Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for similar to 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) h as altered redox activities and is toxic to motor neurons in vitro. Using b ovine SOD, we studied the effects of hydrogen peroxide (H2O2) on Cu,E SOD a nd Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc bind ing activity six times faster than superoxide dismutase activity, whereas i nactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xa nthine. Although urate, xanthine, and ascorbate can protect superoxide dism utase activity of Cu,Zn SOD from inactivation, they were not effective at p rotecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiar y structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydr ogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible. (C) 2 001 Academic Press.