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

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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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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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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