Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. S. pneumoniae has a relatively high manganese requirement and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, that contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.

A disulfide-bridged peptide containing two Ni2+ binding sites based on the nickel superoxide dismutase protein, {Ni2(SODmds)} has been prepared. At physiological pH (7.4), it was found that the metal sites are mononuclear with a square planar NOS2 coordination environment with the two sulfur-based ligands derived from cysteinate residues, the nitrogen ligand derived from the amide backbone, and a water ligand. Furthermore, S K-edge X-ray absorption spectroscopy indicated that the two cysteinate sulfur atoms ligated to nickel are each protonated. Elevation of the pH to 9.6 results in the deprotonation of the cysteinate sulfur atoms, and yields a binuclear, cysteinate bridged Ni22+ center with each nickel contained in a distorted square planar geometry. At both pH = 7.4 and 9.6, the nickel sites are moderately air sensitive, yielding intractable oxidation products. However, at pH = 9.6, {Ni2(SODmds)} reacts with O2 at an ~3.5-fold faster rate than at pH = 7.4. Electronic structure calculations indicate that the reduced reactivity at pH = 7.4 is a result of a reduction in S(3p) character and deactivation of the nucleophilic frontier molecular orbitals upon cysteinate sulfur protonation.