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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.

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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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