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The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster

Nature Chemistry volume 6, pages 336–342 (2014)Cite this article

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An Author Correction to this article was published on 24 September 2019

Abstract

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

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Figure 1: Structure of the H-cluster.
Figure 2: Chronoamperometric data obtained with a film of Cr hydrogenase covalently attached to a rotating graphite electrode.
Figure 3: Rate constants for the inactivation/reactivation processes determined from electrochemical experiments.
Figure 4: Thermal fluctuations of Dd FeFe hydrogenase Phe296 probed by MD.
Figure 5: States of the active sites involved in catalysis (within the green triangle) and in high-potential inactivation.
Figure 6: Structures of 1H2 and 2H2, as predicted by DFT calculations.

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References

  1. Carroll, M. E., Barton, B. E., Rauchfuss, T. B. & Carroll, P. J. Synthetic models for the active site of the [FeFe]-hydrogenase: catalytic proton reduction and the structure of the doubly protonated intermediate. J. Am. Chem. Soc. 134, 18843–18852 (2012).

    Article  CAS  Google Scholar 

  2. Tard, C. & Pickett, C. J. Structural and functional analogues of the active sites of the [Fe]-, [NiFe]-, and [FeFe]-hydrogenases. Chem. Rev. 109, 2245–2274 (2009).

    Article  CAS  Google Scholar 

  3. Liu, T., Dubois, D. L. & Bullock, R. M. An iron complex with pendent amines as a molecular electrocatalyst for oxidation of hydrogen. Nature Chem. 5, 228–233 (2013).

    Article  CAS  Google Scholar 

  4. Fontecilla-Camps, J. C., Volbeda, A., Cavazza, C. & Nicolet, Y. Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chem. Rev. 107, 4273–4303 (2007).

    Article  CAS  Google Scholar 

  5. Hsieh, C-H. et al. Structural and spectroscopic features of mixed valent FeIIFeI complexes and factors related to the rotated configuration of diiron hydrogenase. J. Am. Chem. Soc. 134, 13089–13102 (2012).

    Article  CAS  Google Scholar 

  6. Greco, C., Bruschi, M., Fantucci, P., Ryde, U. & De Gioia, L. Isocyanide in biochemistry? A theoretical investigation of the electronic effects and energetics of cyanide ligand protonation in [FeFe]-hydrogenases. Chem. Eur. J. 17, 1954–1965 (2011).

    Article  CAS  Google Scholar 

  7. Knörzer, P. et al. Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases. J. Biol. Chem. 287, 1489–1499 (2012).

    Article  Google Scholar 

  8. Roseboom, W., De Lacey, A. L., Fernandez, V. M., Hatchikian, E. C. & Albracht, S. P. J. The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy. J. Biol. Inorg. Chem. 11, 102–118 (2006).

    Article  CAS  Google Scholar 

  9. De Lacey, A. L., Fernandez, V. M., Rousset, M. & Cammack, R. Activation and inactivation of hydrogenase function and the catalytic cycle: spectroelectrochemical studies. Chem. Rev. 107, 4304–4330 (2007).

    Article  CAS  Google Scholar 

  10. Parkin, A., Cavazza, C., Fontecilla-Camps, J. C. & Armstrong, F. A. Electrochemical investigations of the interconversions between catalytic and inhibited states of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. J. Am. Chem. Soc. 128, 16808–16815 (2006).

    Article  CAS  Google Scholar 

  11. Baffert, C. et al. Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer. Anal. Chem. 84, 7999–8005 (2012).

    Article  CAS  Google Scholar 

  12. Léger, C. & Bertrand, P. Direct electrochemistry of redox enzymes as a tool for mechanistic studies. Chem. Rev. 108, 2379–2438 (2008).

    Article  Google Scholar 

  13. Fourmond, V., Infossi, P., Giudici-Orticoni, M-T., Bertrand, P. & Léger, C. ‘Two-step’ chronoamperometric method for studying the anaerobic inactivation of an oxygen tolerant NiFe hydrogenase. J. Am. Chem. Soc. 132, 4848–4857 (2010).

    Article  CAS  Google Scholar 

  14. Greco, C., Bruschi, M., Fantucci, P., Ryde, U. & De Gioia, L. Mechanistic and physiological implications of the interplay among iron–sulfur clusters in [FeFe]-hydrogenases. A QM/MM perspective. J. Am. Chem. Soc. 133, 18742–18749 (2011).

    Article  CAS  Google Scholar 

  15. Barton, B. E., Olsen, M. T. & Rauchfuss, T. B. Aza- and oxadithiolates are probable proton relays in functional models for the [FeFe]-hydrogenases. J. Am. Chem. Soc. 130, 16834–16835 (2008).

    Article  CAS  Google Scholar 

  16. Camara, J. M. & Rauchfuss, T. B. Combining acid–base, redox and substrate binding functionalities to give a complete model for the [FeFe]-hydrogenase. Nature Chem. 4, 26–30 (2011).

