Cu,Zn-Superoxide Dismutase (SOD1) has two metal ions on its active site: Cu which plays catalytic role and Zn that has a structural function. Cu ion is placed at SOD1 site through action of CCS (Copper chaperone for SOD) but Zn entrance in SOD1 is still unknown. In this work, we used the native wild type SOD1 and we constructed a mutant enzyme using site directed mutation approach to study Zn complexation and its influence on enzyme activity. This mutant SOD1 resulted in a double amino acid mutation around Zn ion T135S and K136E, resulting in a metal non-stabilization coordination, and a lower superoxide dismutase activity. The CD spectra with metal titration showed a huge difference between the two entries of Zn in the native enzyme, highly different from the binding event of Cu ions in the two metal sites of SOD1, since Cu presents a simultaneous entrance in the protein. Nevertheless, the mutant T135S,K136E-SOD1 showed a metal complexation instability. This found is a plausible explanation for the observed not fully folded SOD1 in absence of zinc and its lability.