Microcin E492 (MccE492), the hydrophobic and anionic 84-residue antimicrobial peptide from the Klebsiella pneumoniae RYC492 fecal strain, was purified from culture supernatants of the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid. While MccE492 (7886 u) was considered an unmodified peptide, we have isolated it in a posttranslationally modified form (MccE492m, 8717 u), which exhibits a more potent antimicrobial activity and a broader spectrum of activity than MccE492. The structure and ion binding properties of this modification are investigated here. Both MccE492 and MccE492m were produced and purified from culture supernatants. Both microcin samples were submitted to chymotrypsin digestion, and the resulting fragment mixtures were analyzed by RP-HPLC. A single fragment purified from the MccE492m digest (1771.6 u) was shown to contain the entire 831.2 Da posttranslational modification. In order to localize and identify the modification, this peptide fragment was submitted to MS analyses, using both hybrid ESI-QqTOF (Q-Star, Applied Biosystems) and nanoESI-ion trap (ESQUIRE 3000, Bruker Daltonics) instruments operating in the positive and negative ion detection modes. Peptide samples (10 pmol/µl) were prepared in 50% acetonitrile. Formic acid (0.5%) was added for positive ion mode analysis. MSn experiments were undertaken on the 1771.6 u fragment. The CID spectrum of the [M+2H]2+ ion at m/z 886.8 recorded in the positive mode, gave two independent series of b- and y-ions. The y-ions were shifted to upper m/z ratios consistent with a 831.2 Da mass increase, while the b-ions were unshifted, thus allowing to localize the modification on the MccE492 C-terminal serine. In the negative mode, MS/MS experiments performed on the [M-2H]2- ion at m/z 884.8, gave an ion in low abundance at m/z 848.2, which corresponds to the entire modification. MSn experiments were further carried out in negative mode in order to get additional structural information. The modification was shown to carry N-(2,3-dihydroxybenzoyl)-L-serine, which belongs to a group of catechol-type siderophores involved in iron uptake by enterobacteria. Moreover, we investigated the Fe3+ binding properties of the modified fragment by MSn experiments conducted in presence of FeCl3 and showed that the modification was able to capture Fe3+ ions. Recently, we have shown that the receptor of the siderophore/iron complex could be involved in the MccE492 recognition by sensitive bacteria. Thus, we propose that the improvement of MccE492 antimicrobial activity upon modification would result from an increase of the microcin/receptor affinity.