X-ray absorption spectroscopy (XAS) is widely used to explore the coordination environments and structures of metal complexes in aqueous solutions and disordered phases. Although soft-XAS studies on gaseous phases, solid phases and their interfaces have shown that XAS is a versatile tool in studying the functional group composition of organic molecules, the application of XAS to studying aqueous solutions is seriously limited because of experimental difficulties. In this report, using a modified synchrotron endstation geometry, we show how soft-XAS was used to study the changes in electronic states of reactive functional groups in a bacterial macromolecule, desferrioxamine B (desB, a hydroxamate siderophore) and its structural analogue (acetohydroxamic acid (aHa)). We collected C, N, and O near edge X-ray absorption fine structure (NEXAFS) spectra of these molecules in aqueous solutions and complemented their spectral interpretation with calculated X-ray spectra of "hydrated" aHa. The experimental spectra of desB are similar to those for aHa at the C, N, and O K-edges. In addition, the electronic transitions of amide and hydroxamate functional groups in the macromolecule can be distinguished from the N spectra. Small energy differences in the pi*((C=O)NO) and the o*(NO) transitions at the C- and N-edges of aHa and desB indicate that the substituent attached to N in desB ((CH2)(n)) determines the electron density in the (C=O)NO core. As the solution pH increased, the pi((C=O))(NO) transition of the hydroxamate group of these two molecules exhibit energy shifts at the C-, N-, and O-edges, which are consistent with increased electron delocalization in the (C=O) NO core of aHa ( and desB), predicted from the calculations. The spectra of the aHa(H2O)(3)(-) anion also provide evidence for partial N-deprotonation at pH values usually attributed to an O-acid. These results indicate that soft-XAS is well suited for studying the electronic states of different functional groups in aqueous organic macromolecules.