Iron–sulfur (Fe–S) clusters are essential metallocofactors that perform a multitude of biological functions1–7. Their synthesis is tightly regulated and defects in this process lead to severe diseases8–10, such as Friedreich’s ataxia, which is caused by defective expression of frataxin (FXN)11. However, the underlying mechanisms that regulate this process remain unclear. Here we show that efficient Fe–S cluster assembly requires a fine-tuned balance in the ratio of FXN and ferredoxin-2 (FDX2). Fe–S clusters are assembled on the scaffold protein ISCU2; sulfur is provided as a persulfide by NFS1, and the persulfide is cleaved into sulfide by FDX2 (refs. 12,13). FXN stimulates the whole process by accelerating the transfer of persulfide to ISCU2 (refs. 12,14,15). Using an in-vitro-reconstituted human system, we show that any deviation from a close-to-equal amount of FXN and FDX2 downregulates Fe–S cluster synthesis. Structure–function investigation reveals that this is due to competition between FXN and FDX2 and their similar affinities for the same binding site on the NFS1–ISCU2 complex, with higher levels of FXN impairing the persulfide reductase activity of FDX2 and higher levels of FDX2 slowing the FXN-accelerated transfer of persulfide to ISCU2. We also find that FDX2 directly hinders persulfide generation and transfer to ISCU2 by interacting with the persulfide-carrying mobile loop of NFS1. We further show that knocking down the expression of FDX2 increases fly lifespan in a Drosophila model of Friedreich’s ataxia. Together, this work highlights a direct regulation of Fe–S cluster biosynthesis through antagonistic binding of FXN and FDX2, and suggests that decreasing FDX2 in the context of FXN deficiency in Friedreich’s ataxia might constitute a novel therapeutic axis.
