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Transcription factor NNR from Paracoccus denitrificans is a sensor of both nitric oxide and oxygen: isolation of nnr* alleles encoding effector-independent proteins and evidence for a haem-based sensing mechanism

Neil Shearer^1,

¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
² School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332-0230, USA

Correspondence
Stephen Spiro
stephen.spiro{at}biology.gatech.edu

The nitrite reductase and nitric oxide reductase regulator (NNR) from Paracoccus denitrificans activates transcription in response to nitric oxide (NO). The mechanism of NO sensing has not been elucidated for NNR, or for any of its orthologues from the FNR/CRP family of transcriptional regulators. Using regulated expression of the nnr gene in Escherichia coli, evidence has now been obtained to indicate that activation of NNR by NO does not require de novo synthesis of the NNR polypeptide. In anaerobic cultures, NNR is inactivated slowly following removal of the source of NO. In contrast, exposure of anaerobically grown cultures to oxygen causes rapid inactivation of NNR, suggesting that the protein is inactivated directly by oxygen. By random and site-directed mutagenesis, two variants of NNR were isolated (with substitutions of arginine at position 80) that show high levels of activity in anaerobic cultures in the absence of NO. These proteins remain substantially inactive in aerobic cultures, suggesting that the substitutions uncouple the NO- and oxygen-signalling mechanisms, thus providing further evidence that NNR senses both molecules. Structural modelling suggested that Arg-80 is close to the C-helix that forms the monomer–monomer interface in other members of the FNR/CRP family and plays an important role in transducing the activating signal between the regulatory and DNA binding domains. Assays of NNR activity in a haem-deficient mutant of E. coli provided preliminary evidence to indicate that NNR activity is haem dependent.

Present address: John Innes Centre, Colney, Norwich NR4 7UH, UK.

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