Purification and Biophysical Characterization of a New [2Fe-2S] Ferredoxin from Azotobacter vinelandii, a Putative [Fe-S] Cluster Assembly/Repair Protein

The rsmA gene of Streptomyces coelicolor lies directly upstream of the gene encoding the group 3 sigma factor M . The RsmA protein is a putative member of the HATPase_c family of anti-sigma factors but is unusual in that it contains seven cysteine residues. Bacterial two-hybrid studies demonstrate that it interacts specifically with M , and in vitro studies of the purified proteins by native PAGE and transcription assays confirmed that they form a complex. Characterization of RsmA revealed that it binds ATP and that, as isolated, it contains significant quantities of iron and inorganic sulfide, in equal proportion, with spectroscopic properties characteristic of a [2Fe-2S] cluster-containing protein. Importantly, the interaction between RsmA and M is dependent on the presence of the iron-sulfur cluster. We propose a model in which RsmA regulates the activity of M . Loss of the cluster, in response to an as yet unidentified signal, activates M by abolishing its interaction with the anti-sigma factor. This represents a major extension of the functional diversity of iron-sulfur cluster proteins.Iron-sulfur proteins constitute a ubiquitous and hugely functionally diverse family of proteins that contain clusters of iron atoms bridged by acid-labile sulfides. Protein cysteine residue side chains normally complete the tetrahedral coordination of each iron. The capacity to delocalize electrons over both iron and sulfur ions makes iron-sulfur clusters ideally suited to roles in electron transfer pathways such as the respiratory and photosynthetic electron transfer chains and in nitrogen fixation. They are also involved in substrate binding and activation (e.g. in dehydratases such as aconitase and the radical S-adenosylmethionine family of enzymes), sulfur donation (e.g. biotin synthase), and the regulation of enzyme activity (e.g. phosphoribosylpyrophosphate amidotransferase (see Ref. 1 and references therein).In addition to these roles, there are also several well characterized examples of iron-sulfur clusters involved in regulation of gene expression in bacteria. SoxR, which contains a redoxactive [2Fe-2S] cluster (2), responds to oxidative stress and activates SoxS, another transcription factor that, in turn, activates the transcription of the target genes (3, 4). FNR is a CRP-like transcription regulator that controls the expression of genes involved in the adaptation to anoxia (5). The sensory domain (N terminus) binds a [4Fe-4S] cluster that degrades in the presence of O 2 to a [2Fe-2S] cluster via a two-step mechanism involving a [3Fe-4S] cluster intermediate (6), with a concomitant loss of DNA-binding activity (7,8). IscR, a transcription regulator involved in iron-cluster synthesis, requires the presence of a [2Fe-2S] cluster for its activity (9).Transcriptional regulation also occurs through the composition of RNA polymerase holoenzyme (10). In addition to the principle sigma factor, essential for housekeeping functions, many bacteria contain several alternative sigma factors that are required for cellular res...

Section: Resultsmentioning

confidence: 59%

RsmA Is an Anti-sigma Factor That Modulates Its Activity through a [2Fe-2S] Cluster Cofactor

Gaskell

Crack²,

Kelemen

et al. 2007

“…The function of ferredoxin encoded by the gene fdx is not known, although the gene fdx has an essential role in overall assembly of iron-sulfur clusters (28). It has also been postulated that ferredoxin is important for biogenesis or repair of iron-sulfur clusters in Azotobacter vinelandii (33) and in Saccharomyces cerevisiae (34). Nevertheless, none of these proteins seem to have the activity that can directly remove the damaged iron-sulfur clusters from proteins.…”

Section: Discussionmentioning

confidence: 99%

“…The E. coli ferredoxin [2Fe-2S] cluster has been used as an example in this study, because the gene encoding ferredoxin is highly conserved in both eukaryotic and prokaryotic organisms (27,28,33,34). To increase the amount of ferredoxin in E. coli cells, the protein was partially overproduced using the expression plasmid pTFDX, in which a His tag was fused to the C terminus of the ferredoxin polypeptide.…”

Section: Ferredoxin [2fe-2s] Clusters Are Transiently Modified When Ementioning

confidence: 99%

l-Cysteine-mediated Destabilization of Dinitrosyl Iron Complexes in Proteins

Rogers

Ding

2001

Nitric oxide is a signaling molecule in intercellular communication as well as a powerful weapon used by macrophages to kill tumor cells and pathogenic bacteria. Here, we show that when Escherichia coli cells are exposed to nitric oxide, its ferredoxin [2Fe-2S] cluster is nitrosylated, forming the dinitrosyl iron complex with a characteristic EPR signal at g av ‫؍‬ 2.04. Such formed ferredoxin dinitrosyl iron complex is efficiently repaired in E. coli cells even in the absence of new protein synthesis. However, the repair activity is completely inactivated once E. coli cells are disrupted, indicating that repairing the ferredoxin dinitrosyl iron complex requires cellular reducing equivalents. In search of such cellular factors, we find that L-cysteine can effectively eliminate the EPR signal of the ferredoxin dinitrosyl iron complex and release the ferrous iron from the complex. In contrast, N-acetyl-L-cysteine and reduced glutathione are much less effective. L-Cysteine seems to have a general function, since it can also remove the otherwise stable dinitrosyl iron complexes from proteins in the cell extracts prepared from the E. coli cells treated with nitric oxide. We propose that L-cysteine is responsible for removing the dinitrosyl iron complexes from the nitric oxide-modified proteins into which a new iron-sulfur cluster will be reassembled.

“…As a matter of fact, several recent studies have shown that Fdx is absolutely required for proper cellular function. In A. vinelandii, disruption of the corresponding gene is lethal (31). In yeast, the corresponding protein, named Yah1p, is also essential, and depletion of Yah1p resulted in accumulation of iron within mitochondria and defects in the incorporation of Fe-S clusters in both mitochondrial and cytosolic Fe-S enzymes (15).…”

Section: Discussionmentioning

confidence: 99%

Iron-Sulfur Cluster Assembly

Choudens

Mattioli

Takahashi

et al. 2001

192

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...