This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1 WT In a pair of publications, Hodgson and Fridovich demonstrated that bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD or SOD1) 1 exhibits a nonspecific peroxidase activity (1, 2). They provided evidence for formation of a potent oxidant in the presence of H 2 O 2 that was ascribed to a copper-bound hydroxyl radical, at the active site of bovine SOD1. Although the rate constant for the reaction between SOD1 and H 2 O 2 is very low (3), it was shown that the bovine SOD1 peroxidase activity could oxidize a variety of small molecular weight anionic ligands (azide, nitrite, formate, etc.) that are accessible to the active site (1, 2, 4, 5). However, a perplexing aspect of this peroxidase activity was that even larger molecules (e.g. 2,2Ј-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid, urate) that are not accessible to the active site of SOD1 were also oxidized (6). It was later discovered that the bicarbonate anion (HCO 3 Ϫ ) that is present in the buffer was responsible for Cu,Zn-SOD peroxidase-dependent oxidations of substrates in the bulk solution (7,8). It was proposed that the copper-bound oxidant (Cu 2ϩ -OH or Cu III O) could oxidize the HCO 3 Ϫ anion (a physiologically relevant molecule) to the carbonate anion radical (CO 3 . ), a potent oxidant that diffuses out of the active site and causes substrate oxidation (7, 8). The fact that CO 3 . , and not hydroxyl radical, is the primary oxidant produced by SOD1-mediated peroxidase activity is physiologically significant, because CO 3 . has a much longer half-life (than hydroxyl radical) and can, therefore, diffuse away from the active site and oxidatively modify critical cellular targets. Liochev et al. (7) suggested that the nitrone spin trap, 5,5Ј-dimethyl-1-pyrroline-N-oxide (DMPO), is oxidized and hydrolyzed by CO 3 . . Zhang et al. (6) provided the experimental proof for this hypothesis. Using electron spin resonance (ESR) spin trapping in the presence of oxygen-17-labeled water, the investigators showed that the oxygen atom in the DMPO-OH adduct is derived totally from water. Zhang et al. (6) also reported that HCO 3 Ϫ -dependent peroxidase activity can be measured using a variety of methods including fluorescence and optical spectroscopy. More recently, Hink et al. (9) demonstrated that an extracellular SOD (SOD3 or ecSOD) enhanced the hydroxylation of a cyclic nitrone spin trap in the presence of H 2 O 2 and HCO 3 Ϫ . In this work, we report that human Cu,Zn-superoxide dismutase (hSOD1 WT ) also exhibits a HCO 3 Ϫ -dependent peroxi-* This work was supported by National Institutes of Health Grants RR01008, NS40494, and NS40819 (to J. P. C.) and the ALS Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. . , carbonate radical anion; DMPO, 5,5Ј-dimethyl-1-pyrroline-N...