Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The atomic-resolution crystal structure of activated [Fe]-hydrogenase

Abstract

Hydrogenases are promising templates for constructing new H2-based catalysts. [Fe]-hydrogenase, which features an iron-guanylylpyridinol (FeGP) cofactor, catalyses a reversible hydride transfer from H2 to methenyl-tetrahydromethanopterin (methenyl-H4MPT+, a C1 carrier in methanogens). Here, we present a detailed mechanistic scenario of this reaction based on the 1.06 Å resolution structure of [Fe]-hydrogenase in a closed active form, in which the Fe of the FeGP cofactor is positioned near the hydride-accepting C14a of a remarkably distorted methenyl-H4MPT+. The open-to-closed transition generates an unsaturated pentacoordinated Fe on expulsion of a water ligand. Quantum mechanics/molecular mechanics computations based on experimental models indicate that a deprotonated 2-OH group on the FeGP cofactor acts as a catalytic base and provides a fairly complete picture of H2 activation: H2 binding on the empty Fe site was found to be nearly thermo-neutral while H2 cleavage and hydride transfer proceed smoothly. The overall reaction involves a repositioning and relaxation of the distorted methenyl-H4MPT+.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The catalytic reaction and chemical structures.
Fig. 2: Structures of the open and closed conformations of [Fe]-hydrogenase.
Fig. 3: Electron density map and model of the cofactor and substrate.
Fig. 4: The proposed catalytic cycle of [Fe]-hydrogenase.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors on reasonable request. X-ray crystallographic data are available in the RCSB-Protein Data Bank under accession numbers 6HAC (open conformation), 6HAV (closed conformation form A) and 6HAE (closed conformation form B).

References

  1. 1.

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

    CAS  Article  Google Scholar 

  2. 2.

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

    CAS  Article  Google Scholar 

  3. 3.

    Thauer, R. K. et al. Hydrogenases from methanogenic archaea, nickel, a novel cofactor and H2 storage. Annu. Rev. Biochem. 79, 507–536 (2010).

    CAS  Article  Google Scholar 

  4. 4.

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

    CAS  Article  Google Scholar 

  5. 5.

    Zirngibl, C. et al. H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron–sulfur clusters in methanogenic archaea. Eur. J. Biochem. 208, 511–520 (1992).

    CAS  Article  Google Scholar 

  6. 6.

    Shima, S. & Ermler, U. Structure and function of [Fe]-hydrogenase and its iron-guanylylpyridinol (FeGP) cofactor. Eur. J. Inorg. Chem. 2011, 963–972 (2011).

    Article  Google Scholar 

  7. 7.

    Vogt, S., Lyon, E. J., Shima, S. & Thauer, R. K. The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases. J. Biol. Inorg. Chem. 13, 97–106 (2008).

    CAS  Article  Google Scholar 

  8. 8.

    Hartmann, G. C., Santamaria, E., Fernández, V. M. & Thauer, R. K. Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O. J. Biol. Inorg. Chem. 1, 446–450 (1996).

    CAS  Article  Google Scholar 

  9. 9.

    Thauer, R. K., Klein, A. R. & Hartmann, G. C. Reactions with molecular hydrogen in microorganisms: evidence for a purely organic hydrogenation catalyst. Chem. Rev. 96, 3031–3042 (1996).

    CAS  Article  Google Scholar 

  10. 10.

    Schleucher, J., Schwörer, B., Thauer, R. K. & Griesinger, C. Elucidation of the stereochemical course of chemical reactions by magnetic labeling. J. Am. Chem. Soc. 117, 2941–2942 (1995).

    CAS  Article  Google Scholar 

  11. 11.

    Pilak, O. et al. The crystal structure of the apoenzyme of the iron–sulfur cluster-free hydrogenase. J. Mol. Biol. 358, 798–809 (2006).

    CAS  Article  Google Scholar 

  12. 12.

    Shima, S. et al. The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site. Science 321, 572–575 (2008).

    CAS  Article  Google Scholar 

  13. 13.

    Hiromoto, T. et al. The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl–iron ligation in the active site iron complex. FEBS Lett. 583, 585–590 (2009).

    CAS  Article  Google Scholar 

  14. 14.

    Hiromoto, T., Warkentin, E., Moll, J., Ermler, U. & Shima, S. The crystal structure of an [Fe]-hydrogenase-substrate complex reveals the framework for H2 activation. Angew. Chem. Int. Ed. 48, 6457–6460 (2009).

    CAS  Article  Google Scholar 

  15. 15.

    Lyon, E. J. et al. Carbon monoxide as an intrinsic ligand to iron in the active site of the iron–sulfur-cluster-free hydrogenase H2-forming methylenetetrahydromethanopterin dehydrogenase as revealed by infrared spectroscopy. J. Am. Chem. Soc. 126, 14239–14248 (2004).

    CAS  Article  Google Scholar 

  16. 16.

    Tamura, H. et al. Crystal structures of [Fe]-hydrogenase in complex with inhibitory isocyanides: implications for the H2-activation site. Angew. Chem. Int. Ed. 52, 9656–9659 (2013).

    CAS  Article  Google Scholar 

  17. 17.

    Shima, S. & Ataka, K. Isocyanides inhibit [Fe]-hydrogenase with very high affinity. FEBS Lett. 585, 353–356 (2011).

    CAS  Article  Google Scholar 

  18. 18.

    Dey, A. Density functional theory calculations on the mononuclear non-heme iron active site of Hmd hydrogenase: role of the internal ligands in tuning external ligand binding and driving H2 heterolysis. J. Am. Chem. Soc. 132, 13892–13901 (2010).

