Pro-oxidant copper-binding mode of the Apo form of ALS-linked SOD1 mutant H43R denatured at physiological temperature.

The mutation of Cu,Zn-superoxide dismutase (SOD1), a major antioxidant enzyme, is associated with amyotrophic lateral sclerosis (ALS). In a previous study, we showed that the metal-depleted apo form of an ALS-linked mutant, H43R, undergoes denaturation at physiological temperature (37 °C) in 90 min and acquires pro-oxidant activity in the presence of Cu(2+) and H2O2. In this study, we have examined the Cu(2+)-binding mode of denatured apo-H43R by circular dichroism (CD), fluorescent oxidation, UV Raman spectroscopy, and photooxidation. CD spectroscopy indicates that denatured apo-H43R loses native β-barrel structure and the binding of Cu(2+) to the denatured apo form induces local refolding. Fluorescent-oxidation assays in the absence and presence of Cu(2+) chelators show that denatured apo-H43R contains two Cu(2+)-binding sites with higher and lower Cu(2+) affinities and with pro-oxidant activities in the reverse order. UV Raman spectroscopy gives evidence that His residues are bound to Cu(2+) mainly through the imidazole Nτ atom at the higher-affinity site and through the Nπ atom at the lower-affinity site, sharing one His residue with each other. The Cu(2+)-binding mode of denatured apo-H43R is analogous to but different from the Cu,Zn-binding mode of the native holo form. Photooxidation experiments confirm the involvement of His residues in the pro-oxidant activity. Taken together, it is suggested that the binding of Cu(2+) induces the local refolding of denatured apo-H43R to create toxic catalytic centers that convert the enzyme from antioxidant to pro-oxidant, leading to the pathogenesis of ALS. His residues are essential for both Cu(2+)-binding and pro-oxidant activities.