Carboxyl groups are important for efficient renal uptake of small anionic molecules. [(TCO)-T-99m(ECH)](2-) (ECH = pentaanionic form of (2R,7R)-2,7-dicarboxy-3,6-diaza-1,8-octanedithiol (ECH(6))) is a potentially useful radiopharmaceutical for diagnosing renal function. As typically isolated, the [(TC)-T-99(V)O](3+) and [Re(V)O](3+) derivatives of ECH(6) are neutral trifunctional acids, e.g., (TCO)-T-99(ECH(3)) and ReO(ECH(3)). The ligand has two carboxyl groups; the presence of the oxo group on the metal lowers the symmetry of the coordinated ligand, and one carboxyl group is syn and the other anti to the oxo group. Extremely broad NMR spectra at physiological pH and difficulty in crystallization have precluded complete characterization. Both the neutral species, ReO(ECH(3)) (3), and the ammonium salt of the trianionic species, [NH4](3)[ReO(EC)].C4H8O2 (5), have now been structurally characterized by X-ray diffraction. The coordination geometry of 3 is distorted octahedral, with the notable and unexpected feature that the anti-carboxylate group is coordinated trans to the oxo ligand. The coordination geometry of 5 is distorted square pyramidal, with the unusual feature that both nitrogen donor atoms are deprotonated. To determine the causes for the very broad H-1 and C-13 spectra of the Tc and Re complexes in aqueous solutions near physiological pH, we first prepared the analogous N2S2 ligand (TMECH(6)) incorporating penicillamine (pen), which has no CH coupling. (The ECH(6) ligand is derived from cysteine (cys), which has a coupled three-spin system.) The acid dissociation constants of the Re EC and TMEC complexes were found to be similar, with one NH group partially deprotonated under physiological conditions. We then applied a battery of 2D NMR methods to Re derivatives of both N2S2 ligands in D2O. From these studies, the EC H-1 NMR spin systems were identified and assigned for both acidic and basic aqueous solutions. From the Karplus relationship, the relevant torsion angles were calculated and compared to those determined crystallographically. These studies demonstrated that the anti-carboxyl group was coordinated in aqueous solution at low pH but not at high pH. The NMR results for ReO(TMECH(3)) under physiological conditions are consistent with two forms : (i) and anti-cys NH deprotonated, anti-carboxyl dissociated form and (ii) an anti-cys NH, coordinated anti-carboxyl form. The unusual broadening of ReO(ECH(3)) and TcO(ECH(3)) signals at pH 7 can be very confidently attributed to the coupling of carboxyl coordination and NH proton dissociation. Thus, proton dissociation makes the N a better donor. : (i) and anti-cys NH deprotonated, anti-carboxyl dissociated form and (ii) an anti-cys NH, coordinated anti-carboxyl form. The unusual broadening of ReO(ECH(3)) and TcO(ECH(3)) signals at pH 7 can be very confidently attributed to the coupling of carboxyl coordination and NH proton dissociation. Thus, proton dissociation makes the N a better donor.