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:

Tracing the ‘ninth sulfur’ of the nitrogenase cofactor via a semi-synthetic approach

Nature Chemistry volume 10, pages 568–572 (2018)Cite this article

Subjects

Abstract

The M-cluster is the [(homocitrate)MoFe7S9C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe4S4] clusters concomitant with the insertion of an interstitial carbon and a ‘ninth sulfur’. Combining synthetic [Fe4S4] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-l-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe8S8C] cluster intermediate, the L*-cluster, which is similar to the [Fe8S9C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.

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: Assembly of the M-cluster.
Fig. 2: DT-dependent cluster maturation.
Fig. 3: DT-free cluster maturation.
Fig. 4: Refined model of L-cluster assembly on NifB.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Rees, D. C. et al. Structural basis of biological nitrogen fixation. Phil. Trans. R. Soc. A 363, 971–984 (2005).

    Article  CAS  Google Scholar 

  3. Chan, M. K., Kim, J. & Rees, D. C. The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 Å resolution structures. Science 260, 792–794 (1993).

    Article  CAS  Google Scholar 

  4. Einsle, O. et al. Nitrogenase MoFe-protein at 1.16 Å resolution: a central ligand in the FeMo-cofactor. Science 297, 1696–1700 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Lancaster, K. M. et al. X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron–molybdenum cofactor. Science 334, 974–977 (2011).

    Article  CAS  Google Scholar 

  7. 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 

  8. 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 

  9. Wiig, J. A., Hu, Y. & Ribbe, M. W. Refining the pathway of carbide insertion into the nitrogenase M-cluster. Nat. Commun. 6, 8034 (2015).

    Article  CAS  Google Scholar 

  10. Hu, Y. et al. FeMo cofactor maturation on NifEN. Proc. Natl Acad. Sci. USA 103, 17119–17124 (2006).

    Article  CAS  Google Scholar 

  11. Hu, Y. et al. Nitrogenase Fe protein: a molybdate/homocitrate insertase. Proc. Natl Acad. Sci. USA 103, 17125–17230 (2006).

    Article  CAS  Google Scholar 

  12. Ribbe, M. W., Hu, Y., Hodgson, K. O. & Hedman, B. Biosynthesis of nitrogenase metalloclusters. Chem. Rev. 114, 4063–4080 (2014).

    Article  CAS  Google Scholar 

  13. Fay, A. W., Wiig, J. A., Lee, C. C. & Hu, Y. Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens. Proc. Natl Acad. Sci. USA 112, 14829–14833 (2015).

    Article  CAS  Google Scholar 

  14. Wilcoxen, J. et al. Electron paramagnetic resonance characterization of three iron-sulfur clusters present in the nitrogenase cofactor maturase NifB from Methanocaldococcus infernus. J. Am. Chem. Soc. 138, 7468–7471 (2016).

    Article  CAS  Google Scholar 

  15. Averill, B. A., Herskovitz, T., Holm, R. H. & Ibers, J. A. Synthetic analogs of the active sites of iron–sulfur proteins. II. Synthesis and structure of the tetra(mercapto-μ3-sulfido-iron) clusters, (Fe4S4(SR)4)2–. J. Am. Chem. Soc. 95, 3523–3534 (1973).

    Article  CAS  Google Scholar 

  16. Barclay, J. E., Davies, S. C., Evans, D. J. & Hughes, D. L. Lattice effects in the Mössbauer spectra of salts of [Fe4S4{S(CH2)nOH}4]2−. Crystal structures of [PPh4]2[Fe4S4{S(CH2)nOH}4] (n = 2, 3 and 4). Inorg. Chim. Acta 291, 101–108 (1999).

    Article  CAS  Google Scholar 

  17. de Carvalho, L. M. & Schwedt, G. Sulfur speciation by capillary zone electrophoresis. Determination of dithionite and its decomposition products sulfite, sulfate and thiosulfate in commercial bleaching agents. J. Chromatogr. A 1099, 185–190 (2005).

    Article  Google Scholar 

  18. Münchow, V. & Steudel, R. The decomposition of aqueous dithionite and its reactions with polythionates SnO2− 6 (n = 3–5) studied by ion-pair chromatography. Z. Anorg. Allg. Chem. 620, 121–126 (1994).

    Article  Google Scholar 

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

    Article  Google Scholar 

  20. 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 

  21. 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 

  22. Brychkova, G., Grishkevich, V., Fluhr, R. & Sagi, M. An essential role for tomato sulfite oxidase and enzymes of the sulfite network in maintaining leaf sulfite homeostasis. Plant Physiol. 161, 148–164 (2013).

