%0 Journal Article %A Lewin, Allison C. %A Doughty, Phillip A. %A Flegg, Lynda %A Moore, Geoffrey R. %A Spiro, Stephen %T The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion %D 2002 %J Microbiology, %V 148 %N 8 %P 2449-2456 %@ 1465-2080 %R https://doi.org/10.1099/00221287-148-8-2449 %K Fur %K Gdn . HCl, guanidinium hydrochloride %K CD, circular dichroism %K MBP, maltose-binding protein %K iron regulation %I Microbiology Society, %X The ferric uptake regulator (Fur) of Pseudomonas aeruginosa was expressed in Escherichia coli in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn2+ that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn2+ ion per monomer. Thus, the P. aeruginosa Fur can probably accommodate a single Zn2+ ion bound to the metal-sensing site. The single cysteine residue of P. aeruginosa Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the E. coli protein, and is believed to provide one of the ligands to a structural Zn2+ ion. This cysteine residue was shown to be dispensable for the in vivo activity of P. aeruginosa Fur, which is consistent with the suggestion that the P. aeruginosa protein does not contain a structural Zn2+ ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of P. aeruginosa Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-148-8-2449