Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster

The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biologically relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu4(GS)6] cluster that is sufficiently stable to limit the effective free aquacopper(I) concentration to the sub-femtomolar regime. Combined spectrophotometric–potentiometric titrations at biologically realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity standards and corroborated by low-temperature phosphorescence studies, established cooperative assembly of [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equilibrium system sets a firm upper limit on the thermodynamic availability of intracellular copper that is 3 orders of magnitude lower than previously estimated. Taking into account their ability to catalyze the production of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiological conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity determinations of Cu(I) chaperones.