Iron-Sulfur Cluster Assembly

Abstract

The synthesis of iron-sulfur clusters inEscherichia 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 UV-visible 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. The synthesis of iron-sulfur clusters inEscherichia 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 UV-visible 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. ferredoxin maltose-binding protein nitrilotriacetic acid 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 (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1486) Google Scholar, 2Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar). 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 vivosynthesis of these clusters, which is believed to require a complex machinery encoded in prokaryotes by the isc (iron-sulfur cluster) operon (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). 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 (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 4Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar, 6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar, 7Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (225) Google Scholar, 8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (76) Google Scholar). 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 bacteriumAzotobacter vinelandii (9Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6735-6744Google Scholar, 10Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (496) Google Scholar). In eukaryotes, mitochondrial proteins, which are highly homologous to the bacterial iscoperon gene products, have been shown to be involved in Fe-S cluster assembly on the basis of biochemical and genetic evidence (11Lill R. Diekert K. Kaut A. Lange H. Pelzer W. Prohl C. Kispal G. Biol. Chem. 1999; 380: 1157-1166Crossref PubMed Scopus (124) Google Scholar, 12Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Mol. Chem. 1999; 274: 33025-33034Scopus (173) Google Scholar, 13Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (582) Google Scholar, 14Garland S.A. Hoff K.G. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (165) Google Scholar, 15Lange H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (242) Google Scholar, 16Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 17Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (154) Google Scholar, 18Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar).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 (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar, 6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar, 8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (76) Google Scholar, 20Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The function of IscS resides in the production of sulfide from cysteine, resulting from a pyridoxal phosphate-dependent cysteine desulfurase activity (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 20Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar,6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar). In contrast, very little is known on IscA. Here we report the first purification and characterization of IscA from Escherichia coli and show that IscA has a specific relationship with ferredoxin (Fdx),1 another member of the isc operon.DISCUSSIONIt has been recently shown that E. coliIscA greatly improves the efficiency of the maturation of overexpressed ferredoxins within E. coli cells (7Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (225) Google Scholar). Furthermore, in yeast, mitochondrial proteins Isa1p and Isa2p, exhibiting strong homology to IscA, were shown to be important for normal mitochondrial and cytosolic iron metabolism and to play a crucial role in the maturation of iron-sulfur proteins (17Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (154) Google Scholar, 18Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar). Although not lethal, deletion ofisa1 and isa2 results in retarded growth on nonfermentable carbon sources, accumulation of iron in mitochondria, and a drastic decrease in the activities of both mitochondrial and cytosolic Fe-S enzymes. The same phenotypes were observed with mutatedisa genes in which each of the 3 cysteines, conserved among IscA-like proteins from bacteria, fungi, plants, and mammals, has been changed to serine residues. These results have suggested that these cysteines constitute an iron binding motif and that IscA proteins participate in Fe-S cluster assembly through the binding of an intermediate Fe-S cluster that is subsequently transferred to target polypeptides.The experiments reported here indeed show that recombinant IscA isolated from E. coli is able to assemble unstable iron-sulfur centers that lose iron and sulfide during prolonged exposure to air. Although the protein did not contain any iron or sulfide after purification it is able to bind iron and sulfide under anaerobic conditions in vitro. Light absorption and resonance Raman spectroscopy showed that IscA could assemble a [2Fe-2S] cluster per protein, with the that the IscA could not bind iron and sulfur The resonance Raman of is of a The observed of the and of the cluster at and all within the of observed for clusters with cysteine and S. Raman of and Scholar, L. in Scholar, J. J. J. M. Biochemistry. PubMed Scopus Google Scholar, J. M. Biochem. PubMed Scopus Google Scholar). However, the IscA 3 cysteines that are all conserved within the IscA and are thus to be to the by 3 cysteines and one is not with the resonance Raman and be the of the of the experiments are to the iron in IscA. the observed of the Fe-S cluster to IscA is with IscA as a for synthesis of intermediate Fe-S which be transferred to polypeptides as previously suggested is by the that a complex with ferredoxin, the product of gene present in the isc operon, as shown by the of an IscA to from E. coli the operon. suggest that the complex has a functional formation of the complex on the of an Fe-S cluster in IscA the of the of the cluster to the can be by transfer of iron and sulfide from to the The that and the of the transfer not form a complex the that one of IscA to the formation of several of The role of IscA in formation of is because is with iron and sulfide in the of IscA. experiments are required to understand this cluster transfer at the molecular and In the transfer the or and of the into the target is an that to be The that could be in the of IscA an of clusters be in of the first are a of other that which iron and sulfur to and is cluster transfer from IscA to by other these results we that the specific of the Fe-S cluster to could be a step in the biosynthesis of iron-sulfur proteins in the a of several recent have shown that is required for cellular In A. of the gene is J. Dean D.