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 spectrom etry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein, Fur is a metalloregulatory protein involved in th e regulation of almost all bacterial genes related to iron uptake in Gram-n egative bacteria such as Escherichia coli. In addition to the iron site, Fu r also possesses a tight- binding zinc site that likely comprises two cyste ines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present i n 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 a lkylation by mass spectrometry, Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine wh ich of the cysteines were protected from alkylation by the zinc atom. Enzym atic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95. were thus assigned as zinc li gands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 wa s shown to be the fastest reacting cysteine, implying it is a surface-expos ed residue.