title: The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster.
abstract: 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.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom. nick.tucker@bbsrc.ac.uk
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Tucker
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Hicks
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Clarke
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.forename: Govind
authors.initials: G
authors.lastname: Chandra
authors.forename: Nick E
authors.initials: NE
authors.lastname: Le Brun
authors.forename: Ray
authors.initials: R
authors.lastname: Dixon
authors.forename: Matthew I
authors.initials: MI
authors.lastname: Hutchings
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D012097:Repressor Proteins; D009569:Nitric Oxide; D004247:DNA