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Transcription Factor FnrP from Paracoccus denitrificans Contains an Iron-Sulfur Cluster and Is Activated by Anoxia: Identification of Essential Cysteine Residues

Matthew I. Hutchings,^1, Jason C. Crack,² Neil Shearer,¹ Benjamin J. Thompson,¹ Andrew J. Thomson,² and Stephen Spiro^1*

School of Biological Sciences,¹ School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom²

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.

Present address: The John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom.

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