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.

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase

An Erratum to this article was published on 17 August 2011

An Erratum to this article was published on 17 August 2011

This article has been updated

Abstract

Hydrogenases are essential for H2 cycling in microbial metabolism and serve as valuable blueprints for H2-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O2. The O2-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H2 uptake in the presence of ambient O2. Here we show that O2 tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O2 as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O2 tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O2 to the active site.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Structural model of the membrane-bound hydrogenase (MBH) from R. eutropha based on multiple sequence alignment using the crystal structure of the D. gigas hydrogenase25 as a template.
Figure 2: Lithoautotrophic growth of Ralstonia derivatives on H2 and CO2 under high and low O2 concentrations.
Figure 3: Immunological detection of the MBH subunits HoxG and HoxK in cell fractions.
Figure 4: FTIR spectra of purified MBHwt and MBHC19G/C120G.
Figure 5: EPR X-band spectra of purified MBHwt and MBHC19G/C120G.
Figure 6: Effect of O2 on MBHwt and MBHC19G/C120G immobilized onto graphite.
Figure 7: Model showing the reactions of the R. eutropha membrane-bound [NiFe]-hydrogenase and its C19G/C120G derivative.

Change history

  • 01 July 2011

    In the version of this article initially published, an arrow was inadvertently omitted from Figure 7. The error has been corrected in the HTML and PDF versions of the article.

  • 01 August 2011

    In the previous version of this article, a water molecule was mislabeled in Figure 7 and an error was inadvertently introduced into the journal title. These errors have been corrected in the HTML and PDF versions of the article.

References

  1. 1

    Lubner, C.E., Grimme, R., Bryant, D.A. & Golbeck, J.H. Wiring photosystem I for direct solar hydrogen production. Biochemistry 49, 404–414 (2010).10.1021/bi901704v

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Krassen, H. et al. Photosynthetic hydrogen production by a hybrid complex of photosystem I and [NiFe]-hydrogenase. ACS Nano 3, 4055–4061 (2009).10.1021/nn900748j

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Esswein, A.J. & Nocera, D.G. Hydrogen production by molecular photocatalysis. Chem. Rev. 107, 4022–4047 (2007).10.1002/chin.200750272

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Tran, P.D., Artero, V. & Fontecave, M. Water electrolysis and photoelectrolysis on electrodes engineered using biological and bio-inspired molecular systems. Energy Environ. Sci. 3, 727–747 (2010).10.1039/B926749B

    CAS  Article  Google Scholar 

  5. 5

    Lubitz, W., Reijerse, E. & van Gastel, M. [NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques. Chem. Rev. 107, 4331–4365 (2007).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    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 

  8. 8

    Happe, R.P., Roseboom, W., Pierik, A.J., Albracht, S.P. & Bagley, K.A. Biological activation of hydrogen. Nature 385, 126 (1997).

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

    De Lacey, A.L., Fernández, V., Rousset, M. & Cammack, R. Activation and inactivation of hydrogenase function and the catalytic cycle: spectroelectrochemical studies. Chem. Rev. 107, 4304–4330 (2007).10.1002/chin.200751260

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Schwartz, E. & Friedrich, B. The H2-Metabolizing Prokaryotes. in The Prokaryotes, Vol. 3 (eds. Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H. & Stackebrandt, E.) 496–563 (Springer-Verlag., 2006).

    Google Scholar 

  12. 12

    Burgdorf, T. et al. [NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation. J. Mol. Microbiol. Biotechnol. 10, 181–196 (2005).10.1159/000091564

    CAS  Article  PubMed  Google Scholar 

  13. 13

    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).10.1002/cphc.200901002

    CAS  Article  PubMed  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    Vincent, K.A. et al. Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels. Proc. Natl. Acad. Sci. USA 102, 16951–16954 (2005).