    CAS  Article  Google Scholar 

  19. 19.

    Hedegård, E. D., Kongsted, J. & Ryde, U. Multiscale modeling of the active site of [Fe] hydrogenase: the H2 binding site in open and closed protein conformations. Angew. Chem. Int. Ed. 54, 6246–6250 (2015).

    Article  Google Scholar 

  20. 20.

    Yang, X. Z. & Hall, M. B. Monoiron hydrogenase catalysis: hydrogen activation with the formation of a dihydrogen, Fe-Hδ−–Hδ+-O, bond and methenyl-H4MPT+ triggered hydride transfer. J. Am. Chem. Soc. 131, 10901–10908 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Berkessel, A. Activation of dihydrogen without transition metals. Curr. Opin. Chem. Biol. 5, 486–490 (2001).

    CAS  Article  Google Scholar 

  22. 22.

    Berkessel, A. & Thauer, R. K. On the mechanism of catalysis by a metal-free hydrogenase from methanogenic archaea: enzymatic transformation of H2 without a metal and its analogy to the chemistry of alkanes in superacidic solution. Angew. Chem. Int. Ed. Engl. 34, 2247–2250 (1995).

    CAS  Article  Google Scholar 

  23. 23.

    Shima, S., Lyon, E. J., Thauer, R. K., Mienert, B. & Bill, E. Mössbauer studies of the iron–sulfur cluster-free hydrogenase: the electronic state of the mononuclear Fe active site. J. Am. Chem. Soc. 127, 10430–10435 (2005).

    CAS  Article  Google Scholar 

  24. 24.

    Korbas, M. et al. The iron–sulfur cluster-free hydrogenase (Hmd) is a metalloenzyme with a novel iron binding motif. J. Biol. Chem. 281, 30804–30813 (2006).

    CAS  Article  Google Scholar 

  25. 25.

    Gütlich, P., Bill, E. & Trautwein, A. X. Mössbauer Spectroscopy and Transition Metal Chemistry (Springer Verlag, 2011).

  26. 26.

    Chen, D. F., Scopelliti, R. & Hu, X. L. A five-coordinate iron center in the active site of [Fe]-hydrogenase: hints from a model study. Angew. Chem. Int. Ed. 50, 5671–5673 (2011).

    CAS  Article  Google Scholar 

  27. 27.

    Shima, S. et al. Reconstitution of [Fe]-hydrogenase using model complexes. Nat. Chem. 7, 995–1002 (2015).

    CAS  Article  Google Scholar 

  28. 28.

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  29. 29.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  30. 30.

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

    CAS  Article  Google Scholar 

  31. 31.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  32. 32.

    BUSTER version 2.10.1. (Global Phasing, 2016); https://www.globalphasing.com/buster/

  33. 33.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  34. 34.

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

    CAS  Article  Google Scholar 

  35. 35.

    Krebs, W. G. & Gerstein, M. The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. Nucleic Acids Res. 28, 1665–1675 (2000).

    CAS  Article  Google Scholar 

  36. 36.

    Chung, L. W. et al. The ONIOM method and its applications. Chem. Rev. 115, 5678–5796 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Frisch, M. J. et al. Gaussian 09, Revision D.01 (Gaussian, 2016).

  38. 38.

    Zhao, Y. & Truhlar, D. G. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Acc. 120, 215–241 (2008).

    CAS  Article  Google Scholar 

  39. 39.

    Zhao, Y. & Truhlar, D. G. Density functionals with broad applicability in chemistry. Acc. Chem. Res. 41, 157–167 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Max Planck Society (to S.S., E.B. and U.E.), Deutsche Forschungsgemeinschaft (Iron-Sulfur for Life, SH 87/1-1 to S.S.) and the Swiss National Science Foundation (to X.H.). M.D.W. thanks C. Corminboeuf (École Polytechnique Fédérale de Lausanne, Switzerland) for financial support and the Laboratory for Computational Molecular Design (EPFL) for providing computing resources. The authors are grateful to H. Michel for continuous support and the staff of the PX II beamline at the Swiss Light Source (SLS) for help with data collection. The authors also thank the staff from the BM30A (FIP) beamline at the European Synchrotron Radiation Facility (ESRF). G.H. was supported by a fellowship from China Scholarship Council (CSC).

Author information

Affiliations

Authors

Contributions

S.S. directed and designed research. G.H. performed cultivation, enzyme purification and crystallization. T.W. and U.E. collected X-ray data. T.W. solved, refined and deposited the structure. M.D.W. and X.H. performed and analysed the QM/MM computations. K.A. performed infrared spectroscopy and E.B. performed Mössbauer spectroscopy. All authors contributed to writing the paper.

Corresponding author

Correspondence to Seigo Shima.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary Information

Supplementary Methods, Supplementary Figs. 1–15, Supplementary Tables 1–3, Supplementary references

Reporting Summary

Supplementary Data 1

Structure 2

Supplementary Data 2

Structure 3

Supplementary Data 3

Structure 4

Supplementary Data 4

Structure 5

Supplementary Data 5

TS3,4

Supplementary Data 6

TS4,5

Supplementary Video 1

Open/closed conformational change

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, G., Wagner, T., Wodrich, M.D. et al. The atomic-resolution crystal structure of activated [Fe]-hydrogenase. Nat Catal 2, 537–543 (2019). https://doi.org/10.1038/s41929-019-0289-4

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing