Iron-Sulfur Cluster Assembly

Abstract

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon. The product of one member of this operon, IscA, has been overexpressed, purified, and characterized. It can assemble an air-sensitive [2Fe-2S] cluster as shown by UVvisible and resonance Raman spectroscopy. The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the [2Fe-2S] holoferredoxin. These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation. This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.Iron-sulfur proteins are present in all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, stabilization of proteins, and sensing for regulatory processes (1, 2). They contain clusters of iron and sulfur atoms with variable nuclearity and complexity, the most extensively studied versions being the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. Still very little is known about the in vivo synthesis of these clusters, which is believed to require a complex machinery encoded in prokaryotes by the isc (ironsulfur cluster) operon (3). In addition to several open reading frames of unknown function, this operon contains genes for molecular chaperones (hscA and hscB), an electron transferring [2Fe-2S] ferredoxin (fdx), and three isc genes, iscS, iscU, and iscA, which have been the targets of recent investigations (3-8). IscS and IscU exhibit strong homology to proteins NifS and NifU, which are encoded by genes present in the nif operon and are responsible for nitrogenase Fe-S cluster biosynthesis in the nitrogen-fixing bacterium Azotobacter vinelandii (9, 10). In eukaryotes, mitochondrial proteins, which are highly homologous to the bacterial isc operon gene products, have been shown to be involved in Fe-S cluster assembly on the basis of biochemical and genetic evidence (11)(12)(13)(14)(15)(16)(17)(18)(19).The process of Fe-S cluster assembly into polypeptides needs to be studied at the molecular level to understand how all these proteins cooperate. The first step is the isolation and characterization of each member of the isc operon. Two gene products, IscS and IscU, which are essential for cluster assembly, have been previously purified and extensively characterized (3,5,6,8,20). The function of IscS resides in the production of sulfide from cysteine, resulting from a pyridoxal phosphate-dependent cysteine desulfurase activity (3,20). The function of IscU is less clear. However, it has been proposed that IscU provides a scaffold for assembly of Fe-S clusters that can be subsequently used for maturation of apo Fe-S proteins (5, 6). In contrast, very little is known on IscA. Here we report the first purification and characterization of IscA from Escherichia coli and show that Isc...

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

“…coli contains both the ISC and SUF systems (2)(3)(4) clusters and transfer them to apoproteins (14)(15)(16)(17)(18). Three conserved Cys residues could act as ligands for these clusters, as indicated by mutagenesis studies and by the recently obtained 3D structure of IscA (26)(27)(28)(29).…”

“…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).…”

Subcellular compartmentalization of human Nfu, an iron–sulfur cluster scaffold protein, and its ability to assemble a [4Fe–4S] cluster

“…Genetic and biochemical studies have shown that two gene products, NifS and NifU, are essential for assembly of the Fe-S clusters in nitrogenase (3)(4)(5)(6). Subsequent studies in a wide variety of prokaryotes resulted in the identification of the isc (iron-sulfur cluster assembly) operon (7,8), which encodes the translational regulator IscR (9), cysteine desulfurase IscS (7), scaffold proteins IscU (10) and IscA (11,12), chaperone proteins HscA and HscB (13), and ferredoxin Fdx. IscS is a cysteine desulfurase that generates alanine and elemental sulfur by means of the formation of a persulfide intermediate on a cysteine residue (4,14).…”

The SufE Protein and the SufBCD Complex Enhance SufS Cysteine Desulfurase Activity as Part of a Sulfur Transfer Pathway for Fe-S Cluster Assembly in Escherichia coli

“…A conserved cysteine residue on IscU accepts sulfur from IscS during Fe-S cluster building (10 -12). IscA also can form Fe-S clusters and may function as an alternate scaffold (13,14). Downstream of the isc operon is the hscB-hscA-fdx operon.…”