Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.

Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkyl...