Abstract
Most zinc in cells is bound to proteins, but a tiny portion is bound to non-proteinaceous Zn complexes coordinated by unidentified metabolites. Such low-mass pools likely play a central role in Zn homeostasis in cells, yet their chemical compositions and concentrations are unestablished. Previous investigations suggest that the collective Zn concentration of so-called "free" or "labile" pools is femtomolar to picomolar. Here the low-mass Zn pool in Escherichia coli was investigated by isolating cytoplasm from cells grown in media supplemented with increasing concentrations of Zn(acetate)(2). Gentle cell lysis was demonstrated using Mössbauer spectroscopy. Corresponding isolated cytoplasm contained increasing concentrations of Zn. Equivalent samples were subjected to liquid chromatography with inline ICP-MS detection. Many Zn and S peaks were detected, due to both proteins and the low-mass pool. Zn complexes were largely stable over several days. Cytoplasm had a large binding capacity for aqueous Zn, implying that cells are essentially devoid of aqueous Zn. Other samples were treated with the chelator N,N,N'N'-tetrakis[(pyridin-2-yl)methyl]ethane-1,2-diamine (TPEN) to evaluate the binding strength of the detected species. TPEN removed Zn from both proteins and pool complexes with about equal propensity. Low-mass zinc complexes were present at μM collective concentrations, orders of magnitude higher than previous reports for "free" or "labile" pools. Two Zn-glutathione (GSH) complexes were identified by electrospray ionization mass spectrometry (ESI-MS) as dominant members of the pool under Zn-replete conditions. Cells do not significantly increase GSH concentrations when exposed to high levels of media Zn; rather, excess imported Zn is sequestered by available abundant GSH.