    Article  Google Scholar 

  17. Camara, J. M. & Rauchfuss, T. B. Mild redox complementation enables H2 activation by [FeFe]-hydrogenase models. J. Am. Chem. Soc. 133, 8098–8101 (2011).

    Article  CAS  Google Scholar 

  18. Fernandez, V. M., Aguirre, R. & Hatchikian, E. C. Reductive activation and redox properties of hydrogenase from Desulfovibrio gigas. Biochim. Biophys. Acta 790, 1–7 (1984).

    Article  CAS  Google Scholar 

  19. Dijk, C. v., Berkel-Arts, A. v. & Veeger, C. The effect of re-oxidation on the reduced hydrogenase of Desulfovibrio vulgaris strain Hildenborough and its oxygen stability. FEBS Lett. 156, 340–344 (1983).

    Article  Google Scholar 

  20. Fernandez, V. M., Hatchikian, E. C., Patil, D. S. & Cammack, R. ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase. Biochim. Biophys. Acta 883, 145–154 (1986).

    Article  CAS  Google Scholar 

  21. Olsen, M. T., Rauchfuss, T. B. & Wilson, S. R. Role of the azadithiolate cofactor in models for [FeFe]-hydrogenase: novel structures and catalytic implications. J. Am. Chem. Soc. 132, 17733–17740 (2010).

    Article  CAS  Google Scholar 

  22. Miyake, T. et al. Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory. J. Biol. Inorg. Chem. 18, 693–700 (2013).

    Article  CAS  Google Scholar 

  23. Zaffaroni, R., Rauchfuss, T. B., Fuller, A., De Gioia, L. & Zampella, G. Contrasting protonation behavior of diphosphido vs dithiolato diiron(I) carbonyl complexes. Organometallics 32, 232–238 (2012).

    Article  Google Scholar 

  24. Apfel, U.-P. et al. Models for the active site in [FeFe] hydrogenase with iron-bound ligands derived from bis-, tris-, and tetrakis(mercaptomethyl)silanes. Inorg. Chem. 49, 10117–10132 (2010).

    Article  CAS  Google Scholar 

  25. Dong, W. et al. An insight into the protonation property of a diiron azadithiolate complex pertinent to the active site of Fe-only hydrogenases. Chem. Commun. 305–307 (2006).

  26. Ezzaher, S., Gogoll, A., Bruhn, C. & Ott, S. Directing protonation in [FeFe] hydrogenase active site models by modifications in their second coordination sphere. Chem. Commun. 46, 5775–5777 (2010).

    Article  CAS  Google Scholar 

  27. Silakov, A., Kamp, C., Reijerse, E., Happe, T. & Lubitz, W. Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii. Biochemistry 48, 7780–7786 (2009).

    Article  CAS  Google Scholar 

  28. Vignais, P. M. & Billoud, B. Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 107, 4206–4272 (2007).

    Article  CAS  Google Scholar 

  29. Fritsch, J. et al. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron–sulphur centre. Nature 479, 249–252 (2011).

    Article  CAS  Google Scholar 

  30. Shomura, Y., Yoon, K-S., Nishihara, H. & Higuchi, Y. Structural basis for a [4Fe–3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase. Nature 479, 253–256 (2011).

    Article  CAS  Google Scholar 

  31. Mouesca, J-M., Fontecilla-Camps, J. C. & Amara, P. The structural plasticity of the proximal [4Fe3S] cluster is responsible for the O2 tolerance of membrane-bound [NiFe] hydrogenases. Angew. Chem. Int. Ed. 52, 2002–2006 (2013).

    Article  CAS  Google Scholar 

  32. Bruschi, M., Fantucci, P. & De Gioia, L. DFT investigation of structural, electronic, and catalytic properties of diiron complexes related to the [2Fe]H subcluster of Fe-only hydrogenases. Inorg. Chem. 41, 1421–1429 (2002).

    Article  CAS  Google Scholar 

  33. Bruschi, M., Fantucci, P. & De Gioia, L. Density functional theory investigation of the active site of [Fe]-hydrogenases: effects of redox state and ligand characteristics on structural, electronic, and reactivity properties of complexes related to the [2Fe]H subcluster. Inorg. Chem. 42, 4773–4781 (2003).

    Article  CAS  Google Scholar 

  34. Sybirna, K. et al. Shewanella oneidensis: a new and efficient system for expression and maturation of heterologous [Fe–Fe] hydrogenase from Chlamydomonas reinhardtii. BMC Biotechnol. 8, 1–8 (2008).

    Article  Google Scholar 

  35. Baffert, C. et al. CO disrupts the reduced H-cluster of FeFe hydrogenase. A combined DFT and protein film voltammetry study. J. Am. Chem. Soc. 133, 2096–2099 (2011).

    Article  CAS  Google Scholar 

  36. Girbal, L. et al. Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl. Environ. Microbiol. 71, 2777–2781 (2005).

    Article  CAS  Google Scholar 

  37. Lautier, T. et al. The quest for a functional substrate access tunnel in FeFe hydrogenase. Faraday Discuss. 148, 385–407 (2011).

