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:

Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor

Nature Catalysis volume 5, pages 443–454 (2022)Cite this article

Subjects

Abstract

Molybdenum nitrogenase catalyses the ambient reduction of N2 to NH3 at the M-cluster, a complex cofactor that comprises two metal-sulfur partial cubanes ligated by an interstitial carbide and three belt-sulfurs. A recent crystallographic study suggests binding of N2 via displacement of the belt-sulfur(s) of the M-cluster upon turnover. However, direct proof of N2 binding and belt-sulfur mobilization during catalysis remains elusive. Here we show that N2 is captured on the M-cluster via electron and sulfur depletion, and that the N2-captured state is catalytically competent in generating NH3. Moreover, we demonstrate that product release occurs only when sulfite is supplied along with a reductant, that sulfite is inserted as sulfide into the belt-sulfur-displaced positions and that there is a dynamic in-and-out of belt-sulfurs during catalysis. Together, these results establish the mobilization of cofactor belt-sulfurs as a crucial, yet overlooked, mechanistic element of the nitrogenase reaction.

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

Fig. 1: GC–MS and frequency-selective NMR analyses of N2-bound Av1*.
Fig. 2: EPR and GC–MS analyses of various Av1 protein species.
Fig. 3: Requirement of sulfite-type species for substrate turnover.
Fig. 4: Crystallographic analysis of Av1*(TOS).
Fig. 5: Catalysis-dependent mobilization of belt-sulfurs.
Fig. 6: XAS/EXAFS analysis of Av1*(TOSe).
Fig. 7: Coordination and reduction of SO32−.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files, or from the corresponding authors upon reasonable request. The structural data related to this work are available at the Worldwide Protein Data Bank (http://www.wwpdb.org) with PDB accession codes 7MCI, 6UG0 and 6VXT. Source data are provided with this paper.

References

  1. Burgess, B. K. & Lowe, D. J. Mechanism of molybdenum nitrogenase. Chem. Rev. 96, 2983–3012 (1996).

    Article  CAS  Google Scholar 

  2. Buscagan, T. M. & Rees, D. C. Rethinking the nitrogenase mechanism: activating the active site. Joule 3, 2662–2678 (2019).

    Article  CAS  Google Scholar 

  3. Rutledge, H. L. & Tezcan, F. A. Electron transfer in nitrogenase. Chem. Rev. 120, 5158–5193 (2020).

    Article  CAS  Google Scholar 

  4. Jasniewski, A. J., Lee, C. C., Ribbe, M. W. & Hu, Y. Reactivity, mechanism, and assembly of the alternative nitrogenases. Chem. Rev. 120, 5107–5157 (2020).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Seefeldt, L. C. et al. Reduction of substrates by nitrogenases. Chem. Rev. 120, 5082–5106 (2020).

    Article  CAS  Google Scholar 

  8. Einsle, O. & Rees, D. C. Structural enzymology of nitrogenase enzymes. Chem. Rev. 120, 4969–5004 (2020).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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  Google Scholar 

  11. Lowe, D. J. & Thorneley, R. N. F. The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation. Biochem. J. 224, 877–886 (1984).

    Article  CAS  Google Scholar 

  12. Lowe, D. J. & Thorneley, R. N. F. The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution. Biochem. J. 224, 895–901 (1984).

    Article  CAS  Google Scholar 

  13. Thorneley, R. N. F. & Lowe, D. J. The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation. Biochem. J. 224, 887–894 (1984).

    Article  CAS  Google Scholar 

  14. Thorneley, R. N. F. & Lowe, D. J. The mechanism of Klebsiella pneumoniae nitrogenase action. Simulation of the dependences of H2-evolution rate on component-protein concentration and ratio and sodium dithionite concentration. Biochem. J. 224, 903–909 (1984).

