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A non-haem iron centre in the transcription factor NorR senses nitric oxide

Nature volume 437, pages 769–772 (2005)Cite this article

Abstract

Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens1,2. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. Regulatory proteins that sense NO mediate the primary response to NO and nitrosative stress3,4,5,6,7,8,9. The only regulatory protein in enteric bacteria known to serve exclusively as an NO-responsive transcription factor is the enhancer binding protein NorR (refs 9, 10–11). In Escherichia coli, NorR activates the transcription of the norVW genes encoding a flavorubredoxin (FlRd) and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity10,12,13,14. The NO-responsive activity of NorR raises important questions concerning the mechanism of NO sensing. Here we show that the regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription by RNA polymerase. The mechanism of NorR reveals an unprecedented biological role for a non-haem mononitrosyl–iron complex in NO sensing.

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Figure 1: Whole-cell EPR spectra and in vivo transcriptional activation by NorR.
Figure 2: EPR spectra of NO-treated purified proteins.
Figure 3: Formation of open promoter complexes and the ATPase activity of NorR.
Figure 4: Schematic of the proposed mechanism for transcriptional activation by NorR.

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References

  1. Griffith, O. W. & Stuehr, D. J. Nitric oxide synthases: properties and catalytic mechanism. Annu. Rev. Physiol. 57, 707–736 (1995)

    Article  CAS  Google Scholar 

  2. MacMicking, J., Xie, Q. W. & Nathan, C. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15, 323–350 (1997)

    Article  CAS  Google Scholar 

  3. Pohlmann, A., Cramm, R., Schmelz, K. & Friedrich, B. A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutropha. Mol. Microbiol. 38, 626–638 (2000)

    Article  CAS  Google Scholar 

  4. Fang, F. C. Antimicrobial reactive oxygen and nitrogen species: Concepts and controversies. Nature Rev. Microbiol. 2, 820–832 (2004)

    Article  CAS  Google Scholar 

  5. D'Autréaux, B., Touati, D., Bersch, B., Latour, J. M. & Michaud-Soret, I. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron. Proc. Natl Acad. Sci. USA 99, 16619–16624 (2002)

    Article  ADS  Google Scholar 

  6. Ding, H. G. & Demple, B. Direct nitric oxide signal transduction via nitrosylation of iron–sulfur centers in the SoxR transcription activator. Proc. Natl Acad. Sci. USA 97, 5146–5150 (2000)

    Article  ADS  CAS  Google Scholar 

  7. Hausladen, A., Privalle, C. T., Keng, T., DeAngelo, J. & Stamler, J. S. Nitrosative stress: activation of the transcription factor OxyR. Cell 86, 719–729 (1996)

    Article  CAS  Google Scholar 

  8. Cruz-Ramos, H. et al. NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. EMBO J. 21, 3235–3244 (2002)

    Article  CAS  Google Scholar 

  9. Mukhopadhyay, P., Zheng, M., Bedzyk, L. A., LaRossa, R. A. & Storz, G. Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species. Proc. Natl Acad. Sci. USA 101, 745–750 (2004)

    Article  ADS  CAS  Google Scholar 

  10. Gardner, A. M., Gessner, C. R. & Gardner, P. R. Regulation of the nitric oxide reduction operon (norRVW) in Escherichia coli. Role of NorR and σ54 in the nitric oxide stress response. J. Biol. Chem. 278, 10081–10086 (2003)

    Article  CAS  Google Scholar 

  11. Studholme, D. J. & Dixon, R. Domain architectures of σ54-dependent transcriptional activators. J. Bacteriol. 185, 1757–1767 (2003)

    Article  CAS  Google Scholar 

  12. Tucker, N. P., D'Autréaux, B., Studholme, D. J., Spiro, S. & Dixon, R. DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse Proteobacteria. J. Bacteriol. 186, 6656–6660 (2004)

    Article  CAS  Google Scholar 

  13. Gomes, C. M. et al. A novel type of nitric-oxide reductase. Escherichia coli flavorubredoxin. J. Biol. Chem. 277, 25273–25276 (2002)

    Article  CAS  Google Scholar 

  14. Hutchings, M. I., Mandhana, N. & Spiro, S. The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species. J. Bacteriol. 184, 4640–4643 (2002)

    Article  CAS  Google Scholar 

  15. Arciero, D. M., Lipscomb, J. D., Huynh, B. H., Kent, T. A. & Munck, E. EPR and Mössbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment. J. Biol. Chem. 258, 14981–14991 (1983)

    CAS  PubMed  Google Scholar 

  16. Clay, M. D., Cosper, C. A., Jenney, F. E. Jr, Adams, M. W. & Johnson, M. K. Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase. Proc. Natl Acad. Sci. USA 100, 3796–3801 (2003)

    Article  ADS  CAS  Google Scholar 

  17. Ray, M. et al. Structure and magnetic properties of trigonal bipyramidal iron nitrosyl complexes. Inorg. Chem. 38, 3110–3115 (1999)