    Article  CAS  Google Scholar 

  38. Liebgott, P-P. et al. Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase. Nature Chem. Biol. 6, 63–70 (2010).

    Article  CAS  Google Scholar 

  39. Fourmond, V. et al. SOAS: a free program to analyze electrochemical data and other one-dimensional signals. Bioelectrochemistry 76, 141–147 (2009).

    Article  CAS  Google Scholar 

  40. Boggs, P. T., Donaldson, J. R., Byrd, R. H. & Schnabel, R. B. Algorithm 676: ODRPACK: software for weighted orthogonal distance regression. ACM Trans. Math. Softw. 15, 348–364 (1989).

    Article  Google Scholar 

  41. Scott, W. R. P. et al. The GROMOS biomolecular simulation program package. J. Phys. Chem. A 103, 3596–3607 (1999).

    Article  CAS  Google Scholar 

  42. Berendsen, H. J. C., Grigera, J. R. & Straatsma, T. P. The missing term in effective pair potentials. J. Phys. Chem. 91, 6269–6271 (1987).

    Article  CAS  Google Scholar 

  43. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  Google Scholar 

  44. Ahlrichs, R., Bär, M., Häser, M., Horn, H. & Kölmel, C. Electronic structure calculations on workstation computers: the program system TURBOMOLE. Chem. Phys. Lett. 162, 165–169 (1989).

    Article  CAS  Google Scholar 

  45. Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098–3100 (1988).

    Article  CAS  Google Scholar 

  46. Perdew, J. P. Density-functional approximation for the correlation energy of the inhomogeneous electron gas. Phys. Rev. B 33, 8822–8824 (1986).

    Article  CAS  Google Scholar 

  47. Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297–3305 (2005).

    Article  CAS  Google Scholar 

  48. Noodleman, L. Valence Bond Description of Anti-Ferromagnetic Coupling in Transition Metal Dimers. J. Chem. Phys. 74, 5737–5743 (1981).

    Article  CAS  Google Scholar 

  49. Greco, C., Fantucci, P., Ryde, U. & De Gioia, L. Fast generation of broken-symmetry states in a large system including multiple iron-sulfur assemblies: investigation of QM/MM energies, clusters charges, and spin populations. Int. J. Quantum Chem. 111, 3949–3960 (2011).

    CAS  Google Scholar 

  50. 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).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank S. Dementin for helpful discussions and critical reading of the manuscript. The authors acknowledge funding from Centre National de la Recherche Scientifique, Aix-Marseille Université and Agence Nationale de la Recherche (ANR-12-BS08-0014 and ANR-2010-BIOE-004). L.D.G. acknowledges support from Ministero dell'Istruzione, dell'Università e della Ricerca (Prin 2010M2JARJ). P.W. acknowledges the Ministry of Education, Republic of China (Taiwan) for a PhD scholarship, and J.B. acknowledges the Engineering and Physical Sciences Research Council, grant EP/J015571/1, and the Royal Society for a University Research Fellowship. The UK's High Performance Computing Materials Chemistry Consortium (funded by the Engineering and Physical Sciences Research Council, EP/ F067496) is acknowledged for access to the high-performance computing facility HECToR.

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Authors and Affiliations

  1. Aix Marseille Université, CNRS, BIP UMR 7281, Marseille, 13402, France

    Vincent Fourmond, Carole Baffert, Pierre Ezanno & Christophe Léger

  2. Department of Earth and Environmental Science, Università degli Studi di Milano-Bicocca, P.zza della Scienza 1, Milan, 20126, Italy

    Claudio Greco & Maurizio Bruschi

  3. iBiTec-S, SB2SM, LMB (UMR CNRS 8221), DSV, CEA, Gif-sur-Yvette, 91191, France

    Kateryna Sybirna & Hervé Bottin

  4. Université de Toulouse, INSA, UPS, INP, LISBP, INRA:UMR792,135 CNRS:UMR 5504, avenue de Rangueil, Toulouse, 31077, France

    Isabelle Meynial-Salles & Philippe Soucaille

  5. Department of Physics and Astronomy, University College London, Gower Street, London, WC1E 6BT, UK

    Po-Hung Wang, Marco Montefiori & Jochen Blumberger

  6. Department of Biotechnology and Biosciences, Università degli Studi di Milano-Bicocca, P.zza della Scienza 2, Milan, 20126, Italy

    Luca De Gioia

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  1. Vincent Fourmond
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Contributions

V.F., C.G., C.B., J.B., L.D.G. and C.L. designed the experiments. V.F., C.G., P-H.W., P.E., M.M. and M.B. performed the experiments. V.F., C.G., J.B., L.D.G. and C.L. analysed the data. K.S. and H.B. provided the enzyme from Cr and constructed the mutants. I.M-S. and P.S. provided the enzyme from Ca. V.F., C.G., C.B., J.B., L.D.G. and C.L. co-wrote the paper.

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Correspondence to Vincent Fourmond.

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Fourmond, V., Greco, C., Sybirna, K. et al. The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster. Nature Chem 6, 336–342 (2014). https://doi.org/10.1038/nchem.1892

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