Bicarbonate-dependent peroxidase activity of human Cu, Zn-superoxide dismutase induces covalent aggregation of protein - Intermediacy of tryptophan-derived oxidation products

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1(WT)) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1(WT) were formed from incubation mixtures containing hSOD1(WT), H2O2, and HCO3-.HCO3--dependent peroxidase activity and covalent aggregation of hSOD1(WT) were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)(5)CO3](+) complex that generates the carbonate radical anion (CO3.-). Human SOD1(WT) has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1(W32F)) that exhibits HCO3--dependent peroxidase activity similar to wildtype enzyme. However, unlike hSOD1(WT), incubations containing hSOD1(W32F), H2O2, and HCO3- did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.- induced covalent aggregation of hSOD1(WT). Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1(WT), but not from hSOD1(W32F). Spin traps also inhibited the covalent aggregation of hSOD1(WT). Fluorescence experiments revealed that Trp-32 was further oxidized by CO3.- forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1(WT), implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1(WT). Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.