Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (O-.(2)-). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H2O2), a by-product of the O-.(2)- scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15 min in vitro incubation with CuZn-SOD and 1 mM H2O2, 76% of the melatonin was oxidized, compared to 52% with 0.25 mM H2O2, and just 9% without H2O2. Pre-incubation with 1 mM H2O2 resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H2O2 levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function. (C) 2004 Elsevier Inc. All rights reserved.