Transcription factor FnrP from Paracoccus denitrificans contains an iron-sulfur cluster and is activated by anoxia: Identification of essential cysteine residues

Hutchings, Matthew I., Crack, Jason C., Shearer, Neil, Thompson, Benjamin J., Thomson, Andrew J. and Spiro, Stephen (2002) Transcription factor FnrP from Paracoccus denitrificans contains an iron-sulfur cluster and is activated by anoxia: Identification of essential cysteine residues. Journal of Bacteriology, 184 (2). pp. 503-508. ISSN 0021-9193

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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.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry
UEA Research Groups:	Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Organisms and the Environment
Depositing User:	EPrints Services
Date Deposited:	01 Oct 2010 13:36
Last Modified:	16 May 2023 10:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/345
DOI:	10.1128/JB.184.2.503-508.2002

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