(823.4) Ferric Uptake Regulator (Fur) Regulates Intracellular Iron Homeostasis via reversible binding of a [2Fe-2S] cluster in Escherichia coli

Poster Board Number: A456

Xialong Ni (Louisiana State University), Chelsey Fontenot (Louisiana State University), Jing Yang (Louisiana State University), Olivia Kluchka (Louisiana State University), Huangen Ding (Louisiana State University)

It has generally been assumed that when intracellular “free” iron content is elevated in bacteria, a global transcription factor Fur (Ferric uptake regulator) binds Fe(II) to regulate expression of its target genes and achieve intracellular iron homeostasis.  Fur is highly conserved among bacteria, and purified Fur proteins from different bacteria may exist as a homodimer or tetramer with each monomer containing three metal binding sites.  While two of the metal binding sites (site 1 and site 2) in purified Fur proteins are often occupied by Zn(II), the iron-bound Fur has never been isolated from any bacteria.  Here we report that Escherichia coli Fur binds a [2Fe-2S] cluster at site 3 (via Cys-93 and Cys-96) in the E. coli mutant cells in which intracellular “free” iron content is elevated due to deficiency of iron-sulfur cluster biogenesis.  Depletion of intracellular "free" iron content effectively removes the [2Fe-2S] cluster from Fur, indicating that binding of a [2Fe-2S] cluster in Fur is determined by intracellular "free" iron content. Fur homolog from Haemophilus influenzae also binds a [2Fe-2S] cluster at site 3 when expressed in E. coli cells, suggesting that binding of a [2Fe-2S] cluster in Fur is highly conserved.  While the Zn(II)-bound E. coli Fur has a basal binding activity for its target DNA sequence known as Fur-box, binding of a [2Fe-2S] cluster significantly enhances its Fur-box binding activity. Similarly, binding of a [2Fe-2S] cluster is required for the strong binding activity of H. influenzae Fur for Fur-box in vitro.  Mutation of Cys-93 to Ala results in an E. coli Fur mutant that fails to bind the [2Fe-2S] cluster, loses the enhanced binding activity for Fur-box, and is inactive to complement the physiological function of E. coli Fur in E. coli cells. Our results suggest that E. coli Fur and H. influenzae Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular “free” iron content in cells.

The work was supported by NSF grant (MCB 2050032).