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 e ffects 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 oxidiz ing (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 less than or equal to 12. In the presence of H2O2 the degree of protection by MT was less and biologica l oxidations and radical formation were inhibited only up to Cu/MT ratios o f 6. Physical interaction of MT and Cu was measured by using low-temperatur e EPR of free Cu2+ in solution. The maximal amount of EPR-silent Cu1+ (pres umably in complex with MT) corresponded to 12 molar equivalents of Cu/MT un der 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 m anner that correlated with the ability of MT to mitigate Cu-redox cycling i n cell-free systems. Thus, optimal binding of Cu to MT is achieved under re ducing conditions; however, a portion of this Cu appears releasable under o xidizing conditions. Release of free Cu from MT during oxidative stress cou ld enhance the formation of reactive oxygen species and potentiate cellular damage. (C) 1999 Academic Press.