Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((OH)-O-.). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (OH)-O-. formation. This dCuZnSOD-dependent (OH)-O-. generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-in dependent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (OH)-O-. generation, implying that Cu may be responsible for dCuZnSOD-induced (OH)-O-. formation. Catalase eliminated (OH)-O-. generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (OH)-O-. production. Furthermore, nitric oxide ((NO)-N-.) completely inhibited dCuZnSOD-induced (OH)-O-. radical generation, indicating that (NO)-N-. is an important (OH)-O-. radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (OH)-O-. formation in biological systems. (c) 2005 Elsevier Inc. All rights reserved.