Iron–sulfur (FeS) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of FeS clusters proceeds via complex pathways involving multiple protein complexes. In the Suf FeS cluster biogenesis system, SufB may be a scaffold for nascent FeS cluster assembly whereas SufA is proposed to act as either a scaffold or an FeS cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB 2 C 2 and SufBC 2 D and the specific functions of these complexes in FeS cluster assembly are not clear. Here we compare the ability of the SufB 2 C 2 and SufBC 2 D complexes as well as SufA to promote in vitro maturation of the [2Fe2S] ferredoxin (Fdx). We found that SufB 2 C 2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed FeS cluster to Fdx. Furthermore, cluster transfer from [4Fe4S] SufB 2 C 2 or SufBC 2 D to Fdx will proceed through a SufA intermediate to Fdx if SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe4S] SufB 2 C 2 to Fdx and from SufBC 2 D to [2Fe2S] SufA or Fdx. These studies indicate that SufB 2 C 2 can serve as a terminal scaffold to load the SufA FeS cluster carrier for in vitro maturation of [2Fe2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction.