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Gramibactin (GBT) is an archetype for the new class of diazeniumdiolate siderophores, produced by Paraburkholderia graminis, a cereal‐associated rhizosphere bacterium, for which a detailed solution thermodynamic study exploring the iron coordination properties is reported. The acid‐base behavior of gramibactin as well as its complexing ability toward Fe3+ was studied over a wide range of pH values (2≤pH≤11). For the latter the ligand‐competition method employing EDTA was used. Only two species are formed: [Fe(GBT)]− (pH 2 to 9) and [Fe(GBT)(OH)2]3− (pH≥9). The formation of [Fe(GBT)]− and its occurrence in real systems was confirmed by LC‐HRESIMS analysis of the bacteria culture broth extract. The sequestering ability of gramibactin was also evaluated in terms of the parameters pFe and pL0.5. Gramibactin exhibits a higher sequestering ability toward Fe3+ than EDTA and of the same order of magnitude as hydroxamate‐type microbial siderophores, but smaller than most of the catecholate‐type siderophores and much higher than the most known phytosiderophores. [ABSTRACT FROM AUTHOR]