Redox-changes associated with the glutathione-dependent ability of the Cu(II)-GSSG complex to generate superoxide

The intracellularly-occurring Cu(I)-glutathione complex (Cu(I)-[GSH]2) has the ability to reduce molecular oxygen into superoxide. Removal of such radicals leads to the irreversible conversion of Cu(I)-[GSH]2 into the redox-inactive Cu(II)-GSSG complex. The present study addressed the potential of reduced glutathione, ascorbate and superoxide to reductively regenerate Cu(I)-[GSH]2 from Cu(II)-GSSG, and investigated the redox changes involved in such process. Results show that: (i) among the three tested reductants, only GSH is able to reduce the Cu(II) bound to GSSG; (ii) during the reduction of Cu(II)-GSSG, a Cu(I)-GSSG intermediate would be formed (supported here by Cu(I) and GSSG recovery data and by NMR studies); (iii) when GSH is present in a molar excess equal or greater than 1:3, the reduction of Cu(II)-GSSG into Cu(I)-[GSH]2 is quantitative and complete. Under such conditions, the Cu(II)-GSSG complex acquires a superoxide-generating capacity which is identical to that seen with the Cu(I)-[GSH]2 complex. Within cells, the concentrations of GSH are at least 2- to 3-fold order of magnitude higher than those expected for the Cu(II)-GSSG complex. Thus, we postulate that the interaction between GSH and Cu(II)-GSSG could be seen as a potential mechanism to regenerate continuously the Cu(I)-[GSH]2 complex and thereby affect the ability of the latter to generate superoxide.