    Article  CAS  Google Scholar 

  15. Lee, S. C., Lo, W. & Holm, R. H. Developments in the biomimetic chemistry of cubane-type and higher nuclearity iron-sulphur clusters. Chem. Rev. 114, 3579–3600 (2014).

    Article  CAS  Google Scholar 

  16. 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  Google Scholar 

  17. 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  Google Scholar 

  18. Kang, W., Lee, C. C., Jasniewski, A. J., Ribbe, M. W. & Hu, Y. Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase. Science 368, 1381–1385 (2020).

    Article  CAS  Google Scholar 

  19. Yates, M. G. In Biological Nitrogen Fixation (eds Stacey, G., Burris, R. H. & Evans, H. J.) 685–735 (Chapman & Hall, 1992).

  20. Jensen, B. B. & Burris, R. H. Effect of high pN2 and high pD2 on NH3 production, H2 evolution, and HD formation by nitrogenases. Biochemistry 24, 1141–1147 (1985).

    Article  CAS  Google Scholar 

  21. Li, J. L. & Burris, R. H. Influence of pN2 and pD2 on HD formation by various nitrogenases. Biochemistry 22, 4472–4480 (1983).

    Article  CAS  Google Scholar 

  22. Burgess, B. K., Wherland, S., Newton, W. E. & Stiefel, E. I. Nitrogenase reactivity: insight into the nitrogen-fixing process through hydrogen-inhibition and HD-forming reactions. Biochemistry 20, 5140–5146 (1981).

    Article  CAS  Google Scholar 

  23. Wherland, S., Burgess, B. K., Stiefel, E. I. & Newton, W. E. Nitrogenase reactivity: effects of component ratio on electron flow and distribution during nitrogen fixation. Biochemistry 20, 5132–5140 (1981).

    Article  CAS  Google Scholar 

  24. Chatt, J. In Nitrogen Fixation (eds Stewart, W. D. P. & Gallon, J. R.) 1–18 (Academic Press, 1980).

  25. Yang, Z.-Y. et al. On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase. Proc. Natl Acad. Sci. USA 110, 16327–16332 (2013).

    Article  CAS  Google Scholar 

  26. Nielander, A. C. et al. A versatile method for ammonia detection in a range of relevant electrolytes via direct nuclear magnetic resonance techniques. ACS Catal. 9, 5797–5802 (2019).

    Article  CAS  Google Scholar 

  27. Sippel, D. et al. A bound reaction intermediate sheds light on the mechanism of nitrogenase. Science 359, 1484–1489 (2018).

    Article  CAS  Google Scholar 

  28. Tanifuji, K. et al. Tracing the incorporation of the “ninth sulfur” into the nitrogenase cofactor precursor with selenite and tellurite. Nat. Chem. 13, 1228–1234 (2021).

    Article  CAS  Google Scholar 

  29. Jasniewski, A. J. et al. Spectroscopic characterization of an eight-iron nitrogenase cofactor precursor that lacks the “9th sulphur”. Angew. Chem. Int. Ed. Engl. 58, 14703–14707 (2019).

    Article  CAS  Google Scholar 

  30. Tanifuji, K. et al. Tracing the “ninth sulphur” of the nitrogenase cofactor via a semi-synthetic approach. Nat. Chem. 10, 568–572 (2018).

    Article  CAS  Google Scholar 

  31. Wei, W. J. & Siegbahn, P. E. M. A mechanism for nitrogenase including loss of a sulphide. Chemistry 28, e202103745 (2022).

  32. Vincent, K. A. et al. Instantaneous, stoichiometric generation of powerfully reducing states of protein active sites using Eu(II) and polyaminocarboxylate ligands. Chem. Commun. 20, 2590–2591 (2003).

    Article  Google Scholar 

  33. Ribbe, M. W., Hu, Y., Guo, M., Schmid, B. & Burgess, B. K. The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features. J. Biol. Chem. 277, 23469–23476 (2002).

