Redox Regulation of Copper–Metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios ≤12. In the presence of H2O2the degree of protection by MT was less and biological oxidations and radical formation were inhibited only up to Cu/MT ratios of 6. Physical interaction of MT and Cu was measured by using low-temperature EPR of free Cu2+in solution. The maximal amount of EPR-silent Cu1+(presumably in complex with MT) corresponded to 12 molar equivalents of Cu/MT under reducing conditions, but only 9 in the presence of H2O2. H2O2modulated the ability of MT to protect HL-60 cells from Cu-induced cell death in a manner that correlated with the ability of MT to mitigate Cu-redox cycling in cell-free systems. Thus, optimal binding of Cu to MT is achieved under reducing conditions; however, a portion of this Cu appears releasable under oxidizing conditions. Release of free Cu from MT during oxidative stress could enhance the formation of reactive oxygen species and potentiate cellular damage.