Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase

Abstract

The localization of Cu,Zn-superoxide dismutase in the mitochondrial intermembrane space suggests a functional relationship with superoxide anion (O₂^•−) released into this compartment. The present study was aimed at examining the functionality of Cu,Zn-superoxide dismutase and elucidating the molecular basis for its activation in the intermembrane space. Intact rat liver mitochondria neither scavenged nor dismutated externally generated O₂^•−, unless the mitochondrial outer membrane was disrupted selectively by digitonin. The activation of the intermembrane space Cu,Zn-superoxide dismutase following the disruption of mitochondrial outer membrane was largely inhibited by bacitracin, an inhibitor of protein disulphide-isomerase. Thiol alkylating agents, such as N-methylmaleimide or iodoacetamide, decreased intermembrane space Cu,Zn-superoxide dismutase activation during, but not after, disruption of the outer membrane. This inhibitory effect was overcome by exposing mitochondria to low micromolar concentrations of H₂O₂ before disruption of the outer membrane in the presence of the alkylating agents. Moreover, H₂O₂ treatment alone enabled intact mitochondria to scavenge externally generated O₂^•−. These findings suggest that intermembrane space Cu,Zn-superoxide dismutase is inactive in intact mitochondria and that an oxidative modification of its critical thiol groups is necessary for its activation.