The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to Friedreich's ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondrial function. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. In this study, a radiolabeling assay and stopped-flow kinetics are used to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. The results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. The authors propose an architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.

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