    Article  CAS  Google Scholar 

  34. Burgess, B. K., Jacobs, D. B. & Stiefel, E. I. Large-scale purification of high activity Azotobacter vinelandii nitrogenase. Biochim. Biophys. Acta 614, 196–209 (1980).

    Article  CAS  Google Scholar 

  35. Lee, C. C., Ribbe, M. W. & Hu, Y. Purification of nitrogenase proteins. Methods Mol. Biol. 1876, 111–124 (2019).

    Article  CAS  Google Scholar 

  36. Gavini, N. & Burgess, B. K. FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity. J. Biol. Chem. 267, 21179–21186 (1992).

    Article  CAS  Google Scholar 

  37. Corbin, J. L. Liquid chromatographic-fluorescence determination of ammonia from nitrogenase reactions: a 2-min assay. Appl. Environ. Microbiol. 47, 1027–1030 (1984).

    Article  CAS  Google Scholar 

  38. Lee, C. C., Hu, Y. & Ribbe, M. W. Tracing the hydrogen source of hydrocarbons formed by vanadium nitrogenase. Angew. Chem. Int. Ed. Engl. 50, 5545–5547 (2011).

    Article  CAS  Google Scholar 

  39. Lee, C. C., Hu, Y. & Ribbe, M. W. Reduction and condensation of aldehydes by the isolated cofactor of nitrogenase. ACS Cent. Sci. 4, 1430–1435 (2018).

    Article  CAS  Google Scholar 

  40. Fulmer, G. R. et al. NMR chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 29, 2176–2179 (2010).

    Article  CAS  Google Scholar 

  41. Wiig, J. A., Hu, Y. & Ribbe, M. W. NifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly. Proc. Natl Acad. Sci. USA 108, 8623–8627 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH-NIGMS grant no. GM141046 (to Y.H. and M.W.R.). We thank P. R. Dennison, Director of the NMR Facility at UC Irvine, for his kind help with frequency-selective NMR analysis of our samples. We also thank the staff at the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Beamline 12-2 (X-ray diffraction) and Beamlines 7-3 and 9-3 (X-ray absorption spectroscopy) for technical support with data collection. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research and by the National Institutes of Health, National Institutes of General Medical Sciences (no. P30GM133894).

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA

    Chi Chung Lee, Wonchull Kang, Andrew J. Jasniewski, Martin T. Stiebritz, Kazuki Tanifuji, Markus W. Ribbe & Yilin Hu

  2. Department of Chemistry, College of Natural Sciences, Soongsil University, Seoul, Republic of Korea

    Wonchull Kang

  3. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Japan

    Kazuki Tanifuji

  4. Department of Chemistry, University of California, Irvine, CA, USA

    Markus W. Ribbe

Authors
  1. Chi Chung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wonchull Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew J. Jasniewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martin T. Stiebritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kazuki Tanifuji
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Markus W. Ribbe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yilin Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.C.L., W.K., A.J.J., M.T.S., K.T., M.W.R. and Y.H. designed experiments. C.C.L., W.K., A.J.J., M.T.S., K.T., M.W.R. and Y.H. analysed data. C.C.L., W.K., A.J.J., M.T.S. and K.T. performed experiments. M.W.R. and Y.H. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Markus W. Ribbe or Yilin Hu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Catalysis thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, Figs. 1–9, Tables 1–9 and references.

Reporting Summary

Source data

Source Data Fig. 1

Source Data Fig. 1a–c.

Source Data Fig. 2

Source Data Fig. 2a–o.

Source Data Fig. 3

Source Data Fig. 3a–c.

Source Data Fig. 5

Source Data Fig. 5a–d.

Source Data Fig. 6

Source Data Fig. 6a–g.

Source Data Fig. 7

Source Data Fig. 7a–d.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, C.C., Kang, W., Jasniewski, A.J. et al. Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor. Nat Catal 5, 443–454 (2022). https://doi.org/10.1038/s41929-022-00782-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-022-00782-7

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