title: Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide.
abstract: NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8-10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry, and.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.affiliation: the School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ and.
authors.forename: John
authors.initials: J
authors.lastname: Munnoch
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry, and.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.affiliation: the School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ and.
authors.forename: Matthew I
authors.initials: MI
authors.lastname: Hutchings
authors.affiliation: From the Centre for Molecular and Structural Biochemistry, School of Chemistry, and 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*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; D009569:Nitric Oxide
keywords: DNA-binding protein; DNA-protein interaction; iron; iron-sulfur protein; nitric oxide; regulator; spectroscopy