Only Zn2+ and Fe2+ out of 12 cations can fold ALS-linked nascent hSOD1

153-residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS, while wild-type hSOD1 aggregation is associated with SALS. So far >180 ALS-causing mutations have been identified within hSOD1, but the underlying mechanism still remains enigmatic. Mature hSOD1 is extremely stable constrained by a disulfide bridge to adopt a Greek-key β-barrel fold that houses copper and zinc cofactors. Conversely, nascent hSOD1 is unfolded and prone to aggregation, requiring Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations disrupting Zn2+-binding correlate with their capacity to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provide not only mechanistic insights, but also therapeutic prospects for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations, encompassing 8 essential for humans (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, exhibited negligible binding or induction for folding of nascent hSOD1. Cu2+ displayed extensive binding to the unfolded state but induced severe aggregation. Unexpectedly, here for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing ability. Surprisingly, Zn2+ failed to induce folding of H80S/D83S-hSOD1, while Fe2+ could. By contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-induced folding of G93A-hSOD1. Comparing with ATP-induced folded state, our results delineate that hSOD1 maturation requires: 1) intrinsic folding capacity encoded by the sequence; 2) specific Zn2+-coordination; 3) disulfide formation and Cu-load catalyzed by hCCS. This study decodes a previously-unknown interplay of cations in controlling the initial folding of hSOD1, thus not only underscoring the critical role of Zn2+ in hSOD1-associated ALS, but also suggesting novel hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity likely relevant to other diseases and aging. ### Competing Interest Statement The authors have declared no competing interest.