    Article  CAS  Google Scholar 

  23. Carbonero, F., Benefiel, A. C., Alizadeh-Ghamsari, A. H. & Gaskins, H. R. Microbial pathways in colonic sulfur metabolism and links with health and disease. Front. Physiol. 3, 448 (2012).

    Article  CAS  Google Scholar 

  24. Kertesz, M. A. Riding the sulfur cycle—metabolism of sulfonates and sulfate esters in Gram-negative bacteria. FEMS Microbiol. Rev. 24, 135–175 (2000).

    CAS  PubMed  Google Scholar 

  25. Integrated Microbial Genomes (Joint Genome Institute); https://img.jgi.doe.gov/cgi-bin/m/main.cgi.

  26. Mishanina, T. V., Libiad, M. & Banerjee, R. Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat. Chem. Biol. 11, 457–464 (2015).

    Article  CAS  Google Scholar 

  27. Frey, P. A., Hegeman, A. D. & Ruzicka, F. J. The radical SAM superfamily. Crit. Rev. Biochem. Mol. Biol. 43, 63–88 (2008).

    Article  CAS  Google Scholar 

  28. Mühlenhoff, U. et al. Compartmentalization of iron between mitochondria and the cytosol and its regulation. Eur. J. Cell Biol. 94, 292–308 (2015).

    Article  Google Scholar 

  29. Mettert, E. L. & Kiley, P. J. Fe–S proteins that regulate gene expression. Biochim. Biophys. Acta 1853, 1284–1293 (2015).

    Article  CAS  Google Scholar 

  30. O’Brien, E. et al. The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport. Science 355, eaag1789 (2017).

    Article  Google Scholar 

  31. Berggren, G. et al. Biomimetic assembly and activation of [FeFe]-hydrogenases. Nature 499, 66–69 (2013).

    Article  CAS  Google Scholar 

  32. Esselborn, J. et al. Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic. Nat. Chem. Biol. 9, 607–609 (2013).

    Article  CAS  Google Scholar 

  33. Shima, S. et al. Reconstitution of [Fe] hydrogenase using model complexes. Nat. Chem. 7, 995–1002 (2015).

    Article  CAS  Google Scholar 

  34. Tanifuji, K. et al. Combining a nitrogenase scaffold and a synthetic compound into an artificial enzyme. Angew. Chem. Int. Ed. 54, 14022–14025 (2015).

    Article  CAS  Google Scholar 

  35. Heinisch, T. & Ward, T. R. Artificial metalloenzymes based on the biotin–streptavidin technology: challenges and opportunities. Acc. Chem. Res. 49, 1711–1721 (2016).

    Article  CAS  Google Scholar 

  36. Lin, Y.-W. Rational design of metalloenzymes: from single to multiple active sites. Coord. Chem. Rev. 336, 1–27 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH-NIGMS grant GM67626 (to M.W.R. and Y.H.), DOE-BES grant DE-DC0014470 (to M.W.R. and Y.H.), a Takeda Science Foundation grant (to Y.O.) and Grant-in-Aids for Scientific Research (nos 23000007 and 16H04116) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.Tat. and Y.O.).

Author information

Authors and Affiliations

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

    Kazuki Tanifuji, Chi Chung Lee, Nathaniel S. Sickerman, Yilin Hu & Markus W. Ribbe

  2. Department of Chemistry, Graduate School of Science and Research Center for Materials Science, Nagoya University, Nagoya, Japan

    Kazuyuki Tatsumi & Yasuhiro Ohki

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

    Markus W. Ribbe

Authors
  1. Kazuki Tanifuji
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chi Chung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathaniel S. Sickerman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuyuki Tatsumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yasuhiro Ohki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yilin Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Markus W. Ribbe
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.Tan., C.C.L., N.S.S., Y.H. and M.W.R. designed and analysed experiments. K.Tan. performed experiments. K.Tat. and Y.O. provided materials. Y.H. and M.W.R. wrote the manuscript with input from all the authors.

Corresponding authors

Correspondence to Yilin Hu or Markus W. Ribbe.

Ethics declarations

Competing interests

The authors declare no competing interests.

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, References, and Figures 1–6

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tanifuji, K., Lee, C.C., Sickerman, N.S. et al. Tracing the ‘ninth sulfur’ of the nitrogenase cofactor via a semi-synthetic approach. Nature Chem 10, 568–572 (2018). https://doi.org/10.1038/s41557-018-0029-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-018-0029-4

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