The ferric uptake regulation (Fur) repressor is a zinc metalloprotein

The Fur protein regulates the expression of a wide variety of iron-responsive genes; however, the interaction of this repressor with its cognate metal ion remains controversial. The iron-bound form of Fur has proved difficult to obtain, and conflicting results have been published using Mn(II) as a probe for in vitro DNA-binding studies. We report here that the purified protein contains tightly bound zinc and propose that Zn(II) is bound to the protein in vivo. Upon purification, Fur retains ca. 2.1 mel of Zn(II)/mol of Fur monomer (Zn(2)Fur). One zinc is easily removed by treatment of Zn2Fur with zinc chelating agents, resulting in Zn(1)Fur with ca. 0.9 mel of Zn(II)/mol of protein. The remaining zinc in Zn(1)Fur can only be removed under denaturing conditions to yield ape-Fur with ca. 0.1 mol of Zn(IT)/mol of protein. Our results suggest that many literature descriptions of purified Fur protein do not correspond to the ape-protein, but to Zn(1)Fur or Zn(2)Fur. Dissociation constants (K-d) Of protein-DNA complexes are ca. 20 nM for both Zn(2)Fur and Zn(1)Fur as determined by electrophoretic mobility shift assays and DNase I footprinting assays. The two metalated forms, however, show qualitative differences in the footprinting assays while ape-Fur does not bind specifically to the operator. The existence of these Zn(II) binding sites in Fur may resolve some discrepancies in the literature and have implications concerning Zur, a Fur homologue in E. coli that regulates zinc-responsive genes.