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.

  • Article
  • Published:

The structure of vanadium nitrogenase reveals an unusual bridging ligand

Nature Chemical Biology volume 13, pages 956–960 (2017)Cite this article

Subjects

Abstract

Nitrogenases catalyze the reduction of dinitrogen (N2) gas to ammonium at a complex heterometallic cofactor. This most commonly occurs at the FeMo cofactor (FeMoco), a [Mo–7Fe–9S–C] cluster whose exact reactivity and substrate-binding mode remain unknown. Alternative nitrogenases replace molybdenum with either vanadium or iron and differ in reactivity, most prominently in the ability of vanadium nitrogenase to reduce CO to hydrocarbons. Here we report the 1.35-Å structure of vanadium nitrogenase from Azotobacter vinelandii. The 240-kDa protein contains an additional α-helical subunit that is not present in molybdenum nitrogenase. The FeV cofactor (FeVco) is a [V–7Fe–8S–C] cluster with a homocitrate ligand to vanadium. Unexpectedly, it lacks one sulfide ion compared to FeMoco, which is replaced by a bridging ligand, likely a μ-1,3-carbonate. The anion fits into a pocket within the protein that is obstructed in molybdenum nitrogenase, and its different chemical character helps to rationalize the altered chemical properties of this unique N2- and CO-fixing enzyme.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of vanadium nitrogenase.
Figure 2: Electron-transferring P-cluster of vanadium nitrogenase.
Figure 3: Structure of FeV cofactor.
Figure 4: The carbonate ligand to FeV cofactor.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Rees, D.C. Dinitrogen reduction by nitrogenase—If N2 isn't broken, it can't be fixed. Curr. Opin. Struct. Biol. 3, 921–928 (1993).

    Article  CAS  Google Scholar 

  2. Rees, D.C. & Howard, J.B. Nitrogenase: standing at the crossroads. Curr. Opin. Chem. Biol. 4, 559–566 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Howard, J.B. & Rees, D.C. Structural basis of biological nitrogen fixation. Chem. Rev. 96, 2965–2982 (1996).

    Article  CAS  PubMed  Google Scholar 

  5. Eady, R.R. Structure–function relationships of alternative nitrogenases. Chem. Rev. 96, 3013–3030 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Einsle, O. Nitrogenase FeMo cofactor: an atomic structure in three simple steps. J. Biol. Inorg. Chem. 19, 737–745 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Bishop, P.E., Jarlenski, D.M.L. & Hetherington, D.R. Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA 77, 7342–7346 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bishop, P.E., Hawkins, M.E. & Eady, R.R. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem. J. 238, 437–442 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bishop, P.E. et al. Nitrogen fixation by Azotobacter vinelandii strains having deletions in structural genes for nitrogenase. Science 232, 92–94 (1986).

    Article  CAS  PubMed  Google Scholar 

  10. Robson, R.L. et al. The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature 322, 388–390 (1986).

    Article  CAS  Google Scholar 

  11. Hales, B.J., Case, E.E., Morningstar, J.E., Dzeda, M.F. & Mauterer, L.A. Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25, 7251–7255 (1986).

    Article  CAS  PubMed  Google Scholar 

  12. Eady, R.R., Robson, R.L., Richardson, T.H., Miller, R.W. & Hawkins, M. The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein. Biochem. J. 244, 197–207 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Joerger, R.D. et al. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J. Bacteriol. 172, 3400–3408 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Eady, R.R. Current status of structure function relationships of vanadium nitrogenase. Coord. Chem. Rev. 237, 23–30 (2003).

    Article  CAS  Google Scholar 

  15. Lee, C.C., Hu, Y. & Ribbe, M.W. Unique features of the nitrogenase VFe protein from Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA 106, 9209–9214 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Miller, R.W. & Eady, R.R. Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase. Biochem. J. 256, 429–432 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lee, C.C., Hu, Y. & Ribbe, M.W. Vanadium nitrogenase reduces CO. Science 329, 642 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang, Q., Cheng, K., Kang, J., Deng, W. & Wang, Y. Fischer-Tropsch catalysts for the production of hydrocarbon fuels with high selectivity. ChemSusChem 7, 1251–1264 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Hu, Y., Lee, C.C. & Ribbe, M.W. Extending the carbon chain: hydrocarbon formation catalyzed by vanadium/molybdenum nitrogenases. Science 333, 753–755 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hu, Y., Lee, C.C. & Ribbe, M.W. Vanadium nitrogenase: a two-hit wonder? Dalton Trans. 41, 1118–1127 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Hwang, J.C., Chen, C.H. & Burris, R.H. Inhibition of nitrogenase-catalyzed reductions. Biochim. Biophys. Acta 292, 256–270 (1973).

