Structural Instability and Cu-Dependent Pro-Oxidant Activity Acquired by the Apo Form of Mutant SOD1 Associated with Amyotrophic Lateral Sclerosis

摘要

Cu,Zn-superoxide dismutase (SOD1) is a cytosolicnantioxidant enzyme, and its mutation has been implicated innamyotrophic lateral sclerosis (ALS), a disease causing a progressivenloss ofmotor neurons. Although the pathogenicmechanismof ALSnremains unclear, it is hypothesized that some toxic propertiesnacquired by mutant SOD1 play a role in the development ofnALS.We have examined the structural and catalytic properties of annALS-linked mutant of human SOD1, His43Arg (H43R), which isncharacterized by rapid disease progression. As revealed by circularndichroism spectroscopy, H43R assumes a stable β-barrel structure in the Cu2þ,Zn2þ-bound holo form, but its metal-depleted aponform is highly unstable and readily unfolds or misfolds into an irregular structure at physiological temperature. The conformationalnchange occurs as a two-state transition froma nativelike apo formto a denatured apo formwith a half-life of∼0.5 h. At the same timenas the denaturation, the apo form of H43R acquires pro-oxidant potential, which is fully expressed in the presence of Cu2þ andnH2O2, as monitored with a fluorogenic probe for detecting pro-oxidant activity. Comparison of du0001d absorption bands suggests thatnthe Cu2þ bindingmode of the denatured apo formis different fromthat of the native holo form. The denatured apo formofH43R isnlikely to provide non-native Cu2þ binding sites where the Cu2þ ion is activated to catalyze harmful oxidation reactions. This studynraises the possibility that the structural instability and the resultant Cu-dependent pro-oxidant activity of the apo form of mutantnSOD1 may be one of the pathogenic mechanisms of ALS.