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 (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.
Zhang
Hao
H
Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Andrekopoulos
Christopher
C
Joseph
Joy
J
Chandran
Karunakaran
K
Karoui
Hakim
H
Crow
John P
JP
Kalyanaraman
B
B
D000690:Amyotrophic Lateral Sclerosis / Q000209:etiology; D001639:Bicarbonates / Q000494:pharmacology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D001639:Bicarbonates; D014364:Tryptophan; D010544:Peroxidases; D013482:Superoxide Dismutase