**5. Oxidative stress by cysteine-oxidized SOD1**

In case that SOD1 is monomerized through conformational destabilization mediated by Cys111 oxidation, Cu coordinated at the ectopic binding site can be redox-active. To see whether SOD1HAC causes an aberrant redox reaction, we measured thiol oxidase activity, a Cu-dependent activity that is reported in human SOD1 (Winterbourn et al., 2002). We found that mutant or Cys111-oxidized wild type SOD1 developed the thiol oxidase activity when it was loaded with Cu, and that the activity was decreased by C111S substitution or intermolecular crosslinking of Cys111. Because SOD1 modified at Cys111 possesses the thiol oxidase activity, it is unlikely that Cys111 itself is the direct binding site for Cu. These results indicate that cysteine-oxidized SOD1 may exert the potentially toxic pro-oxidant activity through ectopic binding of Cu to SOD1HAC at a site within the dimer interface, which becomes exposed upon the dissociation of SOD1. The thiol oxidase activity of mutant SOD1 can promote oxidative stress because of the exhaustion of glutathione, the major free thiol and antioxidant. The activity may also oxidize cysteine residues of other proteins, deteriorating various cell functions (Fig. 1).

Fig. 1. Proposed model of mutant SOD1 toxicity. Modification of Cys111 leads to dissociation of SOD1 dimers into monomers. Cu, either resulting from rearrangement of the active site or from an external source, becomes ectopically bound to the former dimer interface surface, where it can now catalyze thiol oxidase activity (Kishigami et al., 2010).

nitrosoglutathione or hydrogen peroxide in familial ALS-linked mutant SOD1, which is the first step for the substantial monomerization of the protein and increase of the Cu affinity

In case that SOD1 is monomerized through conformational destabilization mediated by Cys111 oxidation, Cu coordinated at the ectopic binding site can be redox-active. To see whether SOD1HAC causes an aberrant redox reaction, we measured thiol oxidase activity, a Cu-dependent activity that is reported in human SOD1 (Winterbourn et al., 2002). We found that mutant or Cys111-oxidized wild type SOD1 developed the thiol oxidase activity when it was loaded with Cu, and that the activity was decreased by C111S substitution or intermolecular crosslinking of Cys111. Because SOD1 modified at Cys111 possesses the thiol oxidase activity, it is unlikely that Cys111 itself is the direct binding site for Cu. These results indicate that cysteine-oxidized SOD1 may exert the potentially toxic pro-oxidant activity through ectopic binding of Cu to SOD1HAC at a site within the dimer interface, which becomes exposed upon the dissociation of SOD1. The thiol oxidase activity of mutant SOD1 can promote oxidative stress because of the exhaustion of glutathione, the major free thiol and antioxidant. The activity may also oxidize cysteine residues of other proteins,

Fig. 1. Proposed model of mutant SOD1 toxicity. Modification of Cys111 leads to

dissociation of SOD1 dimers into monomers. Cu, either resulting from rearrangement of the active site or from an external source, becomes ectopically bound to the former dimer interface surface, where it can now catalyze thiol oxidase activity (Kishigami et al., 2010).

probably by exposing a Cu-accessible interface of the dimer.

**5. Oxidative stress by cysteine-oxidized SOD1** 

deteriorating various cell functions (Fig. 1).
