Nitric oxide signalling by the bacterial enhancer binding protein NorR

Nitric oxide, synthesised in animals and humans by the nitric oxide synthases, has multiple roles in signalling pathways and in protecting against invading bacterial pathogens. Nitric oxide is produced at high concentrations by specialised human cells known as macrophages to poison engulfed bacteria or tumor cells. Bacteria have evolved specific mechanisms to defend themselves against the toxic effects of nitric oxide and to repair some of the damage caused by it. Regulatory proteins that sense nitric oxide provide the primary response by activating the expression of various enzymes that can detoxify this molecule. We have discovered that nitric oxide interacts directly with a bacterial regulatory protein to activate expression of genes required for nitric oxide detoxification. Spectroscopic studies demonstrate that a single molecule of nitric oxide binds to ferrous iron in the protein, rather than to a haem group. We aim to understand how the binding of nitric oxide to the iron centre in the regulatory protein activates the switch that allows bacteria to defend themselves against the toxic effects of this molecule.

This project exploits our recent and novel finding that the GAF domain of the NorR transcriptional activator, a member of the family of bacterial enhancer binding proteins (EBPs), contains a mono-nuclear non-haem iron centre that binds a single molecule of nitric oxide (NO). We have demonstrated that the NO-modified form of NorR activates transcription initiation by RNA polymerase holoenzyme containing the alternative sigma factor sigma 54. The interaction of the GAF domain of NorR with NO apparently relieves intramolecular repression of the AAA+ domain, leading to transcriptional activation. We plan to use a multidisciplinary approach, employing spectroscopy coupled with protein structure -function studies and analysis of nucleoprotein complexes to understand in molecular detail how the binding of NO to the ferrous iron centre in the GAF domain of NorR leads to transcriptional activation by this important regulatory protein.