Streptococcus pneumoniae is a major human pathogen responsible for approximately one million deaths annually, predominantly in children under 5 in developing countries. Our research recently identified that pneumococcal susceptibility to zinc toxicity occurred via competition of high concentrations of zinc for PsaA, the manganese binding protein not by zinc accumulation as previously believed. This results in starvation of the essential metal ion manganese in the pneumococcus, which impairs growth and heightens susceptibility to oxidative stress. Intriguingly, Zn(II) binding to PsaA, unlike Mn(II), was irreversible. However, as the structures of PsaA with Mn(II) or Zn(II) were virtually identical, with both metal ions coordinated by His67, His139, Glu205, and Asp280, the molecular basis for the irreversible Zn(II)-binding remained unclear. Analysis of PsaA using a thermodynamic stability assay showed that Zn(II)-PsaA was more thermostable than Mn(II)-PsaA (+18.2C vs +11.3C) relative to metal free apo-PsaA. To elucidate the interaction of PsaA with the metal ligands, point mutations of the metal-coordinating residues Glu205 (to Gln205) and Asp280 (to Asn280) were generated. Both mutants were observed to be entirely competent for Zn(II) binding, but Mn(II) binding was reduced in PsaA-Gln205 (37%) whilst binding was essentially abrogated in PsaA-Asn280 (10%). This interaction with metal ligands was further supported by thermodynamic stability assays, which showed PsaA-Gln205 and PsaA-Asn280 were stabilised, albeit to a lesser extent, by Zn(II) (+4.7C and +0.9C) whereas Mn(II) could not induce significant stabilisation. Taken together, this data implicates that the metal coordinating carboxylate residues, Glu205 and Asp280, have key roles in facilitating Mn(II) binding to PsaA and reveals a unique target for future drug design studies.