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 crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre

Abstract

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H2 into protons and electrons at high rates1. Those hydrogenases maintaining their activity in the presence of O2 are considered to be central to H2-based technologies, such as enzymatic fuel cells and for light-driven H2 production2. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations3,4,5,6,7,8, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O2 has remained unclear. Here we present the crystal structure of an O2-tolerant [NiFe]-hydrogenase from the aerobic H2 oxidizer Ralstonia eutropha H16 at 1.5 Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H2-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H2 oxidation and as an electron-delivering device upon O2 attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials7,8,9. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H2-converting catalysts that are capable of cycling H2 in air.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overall structure of the membrane-bound hydrogenase from R. eutropha.
Figure 2: Metal cofactors of the MBH.
Figure 3: Architecture of the proximal [4Fe-3S] cluster.
Figure 4: Proposed water/proton transfer pathway from the MBH active site to the protein surface.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structure have been deposited in the Protein Data Bank with the accession code 3RGW.

References

  1. Cammack, R., Frey, M. & Robson, R. Hydrogen as a Fuel, Learning from Nature (Taylor & Francis, 2001)

    Book  Google Scholar 

  2. Friedrich, B., Fritsch, J. & Lenz, O. Oxygen-tolerant hydrogenases in hydrogen-based technologies. Curr. Opin. Biotechnol. 22, 358–364 (2011)

    Article  CAS  Google Scholar 

  3. Ludwig, M., Cracknell, J. A., Vincent, K. A., Armstrong, F. A. & Lenz, O. Oxygen-tolerant H2 oxidation by membrane-bound [NiFe] hydrogenases of Ralstonia species. Coping with low level H2 in air. J. Biol. Chem. 284, 465–477 (2009)

    Article  CAS  Google Scholar 

  4. Saggu, M. et al. Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16. J. Biol. Chem. 284, 16264–16276 (2009)

    Article  CAS  Google Scholar 

  5. Cracknell, J. A., Wait, A. F., Lenz, O., Friedrich, B. & Armstrong, F. A. A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases. Proc. Natl Acad. Sci. USA 106, 20681–20686 (2009)

    Article  ADS  Google Scholar 

  6. Lukey, M. J. et al. How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. J. Biol. Chem. 285, 3928–3938 (2010)

    Article  CAS  Google Scholar 

  7. Goris, T. et al. A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase. Nature Chem. Biol. 7, 310–318 (2011)

    Article  CAS  Google Scholar 

  8. Pandelia, M. E. et al. Characterization of a unique [FeS] cluster in the electron transfer chain of the oxygen tolerant [NiFe] hydrogenase from Aquifex aeolicus. Proc. Natl Acad. Sci. USA 108, 6097–6102 (2011)

    Article  CAS  ADS  Google Scholar 

  9. Schneider, K., Patil, D. S. & Cammack, R. Electron-spin-resonance properties of membrane-bound hydrogenases from aerobic hydrogen bacteria. Biochim. Biophys. Acta 748, 353–361 (1983)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Stripp, S. T. et al. How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proc. Natl Acad. Sci. USA 106, 17331–17336 (2009)

    Article  CAS  ADS  Google Scholar 

  12. Ogata, H., Lubitz, W. & Higuchi, Y. [NiFe] hydrogenases: structural and spectroscopic studies of the reaction mechanism. Dalton Trans. 7577–7587 (2009)

  13. Vincent, K. A., Parkin, A. & Armstrong, F. A. Investigating and exploiting the electrocatalytic properties of hydrogenases. Chem. Rev. 107, 4366–4413 (2007)

    Article  CAS  Google Scholar 

  14. 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 

  15. Schwartz, E. & Friedrich, B. in The Prokaryotes (eds Dworkin, M. et al.) 496–563 (Springer, 2006)

    Book  Google Scholar 

  16. Lenz, O. et al. H2 conversion in the presence of O2 as performed by the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha. ChemPhysChem 11, 1107–1119 (2010)

    Article  CAS  Google Scholar 

  17. Volbeda, A. et al. Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580–587 (1995)

    Article  CAS  ADS  Google Scholar 

  18. Volbeda, A. et al. Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases. J. Biol. Inorg. Chem. 10, 239–249 (2005)

    Article  CAS  Google Scholar 

  19. Matias, P. M. et al. [NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c3 . J. Biol. Inorg. Chem. 6, 63–81 (2001)

    Article  MathSciNet  CAS  Google Scholar 

  20. Ogata, H., Kellers, P. & Lubitz, W. The crystal structure of the [NiFe] hydrogenase from the photosynthetic bacterium Allochromatium vinosum: characterization of the oxidized enzyme (Ni-A state). J. Mol. Biol. 402, 428–444 (2010)

    Article  CAS  Google Scholar 

  21. Seefeldt, L. C., Hoffman, B. M. & Dean, D. R. Mechanism of Mo-dependent nitrogenase. Annu. Rev. Biochem. 78, 701–722 (2009)

    Article  CAS  Google Scholar 

  22. Peters, J. W. et al. Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36, 1181–1187 (1997)

    Article  CAS  Google Scholar 

  23. Knüttel, K. et al. Redox properties of the metal centers in the membrane-bound hydrogenase from Alcaligens eurtophus CH34. Bull. Polish Acad. Sci. 42, 495–511 (1994)

