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Tracing the incorporation of the “ninth sulfur” into the nitrogenase cofactor precursor with selenite and tellurite

Nature Chemistry volume 13, pages 1228–1234 (2021)Cite this article

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Abstract

Molybdenum nitrogenase catalyses the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘ninth sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘ninth sulfur’ insertion remain elusive. Here we trace the ‘ninth sulfur’ insertion using SeO32− and TeO32− as ‘labelled’ SO32−. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the ‘ninth sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2−- and Te2−-like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO32− to S2−, thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.

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Fig. 1: Incorporation of sulfur, selenium and tellurium into the L*-cluster.
Fig. 2: Three-pulse ESEEM spectra of various MaNifB proteins.
Fig. 3: Fe K-edge XAS analysis of of various MaNifB proteins.
Fig. 4: Selenium and tellurium K-edge EXAFS analyses of of various MaNifB proteins.
Fig. 5: Coordination and reduction of XO32− (X = S, Se, Te) at the free binding site of an undercoordinated L*-cluster.
Fig. 6: The role of the ‘ninth sulfur’ in cluster transfer from NifB to NifEN.

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The authors declare that all data supporting the findings of this study are available within the article, the Supplementary Information and the source files published alongside the article. 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. Spatzal, T. et al. Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334, 940 (2011).

    Article  CAS  Google Scholar 

  6. 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 

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

    Article  CAS  Google Scholar 

  8. Ohki, Y. & Tatsumi, K. New synthetic routes to metal–sulfur clusters relevant to the nitrogenase metallo-clusters. Z. Anorg. Allg. Chem. 639, 1340–1349 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Hu, Y. & Ribbe, M. W. Biosynthesis of the metalloclusters of nitrogenases. Annu. Rev. Biochem. 85, 455–483 (2016).

    Article  CAS  Google Scholar 

  11. Tanifuji, K. et al. Tracing the ‘ninth sulfur’ of the nitrogenase cofactor via a semi-synthetic approach. Nat. Chem. 10, 568–572 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Hu, Y. et al. Nitrogenase Fe protein: a molybdate/homocitrate insertase. Proc. Natl Acad. Sci. USA 103, 17125–17130 (2006).

    Article  CAS  Google Scholar 

  15. Hu, Y. & Ribbe, M. W. Maturation of nitrogenase cofactor—the role of a class E radical SAM methyltransferase NifB. Curr. Opin. Chem. Biol. 31, 188–194 (2016).

    Article  CAS  Google Scholar 

  16. Kang, W. et al. Crystallographic analysis of NifB with a full complement of clusters: structural insights into the radical SAM-dependent carbide insertion during nitrogenase cofactor assembly. Angew. Chem. Int. Ed. 60, 2364–2370 (2020).

    Article  Google Scholar 

  17. Rettberg, L. A. et al. Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB. Nat. Commun. 11, 1757 (2020).

    Article  CAS  Google Scholar 

  18. Rettberg, L. A. et al. Probing the coordination and function of Fe4S4 modules in nitrogenase assembly protein NifB. Nat. Commun. 9, 2824 (2018).

    Article  Google Scholar 

  19. Musgrave, K. B., Angove, H. C., Burgess, B. K., Hedman, B. & Hodgson, K. O. All-ferrous titanium(iii) citrate reduced Fe protein of nitrogenase: an XAS study of electronic and metrical structure. J. Am. Chem. Soc. 120, 5325–5326 (1998).

    Article  CAS  Google Scholar 

  20. 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 

  21. Ciurli, S., Yu, S. B., Holm, R. H., Srivastava, K. K. P. & Munck, E. Synthetic nickel–iron NiFe3Q4 cubane-type clusters (S = 3/2) by reductive rearrangement of linear [Fe3Q4(SEt)4]3− (Q = sulfur, selenium). J. Am. Chem. Soc. 112, 8169–8171 (1990).

