Copper, Zinc Superoxide Dismutase as a Univalent NO -Oxidoreductase and as a Dichlorofluorescin Peroxidase

Abstract

Nitroxyl (NO-) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO- to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO- and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O 2.- was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were ∼100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO- caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O2.-, SOD could catalyze the oxidation of NO- to NO. The reverse reaction, i.e. the reduction of NO to NO- by Cu(I),Zn-SOD, followed by the reaction of NO- with O2 would yield ONOO- and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H2O2 caused the HCO3--dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H2O2 production.