Reversible redox- and zinc-dependent dimerization of the Escherichia coli fur protein.

Fur is a bacterial regulator using iron as a cofactor to bind to specific DNA sequences. This protein exists in solution as several oligomeric states, of which the dimer is generally assumed to be the biologically relevant one. We describe the equilibria that exist between dimeric Escherichia coli Fur and higher oligomers. The dissociation constant for the dimer-tetramer equilibrium is estimated to be in the millimolar range. Oligomerization is enhanced at low ionic strength and pH. The as-isolated monomeric form of Fur is not in equilibrium with the dimer and contains two disulfide bridges (C92-C95 and C132-C137). Binding of the monomer to DNA is metal-dependent and sequence specific with an apparent affinity 5.5 times lower than that of the dimer. Size exclusion chromatography, EDC cross-linking, and CD spectroscopy show that reconstitution of the dimer from the monomer requires reduction of the disulfide bridges and coordination of Zn2+. Reduction of the disulfide bridges or Zn2+ alone does not promote dimerization. EDC and DMA cross-links reveal that the N-terminal NH2 group of one subunit is in an ionic interaction with acidic residues of the C-terminal tail and close to Lys76 and Lys97 of the other. Furthermore, the yields of cross-link drastically decrease upon binding of metal in the activation site, suggesting that the N-terminus is involved in the conformational change. Conversely, oxidizing reagents, H2O2 or diamide, disrupt the dimeric structure leading to monomer formation. These results establish that coordination of the zinc ion and the redox state of the cysteines are essential for holding E. coli Fur in a dimeric state.