Deep Mutational Scanning of FDX1 Identifies Key Structural Determinants of Lipoylation and Cuproptosis.

Cuproptosis is a recently described form of regulated cell death triggered by ionophore-induced copper (Cu) overload in mitochondria. It is critically dependent on ferredoxin 1 (FDX1), a mitochondrial iron-sulfur cluster containing protein that acts as an electron shuttle. FDX1 reduces ionophore-bound Cu(II) to Cu(I), thereby triggering its release, and promotes mitochondrial protein lipoylation, which is directly targeted by the released copper to drive cell death. Despite the pivotal role of FDX1 in cuproptosis, the structural determinants underlying its distinct functions remain unclear. To address this, we performed deep mutational scanning on FDX1 and find that two conserved solvent-exposed residues, D136 and D139, on alpha helix 3 are essential for both cuproptosis and lipoylation. Charge-reversal mutations at these positions abolish FDX1's ability to induce cuproptosis and support lipoylation in cells, despite retaining full enzymatic activity in vitro. Guided by structural and genomic analyses, we further identify dihydrolipoamide dehydrogenase (DLD), the E3 subunit of lipoylated complexes as an alternative FDX1 reductase both in cells and in vitro. Together, these findings establish the acidic alpha helix 3 of FDX1 as a critical interface for its upstream regulation and suggest that FDX1's roles in cuproptosis and in lipoylation are both structurally and functionally linked.