The NorR regulatory protein senses nitric oxide (NO) to activate genes required for NO detoxification under anaerobic and microaerobic conditions in Escherichia coli. NorR belongs to the ⁵⁴-dependent family of transcriptional activators and contains an amino-terminal regulatory GAF domain that controls the ATPase activity of the central AAA+ domain to regulate productive interactions with ⁵⁴. Binding of NO to a non-heme iron centre in the GAF domain results in the formation of a mononitrosyl-iron complex and releases intramolecular repression of the AAA+ domain to enable activation of transcription. In this study, we have further characterised NorR spectroscopically, and have substituted conserved residues in the GAF domain. This analysis, in combination with structural modelling of the GAF domain, has identified five candidate ligands to the non-heme iron, and suggests a model in which the metal ion is co-ordinated in a pseudo-octahedral environment by three aspartate residues, an arginine and a cysteine.