DNA Cleavage Mediated By Copper-Containing, Zinc-Deficient Forms Of Copper Zinc Superoxide Dismutase

It has recently been reported that the apo pools and zinc-deficient mutants of Cu,Zn superoxide dismutase (Cu,ZnSOD) exist in vivo, and exposure to either holo or mutant enzymes loaded with copper but lacking zinc is toxic to motor neuron in vitro. To date, the amyotrophic lateral sclerosis (ALS) pathogenesis that is associated with the alterations in the Cu,ZnSOD structures and functions is yet not clear. In this study, in order to get an insight into the aberrant metal ion-mediated chemistry in various forms of the Cu,ZnSOD, we examined the DNA cleavage activity mediated by each member of the Cu²⁺-containing and Zn²⁺-deficient forms of the Cu,ZnSOD (Cu_nSOD, n=1-4) in the presence of added divalent metal ions (Mg²⁺, Mn²⁺). First, it was observed that at least some local conformation of the Cu_nSOD is markedly altered relative to the Cu,ZnSOD and apo SOD. Then, we measured the relative enzyme activity and the steady state kinetic parameters of DNA cleavage, the results indicate that Mn²⁺ can confer a higher nucleolytic activity for each member of the Cu_nSOD than Mg²⁺ among the tested divalent metal ions. Third, using the Cu,ZnSOD and apo SODs as controls shows that each member of the Cu_nSOD activated by Mn²⁺ or Mg²⁺ supports DNA cleavage via a non-oxidative pathway, and the relative DNA cleavage activity is increased in order of Cu,ZnSOD < apo SOD < CuSOD < Cu2SOD Cu4SOD < Cu3SOD. Finally, it has been found that there is not a parallel relationship between the DNA cleavage activity and the main secondary structure fractions of the Cu_nSOD, implying that the secondary structures of the Cu,ZnSOD are not major factor for their nucleolytic activity. It is likely that understanding the details of the nucleolytic chemistry mediated by various forms of the Cu,ZnSOD might yield an avenue for novel therapeutics for neurodegenerative diseases.