Letter | Published:

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

Naturevolume 437pages769772 (2005) | Download Citation



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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

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

  2. 2

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

  3. 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)

  4. 4

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

  5. 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)

  6. 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)

  7. 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)

  8. 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)

  9. 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)

  10. 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)

  11. 11

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

  12. 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)

  13. 13

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

  14. 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)

  15. 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)

  16. 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)

  17. 17

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

  18. 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)

  19. 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)

  20. 20

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

  21. 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)

  22. 22

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

  23. 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)

  24. 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)

  25. 25

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

  26. 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)

  27. 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)

  28. 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 = 1/2S = 3/2 spin equilibrium of [FeLPr(NO)]. Inorg. Chem. 41, 3444–3456 (2002)

  29. 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)

  30. 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)

Download references


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.

Author information


  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


  1. Search for Benoît D'Autréaux in:

  2. Search for Nicholas P. Tucker in:

  3. Search for Ray Dixon in:

  4. Search for Stephen Spiro in:

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ray Dixon or Stephen Spiro.

Supplementary information

  1. 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)

  2. Supplementary Figure Legends

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

  3. Supplementary Methods

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

About this article

Publication history



Issue Date



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.