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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


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.

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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.


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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




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.

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Correspondence to Oliver Lenz.

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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).

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