    Article  CAS  Google Scholar 

  18. Brown, C. A. et al. Spectroscopic and theoretical description of the electronic structure of S = 3/2 iron–nitrosyl complexes and their relation to O2 activation by non-heme iron enzyme active-sites. J. Am. Chem. Soc. 117, 715–732 (1995)

    Article  CAS  Google Scholar 

  19. Hauser, C., Glaser, T., Bill, E., Weyhermuller, T. & Wieghardt, K. The electronic structures of an isostructural series of octahedral nitrosyliron complexes {Fe-NO}6,7,8 elucidated by Mössbauer spectroscopy. J. Am. Chem. Soc. 122, 4352–4365 (2000)

    Article  CAS  Google Scholar 

  20. Enemark, J. H. & Feltham, R. D. Principles of structure, bonding, and reactivity for metal nitrosyl complexes. Coord. Chem. Rev. 13, 339–406 (1974)

    Article  CAS  Google Scholar 

  21. Cannon, W. V., Gallegos, M. T. & Buck, M. Isomerization of a binary sigma-promoter DNA complex by transcription activators. Nature Struct. Biol. 7, 594–601 (2000)

    Article  CAS  Google Scholar 

  22. Zhang, X. et al. Mechanochemical ATPases and transcriptional activation. Mol. Microbiol. 45, 895–903 (2002)

    Article  CAS  Google Scholar 

  23. Austin, S. & Dixon, R. The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J. 11, 2219–2228 (1992)

    Article  CAS  Google Scholar 

  24. Roy, S., Sharma, S., Sharma, M., Aggarwal, R. & Bose, M. Induction of nitric oxide release from the human alveolar epithelial cell line A549: an in vitro correlate of innate immune response to Mycobacterium tuberculosis. Immunology 112, 471–480 (2004)

    Article  CAS  Google Scholar 

  25. Aravind, L. & Ponting, C. P. The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem. Sci. 22, 458–459 (1997)

    Article  CAS  Google Scholar 

  26. D'Autréaux, B. et al. Spectroscopic description of the two nitrosyl–iron complexes responsible for Fur inhibition by nitric oxide. J. Am. Chem. Soc. 126, 6005–6016 (2004)

    Article  Google Scholar 

  27. Zhao, Y., Brandish, P. E., Ballou, D. P. & Marletta, M. A. A molecular basis for nitric oxide sensing by soluble guanylate cyclase. Proc. Natl Acad. Sci. USA 96, 14753–14758 (1999)

    Article  ADS  CAS  Google Scholar 

  28. Li, M. et al. Tuning the electronic structure of octahedral iron complexes [FeL(X)] (L = 1-alkyl-4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclo-nonane, X = Cl, CH3O, CN, NO). The S = 1/2⇔S = 3/2 spin equilibrium of [FeLPr(NO)]. Inorg. Chem. 41, 3444–3456 (2002)

    Article  CAS  Google Scholar 

  29. Serres, R. G. et al. Structural, spectroscopic, and computational study of an octahedral, non-heme [Fe-NO]6–8 series: [Fe(NO)(cyclam-ac)]2+/+/0. J. Am. Chem. Soc. 126, 5138–5153 (2004)

    Article  CAS  Google Scholar 

  30. Little, R., Reyes-Ramirez, F., Zhang, Y., van Heeswijk, W. C. & Dixon, R. Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein. EMBO J. 19, 6041–6050 (2000)

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by a grant from the BBSRC to R.D. and S.S. We are grateful to R. Little, G. Sawers, M. Cheesman, I. Martinez-Argudo, P. Johnson and S. Fairhurst for their assistance and comments at various stages of this project, to M. Naldrett and A. Bottrill for their help with mass spectrometry, and to M. Buttner for comments on the manuscript.

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Authors and Affiliations

  1. John Innes Centre, Colney, NR4 7UH, Norwich, UK

    Benoît D'Autréaux, Nicholas P. Tucker & Ray Dixon

  2. School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Georgia, 30332–0230, Atlanta, USA

    Stephen Spiro

Authors
  1. Benoît D'Autréaux
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  2. Nicholas P. Tucker
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  3. Ray Dixon
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Correspondence to Ray Dixon or Stephen Spiro.

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Supplementary information

Supplementary Figures

Supplementary Figure S1 details SDS–PAGE of purified NorR-Fe(II) and GAFNorR-Fe(II). Supplementary Figure S2 details EPR spectra of purified NorR-Fe(II) and GAFNorR-Fe(II). Supplementary Figure S3 details electrospray/Q-TOF mass spectrometry of NorR-Fe(II). Supplementary Figure S4 details determination of the NorR-Fe(NO) dissociation constant. (PDF 1142 kb)

Supplementary Figure Legends

Contains legends to Supplementary Figures S1–4. (DOC 20 kb)

Supplementary Methods

Contains additional information on the methods used in this study. (PDF 54 kb)

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D'Autréaux, B., Tucker, N., Dixon, R. et al. A non-haem iron centre in the transcription factor NorR senses nitric oxide. Nature 437, 769–772 (2005). https://doi.org/10.1038/nature03953

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