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In yeast, the protein, is and of in accumulation of iron within and in the of Fe-S clusters in both mitochondrial and cytosolic Fe-S H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (242) Google Scholar). The same observed with cells in the essential J. S. Pain D. Dancis A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These suggest that the complex process of biosynthesis of Fe-S clusters on a functional which in an essential electron transfer is functional or in with is an the function of IscA is indeed to and as suggested by the present IscA an important role in for maturation of 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 (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1486) Google Scholar, 2Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar). 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 vivosynthesis of these clusters, which is believed to require a complex machinery encoded in prokaryotes by the isc (iron-sulfur cluster) operon (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). 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 (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 4Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar, 6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar, 7Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (225) Google Scholar, 8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (76) Google Scholar). 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 bacteriumAzotobacter vinelandii (9Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6735-6744Google Scholar, 10Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (496) Google Scholar). In eukaryotes, mitochondrial proteins, which are highly homologous to the bacterial iscoperon gene products, have been shown to be involved in Fe-S cluster assembly on the basis of biochemical and genetic evidence (11Lill R. Diekert K. Kaut A. Lange H. Pelzer W. Prohl C. Kispal G. Biol. Chem. 1999; 380: 1157-1166Crossref PubMed Scopus (124) Google Scholar, 12Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Mol. Chem. 1999; 274: 33025-33034Scopus (173) Google Scholar, 13Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (582) Google Scholar, 14Garland S.A. Hoff K.G. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (165) Google Scholar, 15Lange H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (242) Google Scholar, 16Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 17Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (154) Google Scholar, 18Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar). 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 (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar, 6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar, 8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (76) Google Scholar, 20Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The function of IscS resides in the production of sulfide from cysteine, resulting from a pyridoxal phosphate-dependent cysteine desulfurase activity (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, 20Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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 (5Agar J.N. Krebs C. Frazzon J. Huynh B.H Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (378) Google Scholar,6Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar). In contrast, very little is known on IscA. Here we report the first purification and characterization of IscA from Escherichia coli and show that IscA has a specific relationship with ferredoxin (Fdx),1 another member of the isc operon. has been recently shown that E. coliIscA greatly improves the efficiency of the maturation of overexpressed ferredoxins within E. coli cells (7Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (225) Google Scholar). Furthermore, in yeast, mitochondrial proteins Isa1p and Isa2p, exhibiting strong homology to IscA, were shown to be important for normal mitochondrial and cytosolic iron metabolism and to play a crucial role in the maturation of iron-sulfur proteins (17Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (154) Google Scholar, 18Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar). Although not lethal, deletion ofisa1 and isa2 results in retarded growth on nonfermentable carbon sources, accumulation of iron in mitochondria, and a drastic decrease in the activities of both mitochondrial and cytosolic Fe-S enzymes. The same phenotypes were observed with mutatedisa genes in which each of the 3 cysteines, conserved among IscA-like proteins from bacteria, fungi, plants, and mammals, has been changed to serine residues. These results have suggested that these cysteines constitute an iron binding motif and that IscA proteins participate in Fe-S cluster assembly through the binding of an intermediate Fe-S cluster that is subsequently transferred to target polypeptides.The experiments reported here indeed show that recombinant IscA isolated from E. coli is able to assemble unstable iron-sulfur centers that lose iron and sulfide during prolonged exposure to air. Although the protein did not contain any iron or sulfide after purification it is able to bind iron and sulfide under anaerobic conditions in vitro. Light absorption and resonance Raman spectroscopy showed that IscA could assemble a [2Fe-2S] cluster per protein, with the that the IscA could not bind iron and sulfur The resonance Raman of is of a The observed of the and of the cluster at and all within the of observed for clusters with cysteine and S. Raman of and Scholar, L. in Scholar, J. J. J. M. Biochemistry. PubMed Scopus Google Scholar, J. M. Biochem. PubMed Scopus Google