The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, 2Fe-2S clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into 2Fe-2S apoproteins; third, 4Fe-4S clusters are formed involving the ISC proteins ISCA1–ISCA2–IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial 4Fe-4S aconitase without artificial reductants and verified the 2Fe-2S-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1–ISCA2–IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive 2Fe-2S cluster fusion on ISCA1–ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for 2Fe-2S cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factorassisted 4Fe-4S protein assembly, 2Fe-2S cluster transfer from GLRX5 to 2Fe-2S apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of 2Fe-2S and 4Fe-4S protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial 4Fe-4S cluster synthesis, trafficking, and apoprotein insertion.