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Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase

Nature volume 479, pages 253–256 (2011)Cite this article

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Abstract

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H2-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H2 → 2H+ + 2e− (ref. 1). In contrast to the well-studied O2-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O2-tolerant H2 oxidation activity2,3,4; however, the mechanism of O2 tolerance is unclear5. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues—in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes—and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O2-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O2 tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O2 in preventing the formation of an unready, inactive state of the enzyme.

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Figure 1: Overall structure of MBH heterotetramer.
Figure 2: Structural comparison of the proximal cluster in different redox conditions.
Figure 3: A comparison of charged residues around the proximal Fe-S cluster in MBH and the standard [NiFe]-hydrogenase from D. gigas.
Figure 4: Proposed proton-transfer pathways.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structures have been deposited in the Protein Data Bank with the accession codes 3AYX, 3AYY and 3AYZ.

References

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

    Article  CAS  Google Scholar 

  2. 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)

    Article  CAS  ADS  Google Scholar 

  3. Luo, X., Brugna, M., Tron-Infossi, P., Giudici-Orticoni, M. T. & Lojou, E. Immobilization of the hyperthermophilic hydrogenase from Aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient H2 oxidation. J. Biol. Inorg. Chem. 14, 1275–1288 (2009)

    Article  CAS  Google Scholar 

  4. 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 

  5. 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 

  6. Brugna-Guiral, M. et al. [NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics. Extremophiles 7, 145–157 (2003)

    Article  CAS  Google Scholar 

  7. 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)

    Article  CAS  Google Scholar 

  8. Yoon, K.-S., Fukuda, K., Fujisawa, K. & Nishihara, H. Purification and characterization of a highly thermostable, oxygen-resistant, respiratory [NiFe]-hydrogenase from a marine, aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus. Int. J. Hydrogen Energy 36, 7081–7088 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  10. Higuchi, Y., Yagi, T. & Yasuoka, N. Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis. Structure 5, 1671–1680 (1997)

    Article  CAS  Google Scholar 

  11. 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 

  12. 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 

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

    Article  CAS  Google Scholar 

  14. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Aragão, D., Mitchell, E. P., Frazao, C. F., Carrondo, M. A. & Lindley, P. F. Structural and functional relationships in the hybrid cluster protein family: structure of the anaerobically purified hybrid cluster protein from Desulfovibrio vulgaris at 1.35 Å resolution. Acta Crystallogr. D 64, 665–674 (2008)

    Article  Google Scholar 

  18. Huang, W. et al. Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold. Structure 5, 691–699 (1997)

    Article  CAS  Google Scholar 

  19. 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 

  20. Schneider, K., Patil, D. S. & Cammack, R. ESR properties of membrane-bound hydrogenase from aerobic hydrogen bacteria. Biochim. Biophys. Acta 748, 353–361 (1983)

    Article  CAS  Google Scholar 

  21. 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 

  22. 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 

  23. Dementin, S. et al. A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase. J. Biol. Chem. 279, 10508–10513 (2004)

    Article  CAS  Google Scholar 

  24. Teixeira, V. H., Soares, C. M. & Baptista, A. M. Proton pathways in a [NiFe]-hydrogenase: A theoretical study. Proteins 70, 1010–1022 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Fernandez, V. M., Hatchikian, E. C., Patil, D. S. & Cammack, R. ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase. Biochim. Biophys. Acta 883, 145–154 (1986)

    Article  CAS  Google Scholar 

  27. 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 

  28. Marques, M. C., Coelho, R., De Lacey, A. L., Pereira, I. A. & Matias, P. M. The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, “as-isolated” state. J. Mol. Biol. 396, 893–907 (2010)

    Article  CAS  Google Scholar 

  29. 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)

    Article  CAS  Google Scholar 

  30. Duché, O., Elsen, S., Cournac, L. & Colbeau, A. Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity. FEBS J. 272, 3899–3908 (2005)

    Article  Google Scholar 

  31. Shomura, Y., Hagiya, K., Yoon, K.-S., Nishihara, H. & Higuchi, Y. Crystallization and preliminary X-ray diffraction analysis of membrane-bound respiratory [NiFe] hydrogenase from Hydrogenovibrio marinus. Acta Crystallogr. F 67, 827–829 (2011)

    Article  CAS  Google Scholar 

  32. Ueno, G., Kanda, H., Kumasaka, T. & Yamamoto, M. Beamline Scheduling Software: administration software for automatic operation of the RIKEN structural genomics beamlines at SPring-8. J. Synchrotron Radiat. 12, 380–384 (2005)

    Article  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  35. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  36. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    Article  CAS  ADS  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank K. Hagiya for technical assistance at Ibaraki University. The synchrotron radiation experiments were performed at the BL41XU (proposal no. 2010A1223) and BL44XU (proposal no. 2010A/B6520) with the approval of JASRI. The CCD detector MX225-HE (Rayonix) at BL44XU was financially supported by Academia Sinica and National Synchrotron Radiation Research Center (Taiwan). This work was supported by a grant-in-aid for Scientific Research from MEXT (20051022 (Y.S.), 22770111 (Y.S.), 18GS0207 (Y.H.)), grant-in-aid for Scientific Research from JSPS (20580094 (H.N.), 22370061 (Y.H.) and 22657031 (Y.H.)), grant-in-aid for research and education from University of Hyogo (Y.S.), and grant-in-aid for young scientists from Hyogo Science and Technology Association (Y.S.). This work was also partially supported by the GCOE Program (Y.H.), the Japanese Aerospace Exploration Agency Project (Y.H.), and the basic research programs CREST type, ‘Development of the Foundation for Nano-Interface Technology’ from JST, Japan (Y.H.).

Author information

Author notes

  1. Ki-Seok Yoon

    Present address: Present address: Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.,

Authors and Affiliations

  1. Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan

    Yasuhito Shomura & Yoshiki Higuchi

  2. RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-gun, Sayo-cho, Hyogo 679-5148, Japan

    Yasuhito Shomura & Yoshiki Higuchi

  3. Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chu-ou, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan

    Ki-Seok Yoon & Hirofumi Nishihara

  4. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan

    Yoshiki Higuchi

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Contributions

K.-S.Y. and H.N. performed bacterial culture and protein purification under management by H.N. Y.S. performed crystallization, X-ray data collection and structure determination. Y.S. and Y.H. prepared the manuscript with contributions from all authors. The overall project management was done by Y.H.

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Correspondence to Yoshiki Higuchi.

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Shomura, Y., Yoon, KS., Nishihara, H. et al. Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase. Nature 479, 253–256 (2011). https://doi.org/10.1038/nature10504

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