetic chemistry; M.A. and L.P. contributed to molecular biology; and M.F., G.C., L.P. and E.R. wrote the manuscript with input from all other authors.

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Correspondence to Marc Fontecave.

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Supplementary Results, Supplementary Tables 1–4, Supplementary Figures 1–20 and Supplementary Note 1. (PDF 3746 kb)

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Caserta, G., Pecqueur, L., Adamska-Venkatesh, A. et al. Structural and functional characterization of the hydrogenase-maturation HydF protein. Nat Chem Biol 13, 779–784 (2017). https://doi.org/10.1038/nchembio.2385

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