title: NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.
abstract: The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.affiliation: the School of Biological Sciences, and.
authors.forename: John
authors.initials: J
authors.lastname: Munnoch
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Dodd
authors.affiliation: the School of Biological Sciences, and.
authors.forename: Felicity
authors.initials: F
authors.lastname: Knowles
authors.affiliation: the School of Biological Sciences, and.
authors.forename: Mahmoud M
authors.initials: MM
authors.lastname: Al Bassam
authors.affiliation: the Department of Chemistry, University of California, Davis, California 95616, and.
authors.forename: Saeed
authors.initials: S
authors.lastname: Kamali
authors.affiliation: the Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Holland
authors.affiliation: the Department of Chemistry, University of California, Davis, California 95616, and.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Cramer
authors.affiliation: the School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Hamilton
authors.affiliation: the Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.affiliation: the School of Biological Sciences, and m.hutchings@uea.ac.uk.
authors.forename: Matthew I
authors.initials: MI
authors.lastname: Hutchings
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry, n.le-brun@uea.ac.uk.
authors.forename: Nick E
authors.initials: NE
authors.lastname: Le Brun
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D018085:Regulon / Q000502:physiology; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007506:Iron-Sulfur Proteins; D009569:Nitric Oxide
keywords: DNA-binding Protein; Iron; Iron-Sulfur Protein; Low Molecular Weight Thiol; Nitric Oxide; Nitrosative Stress; Regulator; Spectroscopy