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

• Published in: The Journal of biological chemistry 2003-06, Vol.278 (26), p.24078-24089
• Main Authors: Zhang, Hao, Andrekopoulos, Christopher, Joseph, Joy, Chandran, Karunakaran, Karoui, Hakim, Crow, John P, Kalyanaraman, B
• Format: Article
• Language: English
• Subjects: Amyotrophic Lateral Sclerosis - etiology; Bicarbonates - pharmacology; Dimerization; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Humans; Models, Chemical; Oxidation-Reduction; Peroxidases - chemistry; Peroxidases - drug effects; Peroxidases - metabolism; Protein Conformation; Superoxide Dismutase - chemistry; Superoxide Dismutase - drug effects; Superoxide Dismutase - metabolism; Tryptophan - chemistry
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M302051200

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT 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 (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,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 hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. 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 hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. 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.