Binding of a [2Fe-2S] cluster drives dimerization of ferric uptake regulator (Fur) in Escherichia coli

The ferric uptake regulator (Fur) is a global transcription factor that reversibly binds an iron-sulfur [2Fe-2S] cluster via the cysteine residues (site 3) in response to elevation of intracellular free Fe content in Escherichia coli. Here, we report that when E. coli Fur is expressed in E. coli cells grown in M9 medium supplemented with Fe or zinc (Zn), purified Fur binds a [2Fe-2S] cluster or Zn(II), respectively. While apo-form Fur is a monomer and has no DNA-binding activity, both the [2Fe-2S] cluster-bound Fur and the Zn(II)-bound Fur are homodimers and have a similar binding activity for the DNA sequence known as the Fur-box. The inductively coupled mass spectrometry analyses show that the purified [2Fe-2S] cluster-bound Fur homodimer binds only one [2Fe-2S] cluster per monomer and no other transition cations, and that the Zn(II)-bound Fur homodimer binds only one Zn(II) per monomer. The site-directed mutagenesis studies reveal that Fur binds the [2Fe-2S] cluster or Zn(II) at the same binding site (site 3) via the cysteine residues. While deletion of the Fe-S cluster assembly scaffold protein IscU prevents the [2Fe-2S] cluster assembly in Fur, deletion of IscU has no effect on the Zn(II) binding in Fur. Furthermore, the addition of Zn(II) effectively inhibits the [2Fe-2S] cluster binding in Fur in E. coli cells grown in M9 medium. The results suggest that E. coli Fur dimerizes upon the binding of a [2Fe-2S] cluster at site 3 and that Zn(II) competes with the [2Fe-2S] cluster binding in Fur and disrupts the regulation of intracellular Fe homeostasis.