The Fur protein regulates iron uptake systems in many Gram-negative bacteria. The striking phenotype of the first fur mutants isolated was the overexpression of the outer membrane receptors for siderophore iron transport. In addition, excretion of siderophores under iron-rich growth conditions was observed in these mutants, indicating that the biosynthesis of siderophores is also regulated by Fur.
The Fur protein from E. coli was isolated in one step due to its high affinity for metal-chelate columns loaded with zinc. In DNase footprinting experiments, the Fur protein was shown to bind DNA in the promoter region of several iron-regulated genes. The consensus sequence, called the Fur box, is GATAATGATAATCATTATC. In vitro binding is dependent on the divalent cations Co2+ ^ Mn2+ ^ Cd2+ ^ Cu2+ at 150 ^M, while Fe2+ seemed to be less active at this concentration, probably due to oxidation to Fe3+ (De Lorenzo et al., 1987). The unspecificity for divalent metals observed in vitro shows that the cells have to select the ions transported carefully and have to balance their active concentrations. In addition, it is a caveat for the experimenter to test a hypothesis on metal-ion specificity not only in vitro, but also in vivo.
The important regulatory metal-binding site occupied in vivo by Fe2+ is not well characterized. The metal is bound in this site by at least two histidines and one carboxylate in an axially distorted octahedral environment (Althaus et al., 1999), confirming earlier work (Hamed and Neilands, 1993).
Zn2+ at 100 ^M seems to activate the apo-Fur protein partially, possibly caused by the recently discovered tight binding of zinc to Fur. Two cysteines at positions 92 and 95 have been identified as binding sites by alkylation of cysteines in the presence and absence of zinc (Gonzalez et al., 1999). In addition, from spectroscopic studies two nitrogen/oxygen binding sites were proposed for zinc. The binding of zinc is remarkable since several members of the Fur family, like the Zur (zinc uptake regulator) protein in E. coli, regulate genes for zinc uptake and metabolism (Patzer and Hantke, 1998). Both metal-binding sites seem to be mainly in the carboxy-terminal half of the protein, although iron might also be bound by a histidine in the N-terminal half of the Fur protein (Saito et al., 1991).
The DNA binding site of Fur was predicted and then found to be in the N-terminal domain in an unusual helix-turn-helix motif (Holm et al., 1994; Stojiljkovic and Hantke, 1995). The structure of Fur still remains to be determined and attempts to crystallize the protein have so far been unsuccessful.
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