Multiple Turnover Transfer of [2Fe2S] Clusters by the Iron-Sulfur Cluster Assembly Scaffold Proteins IscU and IscA

Abstract

IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold- [FeS] The biosynthesis of iron-sulfur proteins is a multistep process involving a number of specialized proteins that mediate [FeS] cluster formation and delivery to acceptor proteins (reviewed in Refs. 1-3). One of the key features of the current view of the pathway is the proposed participation of protein scaffolds that function as the initial sites for formation of transient [FeS] clusters; these molecular scaffolds may serve to guide cluster assembly, protect nascent clusters in the cellular environment, and/or assist in cluster transfer to acceptor proteins. The concept of scaffold-mediated metallocluster formation emerged from studies on the nitrogenase MoFe protein (4 -6), and initial formation of [FeS] clusters on scaffold proteins was first proposed for NifU, a protein required for the biogenesis of [FeS] clusters in the nitrogenase Fe-protein (7). Subsequent studies of [FeS] cluster formation on other proteins implicated in ironsulfur protein biogenesis, the NifU-related protein IscU/Isu (8, 9), IscA/Isa (10, 11), and SufA (12), led to the suggestion that these proteins may also function as specific scaffolds for preassembly of [FeS] clusters.In vitro studies demonstrating transfer of preformed scaffold- [FeS] complexes from NifU (13) (21, 22) show that cysteine residues presumed to be involved in binding to iron atoms of the clusters are located at the surface of the apo-forms of the scaffold proteins. The scaffold-bound [FeS] clusters are therefore likely to be at least partially exposed to solvent and accessible for transfer to acceptor proteins. Little is known about how the clusters are released, but there is some evidence for a direct transfer involving scaffold-acceptor complexes. Studies employing a modified form of human Isu containing a 57 Fe-labeled cluster showed that the label did not exchange with ferrous sulfate present in solution during cluster transfer to apo-ferredoxin (15), and studies with Escherichia coli IscA and SufA showed that the iron chelator bathophenanthroline sulfonate does not interfere with cluster transfer to apo-BioB (12). These findings are consistent with a mechanism in which transfer occurs without a significant degree of cluster disassembly and reassembly and suggest that clusters may be captured immediately upon release from the scaffold protein or may be transferred directly in a scaffoldacceptor protein complex. Evidence that complex formation can occur has been obtained for IscA-ferredoxin and SufA-BioB pairs using affinity chromatography (16,19) and for Isu-and Isa-ferredoxin pairs using chemical cross-linking (9, 11). However, the nature of the scaffold-acceptor interactions has not been characterized, and it has not been firmly established that complex formation is required for cluster t...

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

“…The inability to accomplish full activation of HydA DEFG may result from metal content heterogeneity and/or improper folding in purified HydA DEFG , either of which might result from heterologous expression in the absence of the full complement of maturation proteins. In addition, a decreased percentage of HydA DEFG activation is observed at higher HydF EG to HydA-D EFG ratios which is similar to effects observed for other systems in which scaffold proteins operate including the Isc and Nif systems [16,17].…”

The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

“…In addition, the observed reconstitution reaction is too slow to faithfully mimic the situation in vivo. This observation is similar to studies with bacterial Fe/S cluster assembly, which occurred at much slower rates than the in vivo process (18,19,53). We therefore conclude that an experimental system including only Nfs1p and Isu1p is too simple and additional components are required to better reflect the in vivo situation.…”

“…Recent elegant studies showed that E. coli IscU or Azotobacter vinelandii NifU are capable of catalyzing Fe/S cluster transfer to target proteins without the need of further assistance of a chaperone (14,18). Even multiple rounds of cluster transfer were documented.…”

ErpA, an iron–sulfur (Fe–S) protein of the A-type essential for respiratory metabolism in Escherichia coli

“…Cysteine desulfurases provide sulfur from Lcysteine (11). Scaffolds are defined as being able to bind both iron and sulfur so that a Fe-S cluster can form before being transferred to targeted apoproteins (12)(13)(14)(15)(16)(17)(18). The SUF system includes an atypical cytosolic ABC-type transporter that functions with the cysteine desulfurase for increasing the production of sulfur (19,20).…”