The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

• Published in: PloS one Vol. 3; no. 11; p. e3623
• Main Authors: Tucker, Nicholas P, Hicks, Matthew G, Clarke, Thomas A, Crack, Jason C, Chandra, Govind, Le Brun, Nick E, Dixon, Ray, Hutchings, Matthew I
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.11.2008; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Bacillus subtilis; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Binding; Binding Sites; Biochemistry/Protein Chemistry; Blood proteins; Clusters; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA binding; E coli; Electron Spin Resonance Spectroscopy; Genes; Genetic aspects; Genetic research; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - isolation & purification; Iron-Sulfur Proteins - metabolism; Ligands; Microbiology; Molecular Biology/Transcription Initiation and Activation; Molecular Sequence Data; Mycobacterium tuberculosis; Nitrates; Nitric oxide; Nitric Oxide - metabolism; Pharmacy; Proteins; Pseudomonas; Repressor Proteins - chemistry; Repressor Proteins - isolation & purification; Repressor Proteins - metabolism; Repressors; Research parks; Salmonella; Salmonella Typhimurium; Sequence Alignment; Spectroscopy; Spectroscopy, Mossbauer; Streptomyces; Streptomyces coelicolor - metabolism; Transcription (Genetics); Transcription factors; Transcription, Genetic; Tuberculosis; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0003623

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.