title: Transcription factor FnrP from Paracoccus denitrificans contains an iron-sulfur cluster and is activated by anoxia: identification of essential cysteine residues.
abstract: The Paracoccus denitrificans transcription factor FnrP has been characterized using artificial FNR-dependent promoter-lacZ fusion plasmids in Escherichia coli. FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at -41.5 with respect to the transcription start site. FnrP was found to be inactive in an iscS mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP. Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine. Thus, FnrP is a true orthologue of FNR from E. coli and switches on target genes in response to anoxia. Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster. Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo. Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation. The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system. Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO(+)) in defined growth media but are only moderately sensitive to those sources of NO that were tested.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
authors.forename: Matthew I
authors.initials: MI
authors.lastname: Hutchings
authors.forename: Jason C
authors.initials: JC
authors.lastname: Crack
authors.forename: Neil
authors.initials: N
authors.lastname: Shearer
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Thompson
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: Stephen
authors.initials: S
authors.lastname: Spiro
mesh_terms: D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007501:Iron; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009569:Nitric Oxide / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D013455:Sulfur; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; C445305:FnrP protein, Paracoccus denitrificans; D007506:Iron-Sulfur Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D009569:Nitric Oxide; D013455:Sulfur; D007501:Iron; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; D003545:Cysteine