This study shows mitochondrial [2Fe–2S] iron–sulfur cluster synthesis depends on a finely balanced ratio of frataxin (FXN) and ferredoxin-2 (FDX2), which compete for the same site on the NFS1–ISCU2 assembly complex, and that lowering FDX2 extends lifespan in a Friedreich's ataxia fly model. Flow Induced Dispersion Analysis (FIDA), which measures the Taylor dispersion profile and hydrodynamic radius of proteins under laminar flow, was used as one of several biophysical methods to characterize this competitive binding and confirm that FXN and FDX2 do not bind the complex simultaneously. It is an example of using FIDA to probe competitive protein–protein binding in solution.