    Article  CAS  Google Scholar 

  22. Bobrik, M. A. et al. Selenium substitution in [Fe4S4(SR)4]2−: synthesis and comparative properties of [Fe4X4(YC6H5)4]2− (X, Y = sulfur, selenium) and the structure of [(CH3)4N]2[Fe4Se4(SC6H5)4]. Inorg. Chem. 17, 1402–1410 (1978).

    Article  CAS  Google Scholar 

  23. Barbaro, P., Bencini, A., Bertini, I., Briganti, F. & Midollini, S. The tetranuclear trianion [Fe4Te4(SC6H5)4]3−: crystal and molecular structure and magnetic properties. J. Am. Chem. Soc. 112, 7238–7246 (1990).

    Article  CAS  Google Scholar 

  24. Simon, W., Wilk, A., Krebs, B. & Henkel, G. [Fe4Te4(TePh)4]3−, the first telluride–tellurolate complex. Angew. Chem. Int. Ed. 26, 1009–1010 (1987).

    Article  Google Scholar 

  25. Zimmermann, M. D. & Tossell, J. A. Acidities of arsenic(iii) and arsenic(v) thio- and oxyacids in aqueous solution using the CBS-QB3/CPCM method. J. Phys. Chem. A 113, 5105–5111 (2009).

    Article  CAS  Google Scholar 

  26. 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 

  27. 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 

  28. 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 

Download references

Acknowledgements

This work was supported by NIH-NIGMS grants GM67626 (to M.W.R. and Y.H.), GM141046 (to Y.H. and M.W.R.), R35 GM126961 (to R.D.B.), GM110501 (to J.Y.) and GM126289 (to J.K.). Y.O. was supported by Grant-in-Aids for Scientific Research (MEXT Japan) (nos. 19H02733 and 20K21207), International Collaborative Research Program of ICR, Kyoto University, Takeda Science Foundation and Tatematsu Foundation. K.T. recieved support from the Kyoto University Research Fund for Young Scientist (Start-Up). XAS data were collected at Beamlines 7-3 and 9-3 at the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory. SLAC 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 DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P30GM133894).

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Authors and Affiliations

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

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

  2. International Research Center for Elements Science, Institute for Chemical Research, Kyoto University, Kyoto, Japan

    Kazuki Tanifuji & Yasuhiro Okhi

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

    David Villarreal & R. David Britt

  4. Department of Chemistry and Biochemistry, University of Wisconsin, Milwaukee, WI, USA

    Jarett Wilcoxen

  5. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Ruchira Chatterjee, Isabel Bogacz, Junko Yano & Jan Kern

  6. Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA

    Britt Hedman & Keith O. Hodgson

  7. Department of Chemistry, Stanford University, Stanford, CA, USA

    Keith O. Hodgson

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

    Markus W. Ribbe

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  1. Kazuki Tanifuji
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Contributions

K.T., A.J.J., M.T.S., C.C.L, Y.H. and M.W.R. designed experiments. K.T., A.J.J., M.T.S., C.C.L, D.V., J.W., R.C., I.B., J.Y., J.K., R.D.B., Y.H. and M.W.R. analysed data. K.T., A.J.J., M.T.S., C.C.L., D.V., J.W., R.C., I.B., J.Y and J.K. performed experiments. Y.O., B.H. and K.O.H provided material and/or technical resources. Y.H. and M.W.R. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to R. David Britt, Yilin Hu or Markus W. Ribbe.

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The authors declare no competing interests.

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Peer review information Nature Chemistry thanks Benoit D’Autréaux and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary methods, Figs. 1–3, Tables 1–7 and references.

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Tanifuji, K., Jasniewski, A.J., Villarreal, D. et al. Tracing the incorporation of the “ninth sulfur” into the nitrogenase cofactor precursor with selenite and tellurite. Nat. Chem. 13, 1228–1234 (2021). https://doi.org/10.1038/s41557-021-00799-8

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