    Google Scholar 

  24. Cammack, R. “Super-reduction” of chromatium high-potential iron-sulphur protein in the presence of dimethyl sulphoxide. Biochem. Biophys. Res. Commun. 54, 548–554 (1973)

    Article  CAS  Google Scholar 

  25. Thomson, A. J. et al. Low-temperature magnetic circular-dichroism evidence for the conversion of 4-iron-sulfur clusters in a ferredoxin from Clostridium pasteurianum into 3-iron-sulfur clusters. Biochim. Biophys. Acta 637, 423–432 (1981)

    Article  CAS  Google Scholar 

  26. Capozzi, F., Ciurli, S. & Luchinat, C. Coordination sphere versus protein environment as determinants of electronic and functional properties of iron-sulfur proteins. Metal Sites Proteins Models 90, 127–160 (1998)

    Article  CAS  Google Scholar 

  27. Carter, C. W., Jr New stereochemical analogies between iron-sulfur electron transport proteins. J. Biol. Chem. 252, 7802–7811 (1977)

    CAS  PubMed  Google Scholar 

  28. Dey, A. et al. Solvent tuning of electrochemical potentials in the active sites of HiPIP versus ferredoxin. Science 318, 1464–1468 (2007)

    Article  CAS  ADS  Google Scholar 

  29. Heering, H. A., Bulsink, B. M., Hagen, W. R. & Meyer, T. E. Influence of charge and polarity on the redox potentials of high-potential iron-sulfur proteins: evidence for the existence of two groups. Biochemistry 34, 14675–14686 (1995)

    Article  CAS  Google Scholar 

  30. Montet, Y. et al. Gas access to the active site of Ni-Fe hydrogenases probed by X-ray crystallography and molecular dynamics. Nature Struct. Biol. 4, 523–526 (1997)

    Article  CAS  Google Scholar 

  31. Schubert, T., Lenz, O., Krause, E., Volkmer, R. & Friedrich, B. Chaperones specific for the membrane-bound [NiFe]-hydrogenase interact with the Tat signal peptide of the small subunit precursor in Ralstonia eutropha H16. Mol. Microbiol. 66, 453–467 (2007)

    Article  CAS  Google Scholar 

  32. Schink, B. & Schlegel, H. G. The membrane-bound hydrogenase of Alcaligenes eutrophus. I. Solubilization, purification, and biochemical properties. Biochim. Biophys. Acta 567, 315–324 (1979)

    Article  CAS  Google Scholar 

  33. Jancarik, J. & Kim, S.-H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Cryst. 24, 409–411 (1991)

    Article  CAS  Google Scholar 

  34. Flot, D. et al. The ID23-2 structural biology microfocus beamline at the ESRF. J. Synchrotron Radiat. 17, 107–118 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  37. Collaborative Computational Project, Number 4 . The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  40. Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  43. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: A program to check the stereochemical quality of protein structures. Appl. Cryst. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  44. Hooft, R. W., Vriend, G., Sander, C. & Abola, E. E. Errors in protein structures. Nature 381, 272 (1996)

    Article  CAS  ADS  Google Scholar 

  45. McDonald, I. K. & Thornton, J. M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994)

    Article  CAS  Google Scholar 

  46. Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995)

    Article  CAS  Google Scholar 

  47. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    Article  CAS  Google Scholar 

  48. DeLano, W. L. The PyMOL Molecular Graphics System 〈http://www.pymol.org〉 (2002)

Download references

Acknowledgements

We are grateful to U. Müller, M. Weiss and the scientific staff of the BESSY-MX/Helmholtz Zentrum Berlin für Materialien und Energie at beamlines BL 14.1 and BL 14.2, D. von Stetten and A. Royant of the ID29S-Cryobench (ESRF, Grenoble) and the European Synchrotron Radiation Facility (ESRF, Grenoble) at beamlines ID23-1, ID23-2, ID14-1 and ID 14-4, where the data were collected, for support. This work was supported by the EU/FP7 programme Solar-H2 (to J.F.), the DFG Cluster of Excellence ‘Unifying Concepts in Catalysis’ (to S.F., B.F., O.L.), and the Sfb740 (to C.M.T.S.). P.S. acknowledges K. P. Hofmann and his advanced investigator ERC grant (ERC-2009/249910—TUDOR) for support.

Author information

Authors and Affiliations

Authors

Contributions

J.F. and P.S. are joint first authors. J.F. optimized cell growth conditions as well as the MBH purification procedure. P.S. conducted the crystallization screening; P.S., J.F. and S.F. optimized MBH crystallization conditions. P.S. and S.K. collected the X-ray diffraction data. P.S. performed data processing, solved and refined the MBH structure. B.F., O.L. and P.S. coordinated the project. J.F., P.S., S.F. and O.L. analysed data. J.F., P.S., S.F., B.F., O.L. and C.M.T.S. wrote the manuscript.

Corresponding authors

Correspondence to Patrick Scheerer, Oliver Lenz or Christian M. T. Spahn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-9 with legends, Supplementary Table 1 and additional references. (PDF 10591 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fritsch, J., Scheerer, P., Frielingsdorf, S. et al. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 479, 249–252 (2011). https://doi.org/10.1038/nature10505

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10505

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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