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 H2O2 the 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. H2O2 modulated 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. Copyright 1999 Academic Press.