    CAS  Article  Google Scholar 

  16. 16

    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).10.1073/pnas.0905959106

    Article  PubMed  Google Scholar 

  17. 17

    Vincent, K.A. et al. Electrochemical definitions of O2 sensitivity and oxidative inactivation in hydrogenases. J. Am. Chem. Soc. 127, 18179–18189 (2005).10.1021/ja055160v

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Cracknell, J.A. et al. Enzymatic oxidation of H2 in atmospheric O2: the electrochemistry of energy generation from trace H2 by aerobic microorganisms. J. Am. Chem. Soc. 130, 424–425 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Lamle, S.E., Albracht, S.P. & Armstrong, F.A. Electrochemical potential-step investigations of the aerobic interconversions of [NiFe]-hydrogenase from Allochromatium vinosum: insights into the puzzling difference between unready and ready oxidized inactive states. J. Am. Chem. Soc. 126, 14899–14909 (2004).10.1021/ja047939v

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Fontecilla-Camps, J.C., Amara, P., Cavazza, C., Nicolet, Y. & Volbeda, A. Structure-function relationships of anaerobic gas-processing metalloenzymes. Nature 460, 814–822 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Ogata, H., Lubitz, W. & Higuchi, Y. [NiFe] hydrogenases: structural and spectroscopic studies of the reaction mechanism. Dalton Trans. 37, 7577–7587 (2009).10.1039/B903840J

    Article  Google Scholar 

  22. 22

    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).10.1074/jbc.M805690200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Pandelia, M.E. et al. Membrane-bound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance. J. Am. Chem. Soc. 132, 6991–7004 (2010).10.1021/ja910838d

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Lukey, M.J. et al. How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. J. Biol. Chem. 285, 3928–3938 (2010).10.1074/jbc.A109.067751

    CAS  Article  PubMed  Google Scholar 

  25. 25

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

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    Saggu, M. et al. Comparison of the membrane-bound [NiFe] hydrogenases from R. eutropha H16 and D. vulgaris Miyazaki F in the oxidized ready state by pulsed EPR. Phys. Chem. Chem. Phys. 12, 2139–2148 (2010).10.1039/B922236G

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Goldet, G. et al. Hydrogen production under aerobic conditions by membrane-bound hydrogenases from Ralstonia species. J. Am. Chem. Soc. 130, 11106–11113 (2008).

    CAS  Article  Google Scholar 

  29. 29

    Volbeda, A., Montet, Y., Vernede, X., Hatchikian, E.C. & Fontecilla-Camps, J.C. High-resolution crystallographic analysis of Desulfovibrio fructiosovorans [NiFe] hydrogenase. Int. J. Hydrogen Energy 27, 1449–1461 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Buhrke, T., Lenz, O., Krauss, N. & Friedrich, B. Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site. J. Biol. Chem. 280, 23791–23796 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Leroux, F. et al. Experimental approaches to kinetics of gas diffusion in hydrogenase. Proc. Natl. Acad. Sci. USA 105, 11188–11193 (2008).

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Beinert, H., Holm, R.H. & Munck, E. Iron-sulfur clusters: nature's modular, multipurpose structures. Science 277, 653–659 (1997).

    CAS  Article  Google Scholar 

  34. 34

    Knüttel, K. et al. Redox properties of the metal centres in the membrane-bound hydrogenase from Alcaligenes eutrophus CH34. Bull. Pol. Acad. Sci. Chem. 42, 495–511 (1994).

    Google Scholar 

  35. 35

    Schneider, K., Patil, D. & Cammack, R. ESR properties of membrane-bound hydrogenases from aerobic hydrogen bacteria. Biochim. Biophys. Acta 748, 353–361 (1983).10.1016/0167-4838(83)90179-6

    CAS  Article  Google Scholar 

  36. 36

    Cammack, R., Lalla-Maharajh, W. & Schneider, K. EPR studies of some oxygen-stable hydrogenases. in Electron Transport and Oxygen Utilization (ed. Ho, C.) 411–415 (Elsevier, 1982).

