Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant

Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of famili al amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably re tains the gain-of-function activity that leads to disease. We demonstrate t hat CU2+ does not bind to the copper-specific catalytic site of H46R CuZnSO D and that CU2+ competes with other metals for the zinc binding site. Most importantly, CU2+ was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR s pectroscopy, and chemical derivatization. Cysteine ill was pinpointed as th e position of the reactive ligand by tryptic digestion of the modified prot ein and by mutational analysis. This solvent-exposed residue may play a rol e in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine ill residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal i nsertion during biosynthesis of wild-type CuZnSOD in vivo.