Separate FeS scaffold and carrier functions for SufB₂C₂ and SufA during in vitro maturation of [2Fe2S] Fdx

Abstract

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.