The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster.
The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.
Tucker
Nicholas P
NP
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom. nick.tucker@bbsrc.ac.uk
Hicks
Matthew G
MG
Clarke
Thomas A
TA
Crack
Jason C
JC
Chandra
Govind
G
Le Brun
Nick E
NE
Dixon
Ray
R
Hutchings
Matthew I
MI
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D015204:Spectroscopy, Mossbauer; D048372:Streptomyces coelicolor / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D012097:Repressor Proteins; D009569:Nitric Oxide; D004247:DNA