Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization

The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔG_Het) between WT and ALS-mutant SOD1 in mismatched metalation states—where one subunit is metalated and the other is not—have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by “stealing” metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers—expressed as time to reach 30% heterodimer—ranged from t_30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔG_Het = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔG_Het values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn²⁺ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn²⁺ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn²⁺.