Distinct oxidative cleavage and modification of bovine [Cu– Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule

Abstract

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2 kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5 Angstrom from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.

Copper ion / H2O2 oxidation of Cu/Zn-Superoxide dismutase: Implications for enzymatic activity and antioxidant action

“…1F). These observations are in agreement with a previous study where the exogenous supply of Cu 2+ , Fe 2+ , or metal chelator DTPA did not alter H 2 O 2 -driven carbonylation of bovine SOD1 (bSOD1) [42]. Under the conditions studied here, the carbonyl levels detected were higher in the presence of Cu 2+ alone, compared to its absence, but the carbonyl yield was not sensitive to the addition of higher levels of Cu 2+ over the range studied (Fig.…”

“…The same conditions also resulted in the detection of several low-intensity bands of lower mass than the parent ySOD1. The detection of these species are consistent with studies that have mapped the fragmentation pattern of bSOD1in the presence of Cu 2+ and H 2 O 2 [42] as well as glucose [53] in the case of hSOD1. These data indicate that μM concentrations of H 2 O 2 or glucose, in the presence of Cu 2+ , are sufficient to induce fragmentation of ySOD1, in addition to the mM concentrations employed previously [2,42,53].…”

“…The detection of these species are consistent with studies that have mapped the fragmentation pattern of bSOD1in the presence of Cu 2+ and H 2 O 2 [42] as well as glucose [53] in the case of hSOD1. These data indicate that μM concentrations of H 2 O 2 or glucose, in the presence of Cu 2+ , are sufficient to induce fragmentation of ySOD1, in addition to the mM concentrations employed previously [2,42,53]. The presence of AscH − (condition 4) inhibited both the dimer and fragment bands of ySOD1, an observation in contrast to the Cu 2+ -dependent AscH − /H 2 O 2 -mediated fragmentation observed with bSOD1 [42].…”

“…A previous study of bSOD1 has reported maximum carbonylation in reaction mixtures containing 30 μM Cu 2+ , 1 mM H 2 O 2 , and 5 mM AscH − [42]. However, we found that 285 μM Cu 2+ , 950 μM H 2 O 2 , and 950 μM AscH − resulted in lower levels of carbonyls throughout the entire incubation period (Table S2, Fig.…”

“…Several molecular studies have focused on elucidating the metal ion coordination, activity, mutation and structural determinants of metal-ion binding to SOD1, and the role of metal ion–catalyzed oxidation (MCO) on SOD1 aggregation/oligomerization [2,39,[41], [42], [43], [44], [45], [46], [47], [48], [49]]. The significance of the disulfide bond and the impact of oxidative modification of this species on SOD1 structure and function has been studied [35,45,50,51].…”