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.

FIG. 1.

Alignment of the N-terminal regulatory domains of FNR from E. coli (23), HlyX from A. pleuropneumoniae (5), CydR from A. vinelandii (30), FnrL from R. sphaeroides (31), FixK from B. japonicum (1), and FnrP from P. denitrificans (25). The alignment is not intended to be exhaustive but rather to show examples of proteins from the gamma and alpha subdivisions of the proteobacteria for which some biological information is available. Cysteine residues of FNR known to be essential for activity are indicated (+), as are the cysteine residues of FnrP shown in this study to be essential (*). Residues of FNR that can be substituted with effects on oxygen sensitivity and that are discussed in the text are indicated (§).

FIG. 2.

Reconstitution of an iron-sulfur cluster into purified FnrP. FnrP was purified from a GST fusion and was treated with cysteine desulfurase (NifS, from A. vinelandii) under anaerobic conditions in the presence of cysteine, DTT, and ferrous ammonium sulfate. FnrP concentration was approximately 14 μM. The lower spectrum was recorded immediately after the reaction was set up, and the upper spectrum was recorded after a reaction time of 95 min.