Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

Abstract

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular “free” iron content due to deletion of the iron-sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the electron paramagnetic resonance and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is about 31% in the E. coli iscA/sufA mutant cells and is decreased to about 4% in wild-type E. coli cells. Depletion of the intracellular “free” iron content using the membrane-permeable iron chelator 2,2’-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular “free” iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, as the Fur homolog from Haemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

“…FeSO 4 also reduced the activity of the chromosomal reporter strain CFT073 tcpC::gfpmut2 significantly after incubation of 3 h and overnight ( Figure 9 G,H). To further analyze the influence of FeSO 4 on the P2 promoter, we used the iron chelator 2,2’-bipyridine, which is also known to deplete intracellular iron from E. coli [ 22 ]. We found that incubation of the P2-reporter strain with the iron chelator significantly increased the activity of the P2 promoter ( Figure 10 ).…”

Do FeS clusters rule bacterial iron regulation?

“…The new study by Fontenot et al (5) convincingly shows that bacterial Fur proteins isolated from E. coli can bind an all-Cys-coordinated [2Fe-2S] cluster. Therefore, it seems possible that this metallo cofactor rather than the previously thought mononuclear Fe 21 may serve as the iron-regulatory device (Fig.…”

“…1b) (2,9). Finally, Fontenot et al (5) overexpressed the Fur ortholog from the Gramnegative bacterium Haemophilus influenzae in DiscA/sufA E. coli cells. This protein bound even 2-fold more [2Fe-2S] cluster, implying that cluster binding may be a general feature of Fur proteins.…”

“…1b). So far, Fontenot et al (5) have not dwelled on this important question, apart from showing that [2Fe-2S] cluster binding is labile and reversible, undoubtedly two important characteristics of a regulatory device. Future genetic and cell biological studies will have to clarify the relative contribution of [2Fe-2S] clusters and Fe 21 , possibly bound at different sites, to iron homeostasis.…”

Thioredoxin Dependent Changes in the Redox States of FurA from Anabaena sp. PCC 7120

“…The available structures of Fur proteins from different organisms unveil that, in addition to the regulatory metal-binding site, Fur displays a so-called structural zincbinding site which, in most cases, is formed by four cysteines arranged in two CXXC motifs [1]. Furthermore, it has been found that, in E. coli, Fur binds a [2Fe-2S] cluster via conserved cysteine residues [7]. Fur has also been identified as one of the transcription factors that are associated with thioredoxin in a detailed analysis of the thioredoxinlinked E. coli proteome [8], as well as in further studies using E. coli engineered strains to optimize trapping and characterization of the in vivo Trx redox interactome [9].…”