Superoxide dismutase 1 (SOD1), which catalyzes the conversion of superoxide anion to molecular oxygen and hydrogen peroxide, undergoes several maturation events to become an active homodimeric enzyme. Because immature forms of SOD1 have been linked to neurodegenerative disease in humans, Banci et al. set out to understand the mechanisms underlying maturation, particularly events involving the copper chaperone for SOD1 (CCS). Using ESI-MS, the authors showed that, in vitro, full-length CCS loaded with Cu yields mature dimeric SOD1, bound to both Zn and Cu and with appropriate disulfide bonds from the monomeric Zn-loaded form of SOD1. All of the CCS domains are required for complete maturation, with domain 1 (D1) required for Cu transfer, D2 required for the SOD1-CCS interaction and D3 required for disulfide bond formation. CCS showed reduced ability to transfer Cu to disulfide-oxidized SOD1, indicating that maturation is a stepwise process in which Cu loading precedes disulfide bond formation. NMR of wild-type CCS and CCS bearing mutations in the Cys-X-Cys motif of the D3 domain showed that in mutants with singly or doubly mutated cysteine residues, copper transfer occurred but disulfide formation did not, consistent with the process suggested by ESI-MS. Taken together, these data support a stepwise model of SOD1 maturation, involving Zn acquisition, CCS-SOD1 heterodimer formation, Cu transfer from CCS to SOD1, disulfide bond transfer from CCS to SOD1 and dimerization of SOD1.
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Donner, A. Steps to SOD1. Nat Chem Biol 8, 808 (2012). https://doi.org/10.1038/nchembio.1075