Separate Fe - S scaffold and carrier functions for SufB2C2 and SufA during in vitro maturation of [2Fe-2S] Fdx

Iron-sulfur (Fe - S) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of Fe - S clusters proceeds via complex pathways involving multiple protein complexes. In the Suf Fe - S cluster biogenesis system, SufB may be a scaffold for nascent Fe - S cluster assembly whereas SufA is proposed to act as either a scaffold or an Fe - S 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 Fe - S 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 [2Fe - 2S] 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 Fe - S cluster to Fdx. Furthermore, cluster transfer from [4Fe - 4S] 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 [4Fe - 4S] SufB(2)C(2) to Fdx and from SufBC(2)D to [2Fe - 2S] SufA or Fdx. These studies indicate that SufB(2)C(2) can serve as a terminal scaffold to load the SufA Fe - S cluster carrier for in vitro maturation of [2Fe - 2S] 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. (c) 2012 Elsevier Inc. All rights reserved.