    Google Scholar 

  37. 37

    Bernhard, M., Benelli, B., Hochkoeppler, A., Zannoni, D. & Friedrich, B. Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16. Eur. J. Biochem. 248, 179–186 (1997).

    CAS  Article  Google Scholar 

  38. 38

    Hamann, N. et al. A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis. Biochemistry 46, 12875–12885 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Berrisford, J.M. & Sazanov, L.A. Structural basis for the mechanism of respiratory complex I. J. Biol. Chem. 284, 29773–29783 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Chartron, J. et al. Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5′-phosphosulfate reductase. J. Mol. Biol. 364, 152–169 (2006).10.1016/j.jmb.2006.08

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Sazanov, L.A. & Hinchliffe, P. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 311, 1430–1436 (2006).

    CAS  Article  Google Scholar 

  42. 42

    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).10.1021/bi00045a008

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Heering, H.A., Bulsink, Y.B., Hagen, W.R. & Meyer, T.E. Reversible super-reduction of the cubane [4Fe-4S](3+;2+;1+) in the high-potential iron-sulfur protein under non-denaturing conditions. EPR spectroscopic and electrochemical studies. Eur. J. Biochem. 232, 811–817 (1995).

    CAS  Article  Google Scholar 

  44. 44

    The UniProt Consortium. The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 38, D142–D148 (2010).

  45. 45

    Sali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    CAS  Article  Google Scholar 

  46. 46

    Gabdoulline, R.R., Stein, M. & Wade, R.C. qPIPSA: relating enzymatic kinetic parameters and interaction fields. BMC Bioinformatics 8, 373 (2007).10.1186/1471-2105-8-373

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    CAS  Article  Google Scholar 

  48. 48

    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).10.1016/0005-2744(79)90117-7

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Ludwig, M. et al. Concerted action of two novel auxiliary proteins in assembly of the active site in a membrane-bound [NiFe] hydrogenase. J. Biol. Chem. 284, 2159–2168 (2009).10.1074/jbc.M808488200

    CAS  Article  PubMed  Google Scholar 

  50. 50

    Bard, A.J. & Faulkner, L.R. (eds.) Electrochemical Methods: Fundamentals and Applications (Wiley VCH, 2001).

    Google Scholar 

Download references

Acknowledgements

The authors wish to thank J. Priebe for obtaining preliminary EPR results, J. Hamann and A. Strack for excellent technical assistance and P. Hildebrandt and R. Bittl for generous support. This work was supported by the German Federal ministry of Education and Research (T.G.; BMBF project “H2 Design Cells”), the Deutsche Forschungsgemeinschaft (M. Saggu, N.H., I.Z., B.F., O.L.; Cluster of Excellence “UniCat”), the FP7 of the European Union (J.F.; energy network project SOLAR-H2), the Klaus Tschira Foundation and the Max Planck Society for the Advancement of Science (M. Stein), and the Engineering and Physical Sciences Research Council UK (A.F.W., F.A.A; Grant Supergen 5) and the Biotechnology and Biological Sciences Research Council UK (A.F.W., F.A.A; Grant BB/H003878/1).

Author information

Affiliations

Authors

Contributions

T.G. performed the majority of the experiments, including mutant construction, biochemical-physiological analysis and protein purification. J.F. contributed to a great extent to protein purification. A.F.W. performed the electrochemical analysis of the proteins. Bioinformatics and protein structural modeling were carried out by M. Stein. N.H. and I.Z. conducted and analyzed the FTIR spectroscopic measurements. M. Saggu and F.L. performed and analyzed the EPR experiments. T.G., J.F., M. Stein, I.Z., F.L., F.A.A., B.F. and O.L. contributed ideas, evaluated and discussed data and prepared the manuscript.

Corresponding author

Correspondence to Oliver Lenz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 989 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Goris, T., Wait, A., Saggu, M. et al. A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase. Nat Chem Biol 7, 310–318 (2011). https://doi.org/10.1038/nchembio.555

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