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Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase

Nature Chemical Biology volume 10, pages 378–385 (2014)Cite this article

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Abstract

Hydrogenases catalyze the reversible oxidation of H2 into protons and electrons and are usually readily inactivated by O2. However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O2 that enables their host organisms to utilize H2 as an energy source at high O2. This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H2 oxidation at high O2. We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range.

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Figure 1: Cartoon and crystal structure of the MBH from R. eutropha.
Figure 2: 15N Davies-ENDOR on as-isolated MBH indicating a covalent bond between Fe4 and the peptide amide N from C20S found in the superoxidized state.
Figure 3: An oxygen ligand is bound to Fe1 of the [4Fe-3S] cluster in the as-isolated (superoxidized) state.
Figure 4: Detection of the Fe1-bound OH− ligand by RR and pulsed EPR spectroscopy.
Figure 5: Redox-dependent structural changes within the [4Fe-3S] cluster.
Figure 6: Redox-dependent structural changes occurring during high potential redox transition.

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Acknowledgements

We are grateful to D. von Stetten and A. Royant of the ID29S-Cryobench (European Synchrotron Radiation Facility (ESRF), Grenoble) and the scientific staff of the ESRF at beamlines ID14-1, ID 14-4, ID29, ID 23-1 and ID23-2 and 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, BL 14.2 and BL 14.3 operated by the Joint Berlin MX-Laboratory at the BESSY II electron storage ring (Berlin-Adlershof, Germany), where the data were collected, for continuous support. We thank C. Müller-Dieckmann for helpful advice in collecting anomalous data at the ID29 (ESRF, Grenoble). J. Hamann is acknowledged for excellent technical support in molecular biology. J. Newie, S. Wohlfahrt and T. Goris are acknowledged for their help in constructing MBH variants. This work was supported by the German Research Foundation (DFG) Cluster of Excellence 'Unifying Concepts in Catalysis' (to P.S., O.L., P.H., V.P. and R.B.). P.S. acknowledges K.P. Hofmann and his advanced investigator European Research Council grant (ERC-2009/249910-TUDOR) for support.

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Authors and Affiliations

  1. Institut für Biologie–Mikrobiologie, Humboldt-Universität zu Berlin, Berlin, Germany

    Stefan Frielingsdorf, Johannes Fritsch, Tina Jaenicke, Bärbel Friedrich & Oliver Lenz

  2. Institut für Chemie, Sekr. PC14, Technische Universität Berlin, Berlin, Germany

    Stefan Frielingsdorf, Elisabeth Siebert, Yvonne Rippers, Friedhelm Lendzian, Ingo Zebger, Peter Hildebrandt & Oliver Lenz

  3. Institut für Medizinische Physik und Biophysik (CharitéCrossOver), AG Proteinstrukturanalyse, Charité–Universitätsmedizin Berlin, Berlin, Germany

    Andrea Schmidt, Mathias Hammer, Jacqueline Kalms & Patrick Scheerer

  4. Fachbereich Physik, Freie Universität Berlin, Berlin, Germany

    Julia Löwenstein, Christian Teutloff & Robert Bittl

  5. Institut für Chemie, Sekr. C7, Technische Universität Berlin, Berlin, Germany

    Vladimir Pelmenschikov & Martin Kaupp

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Contributions

S.F. and J.F. prepared MBH samples. P.S., J.F. and S.F. optimized MBH crystallization under different conditions and performed soaking experiments. P.S. collected the X-ray diffraction data, performed data processing and solved the MBH structures. A.S., M.H., J.K. and P.S. refined the crystal structures. J.L., C.T., F.L. and R.B. recorded and analyzed EPR data. E.S., I.Z. and P.H. recorded and analyzed RR spectroscopy data. V.P., Y.R. and M.K. performed and analyzed the DFT calculations. S.F. performed and analyzed H-D exchange measurements. T.J. and O.L. constructed the MBH mutant proteins. S.F., J.F., O.L. and P.S. wrote the paper with contributions from all co-authors.

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Correspondence to Oliver Lenz or Patrick Scheerer.

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Frielingsdorf, S., Fritsch, J., Schmidt, A. et al. Reversible [4Fe-3S] cluster morphing in an O2-tolerant [NiFe] hydrogenase. Nat Chem Biol 10, 378–385 (2014). https://doi.org/10.1038/nchembio.1500

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