Scholar). However, the IscA 3 cysteines that are all conserved within the IscA and are thus to be to the by 3 cysteines and one is not with the resonance Raman and be the of the of the experiments are to the iron in IscA. the observed of the Fe-S cluster to IscA is with IscA as a for synthesis of intermediate Fe-S which be transferred to polypeptides as previously suggested is by the that a complex with ferredoxin, the product of gene present in the isc operon, as shown by the of an IscA to from E. coli the operon. suggest that the complex has a functional formation of the complex on the of an Fe-S cluster in IscA the of the of the cluster to the can be by transfer of iron and sulfide from to the The that and the of the transfer not form a complex the that one of IscA to the formation of several of The role of IscA in formation of is because is with iron and sulfide in the of IscA. experiments are required to understand this cluster transfer at the molecular and In the transfer the or and of the into the target is an that to be The that could be in the of IscA an of clusters be in of the first are a of other that which iron and sulfur to and is cluster transfer from IscA to by other these results we that the specific of the Fe-S cluster to could be a step in the biosynthesis of iron-sulfur proteins in the a of several recent have shown that is required for cellular In A. of the gene is J. Dean D.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In yeast, the protein, is and of in accumulation of iron within and in the of Fe-S clusters in both mitochondrial and cytosolic Fe-S H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (242) Google Scholar). The same observed with cells in the essential J. S. Pain D. Dancis A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These suggest that the complex process of biosynthesis of Fe-S clusters on a functional which in an essential electron transfer is functional or in with is an the function of IscA is indeed to and as suggested by the present IscA an important role in for maturation of Fe-S proteins. It has been recently shown that E. coliIscA greatly improves the efficiency of the maturation of overexpressed ferredoxins within E. coli cells (7Takahashi Y. Nakamura M. J. Biochem. 1999; 126: 917-926Crossref PubMed Scopus (225) Google Scholar). Furthermore, in yeast, mitochondrial proteins Isa1p and Isa2p, exhibiting strong homology to IscA, were shown to be important for normal mitochondrial and cytosolic iron metabolism and to play a crucial role in the maturation of iron-sulfur proteins (17Jensen L.T. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (154) Google Scholar, 18Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar). Although not lethal, deletion ofisa1 and isa2 results in retarded growth on nonfermentable carbon sources, accumulation of iron in mitochondria, and a drastic decrease in the activities of both mitochondrial and cytosolic Fe-S enzymes. The same phenotypes were observed with mutatedisa genes in which each of the 3 cysteines, conserved among IscA-like proteins from bacteria, fungi, plants, and mammals, has been changed to serine residues. These results have suggested that these cysteines constitute an iron binding motif and that IscA proteins participate in Fe-S cluster assembly through the binding of an intermediate Fe-S cluster that is subsequently transferred to target The experiments reported here indeed show that recombinant IscA isolated from E. coli is able to assemble unstable iron-sulfur centers that lose iron and sulfide during prolonged exposure to air. Although the protein did not contain any iron or sulfide after purification it is able to bind iron and sulfide under anaerobic conditions in vitro. Light absorption and resonance Raman spectroscopy showed that IscA could assemble a [2Fe-2S] cluster per protein, with the that the IscA could not bind iron and sulfur The resonance Raman of is of a The observed of the and of the cluster at and all within the of observed for clusters with cysteine and S. Raman of and Scholar, L. in Scholar, J. J. J. M. Biochemistry. PubMed Scopus Google Scholar, J. M. Biochem. PubMed Scopus Google Scholar). However, the IscA 3 cysteines that are all conserved within the IscA and are thus to be to the by 3 cysteines and one is not with the resonance Raman and be the of the of the experiments are to the iron in IscA. the observed of the Fe-S cluster to IscA is with IscA as a for synthesis of intermediate Fe-S which be transferred to polypeptides as previously suggested This is by the that a complex with ferredoxin, the product of gene present in the isc operon, as shown by the of an IscA to from E. coli the operon. suggest that the complex has a functional formation of the complex on the of an Fe-S cluster in IscA the of the of the cluster to the can be by transfer of iron and sulfide from to the The that and the of the transfer not form a complex the that one of IscA to the formation of several of The role of IscA in formation of is because is with iron and sulfide in the of IscA. experiments are required to understand this cluster transfer at the molecular and In the transfer the or and of the into the target is an that to be The that could be in the of IscA an of clusters be in of the first are a of other that which iron and sulfur to and is cluster transfer from IscA to by other these results we that the specific of the Fe-S cluster to could be a step in the biosynthesis of iron-sulfur proteins in the a of several recent have shown that is required for cellular In A. of the gene is J. Dean D.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In yeast, the protein, is and of in accumulation of iron within and in the of Fe-S clusters in both mitochondrial and cytosolic Fe-S H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (242) Google Scholar). The same observed with cells in the essential J. S. Pain D. Dancis A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These suggest that the complex process of biosynthesis of Fe-S clusters on a functional which in an essential electron transfer is functional or in with is an the function of IscA is indeed to and as suggested by the present IscA an important role in for maturation of Fe-S proteins.