    Article  CAS  PubMed  Google Scholar 

  22. Cameron, L.M. & Hales, B.J. Investigation of CO binding and release from Mo-nitrogenase during catalytic turnover. Biochemistry 37, 9449–9456 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Spatzal, T., Perez, K.A., Einsle, O., Howard, J.B. & Rees, D.C. Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase. Science 345, 1620–1623 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Spatzal, T., Perez, K.A., Howard, J.B. & Rees, D.C. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor. eLife 4, e11620 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Sippel, D. et al. Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion. J. Biol. Inorg. Chem. 22, 161–168 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Kim, J. & Rees, D.C. Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii. Nature 360, 553–560 (1992).

    Article  CAS  PubMed  Google Scholar 

  27. Zhang, L. et al. The sixteenth iron in the nitrogenase MoFe protein. Angew. Chem. Int. Ed. Engl. 52, 10529–10532 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dyer, D.H. et al. The three-dimensional structure of the core domain of Naf Y from Azotobacter vinelandii determined at 1.8-A resolution. J. Biol. Chem. 278, 32150–32156 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Homer, M.J., Paustian, T.D., Shah, V.K. & Roberts, G.P. The nifY product of Klebsiella pneumoniae is associated with apodinitrogenase and dissociates upon activation with the iron-molybdenum cofactor. J. Bacteriol. 175, 4907–4910 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schmid, B. et al. Structure of a cofactor-deficient nitrogenase MoFe protein. Science 296, 352–356 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Hernandez, J.A. et al. A sterile α-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain. J. Biol. Chem. 286, 6321–6328 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Schindelin, H., Kisker, C., Schlessman, J.L., Howard, J.B. & Rees, D.C. Structure of ADP·AIF4--stabilized nitrogenase complex and its implications for signal transduction. Nature 387, 370–376 (1997).

    Article  CAS  PubMed  Google Scholar 

  33. Tezcan, F.A. et al. Nitrogenase complexes: multiple docking sites for a nucleotide switch protein. Science 309, 1377–1380 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. Tittsworth, R.C. & Hales, B.J. Oxidative titration of the nitrogenase VFe protein from Azotobacter vinelandii: an example of redox-gated electron flow. Biochemistry 35, 479–487 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Rees, J.A. et al. The Fe-V cofactor of vanadium nitrogenase contains an interstitial carbon atom. Angew. Chem. Int. Ed. Engl. 54, 13249–13252 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Spatzal, T. et al. Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334, 940 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kovacs, J.A. & Holm, R.H. Assembly of vanadium-iron-sulfur cubane clusters from mononuclear and linear trinuclear reactants. J. Am. Chem. Soc. 108, 340–341 (1986).

    Article  CAS  Google Scholar 

  39. Varley, J.B., Wang, Y., Chan, K., Studt, F. & Nørskov, J.K. Mechanistic insights into nitrogen fixation by nitrogenase enzymes. Phys. Chem. Chem. Phys. 17, 29541–29547 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Setubal, J.C. et al. Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J. Bacteriol. 191, 4534–4545 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mendel, R.R. The molybdenum cofactor. J. Biol. Chem. 288, 13165–13172 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lake, M.W., Wuebbens, M.M., Rajagopalan, K.V. & Schindelin, H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Nature 414, 325–329 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Wiig, J.A., Hu, Y., Lee, C.C. & Ribbe, M.W. Radical SAM-dependent carbon insertion into the nitrogenase M-cluster. Science 337, 1672–1675 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Lipscomb, J.D. Biochemistry of the soluble methane monooxygenase. Annu. Rev. Microbiol. 48, 371–399 (1994).

    Article  CAS  PubMed  Google Scholar 

  45. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the European Research Council (grant no. 310656 to O.E.) and Deutsche Forschungsgemeinschaft (RTG 1976 and PP 1927 to O.E.) and the BIOSS Centre for Biological Signaling Studies at Albert-Ludwigs-Universität Freiburg (to O.E.). We thank the beamline staff at the Swiss Light Source, Villigen, Switzerland, G. Fritz and A. Brausemann for their excellent assistance with data collection, and S. Andrade for critical reading of the manuscript and helpful discussions.

Author information

Authors and Affiliations

  1. Lehrstuhl Biochemie, Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg Research Institute for Advanced Studies (FRIAS), and BIOSS Centre for Biological Signalling Studies, Freiburg, Germany

    Daniel Sippel & Oliver Einsle

Authors
  1. Daniel Sippel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oliver Einsle
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.S. performed the experiments and built and refined the structural model; O.E. designed the experiments, built and refined the structural model and wrote the manuscript.

Corresponding author

Correspondence to Oliver Einsle.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–7 (PDF 2824 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sippel, D., Einsle, O. The structure of vanadium nitrogenase reveals an unusual bridging ligand. Nat Chem Biol 13, 956–960 (2017). https://doi.org/10.1038/nchembio.2428

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2428

This article is cited by

Search

Advanced 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