Iron-Sulfur Cluster Biosynthesis
2006; Elsevier BV; Volume: 281; Issue: 24 Linguagem: Inglês
10.1074/jbc.m513569200
ISSN1083-351X
AutoresGunhild Layer, Sandrine Ollagnier de Choudens, Yiannis Sanakis, Marc Fontecave,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoThe biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use l-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Nevertheless, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported so far. In this paper we demonstrate for the first time a specific interaction between CyaY and IscS, a cysteine desulfurase participating in iron-sulfur cluster assembly. Analysis of the iron-loaded CyaY protein demonstrated a strong binding of Fe3+ and a weak binding of Fe2+ by CyaY. Biochemical analysis showed that the CyaY-Fe3+ protein corresponds to a mixture of monomer, intermediate forms (dimer-pentamers), and oligomers with the intermediate one corresponding to the only stable and soluble iron-containing form of CyaY. Using spectroscopic methods, this form was further demonstrated to be functional in vitro as an iron donor during [Fe-S] cluster assembly on the scaffold protein IscU in the presence of IscS and cysteine. All of these results point toward a link between CyaY and [Fe-S] cluster biosynthesis, and a possible mechanism for the process is discussed. The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use l-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Nevertheless, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported so far. In this paper we demonstrate for the first time a specific interaction between CyaY and IscS, a cysteine desulfurase participating in iron-sulfur cluster assembly. Analysis of the iron-loaded CyaY protein demonstrated a strong binding of Fe3+ and a weak binding of Fe2+ by CyaY. Biochemical analysis showed that the CyaY-Fe3+ protein corresponds to a mixture of monomer, intermediate forms (dimer-pentamers), and oligomers with the intermediate one corresponding to the only stable and soluble iron-containing form of CyaY. Using spectroscopic methods, this form was further demonstrated to be functional in vitro as an iron donor during [Fe-S] cluster assembly on the scaffold protein IscU in the presence of IscS and cysteine. All of these results point toward a link between CyaY and [Fe-S] cluster biosynthesis, and a possible mechanism for the process is discussed. Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. They are involved in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity reactions (1Beinert H. Holm R.H. Munck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1531) Google Scholar). Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. In Escherichia coli three different types of [Fe-S] cluster biosynthesis systems have been identified so far, namely the iron-sulfur cluster, sulfur mobilization, and cysteine sulfinate desulfinase systems (2Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1110) Google Scholar, 3Barras F. Loiseau L. Py B. Adv. Microb. Physiol. 2005; 50: 41-101Crossref PubMed Scopus (88) Google Scholar). These different machineries have in common the involvement of a cysteine desulfurase that allows utilization of cysteine as source of sulfur atoms (4Mihara H. Esaki N. Appl. Microbiol. Biotechnol. 2002; 60: 12-23Crossref PubMed Scopus (225) Google Scholar). The ISC and SUF systems, furthermore, both contain scaffold proteins that provide an intermediate assembly site for [Fe-S] clusters or [Fe-S] cluster precursors (5Ollagnier-de-Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 6Ollagnier-de Choudens S. Nachin L. Sanakis Y. Loiseau L. Barras F. Fontecave M. J. Biol. Chem. 2003; 278: 17993-18001Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Krebs C. Agar J.N. Smith A.D. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. Biochemistry. 2001; 40: 14069-14080Crossref PubMed Scopus (210) Google Scholar, 8Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar). Important questions related to [Fe-S] cluster biosynthesis include: (i) the molecular mechanism by which iron and sulfide are assembled on the scaffold protein; (ii) how accessory proteins (chaperones in particular) participate in the process; and (iii) how the cluster is transferred from the scaffold to an apo target protein. Another essential question is the identity of iron and sulfur donors for the formation of [Fe-S] clusters. Whereas l-cysteine has been identified as the ultimate source of sulfur, the question "Where does the iron come from?" still remains unanswered. Whereas some preliminary answers have been provided to most of the above issues, very little is known regarding the last question. It is simply assumed that, because of its toxicity, iron has to be stored and transported by proteins from which it can be mobilized for assembly of iron sites. In bacteria, IscA and YggX, which were shown to be able to bind iron and to be, to some extent, involved in [Fe-S] metabolism, are potential candidates requiring further investigations (9Ding B. Smith E.S. Ding H. Biochem. J. 2005; 389: 797-802Crossref PubMed Scopus (48) Google Scholar, 10Gralnick J.A. Downs D.M. J. Biol. Chem. 2003; 278: 20708-20715Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Gralnick J. Downs D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8030-8035Crossref PubMed Scopus (40) Google Scholar, 12Skovran E. Lauhon C.T. Downs D.M. J Bacteriol. 2004; 186: 7626-7634Crossref PubMed Scopus (52) Google Scholar). However, this is controversial because IscA was proposed to be an [Fe-S] scaffold protein (5Ollagnier-de-Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 7Krebs C. Agar J.N. Smith A.D. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. Biochemistry. 2001; 40: 14069-14080Crossref PubMed Scopus (210) Google Scholar), whereas a recent report could not establish iron binding to YggX (13Osborne M.J. Siddiqui N. Landgraf D. Pomposiello P.J. Gehring K. Protein Sci. 2005; 14: 1673-1678Crossref PubMed Scopus (13) Google Scholar). More information concerning a putative iron donor protein is available in eukaryotic systems. In eukaryotes, [Fe-S] cluster assembly requires two biosynthetic protein machineries. One is localized in the mitochondria and functions in the assembly of all cellular [Fe-S] proteins, whereas the other one is cytosolic, specifically involved in the maturation of cytosolic and nuclear [Fe-S] cluster proteins (14Lill R. Fekete Z. Sipos K. Rotte C. IUBMB Life. 2005; 57: 701-703Crossref PubMed Scopus (14) Google Scholar). Several lines of evidence support the proposed role of the frataxin protein as an iron donor for assembly of [Fe-S] clusters in eukaryotes: (i) low expression of the frataxin gene in humans results in Friedreich's ataxia, a neurodegenerative disease characterized by reduced levels of mitochondrial [Fe-S] enzyme activities (15Rotig A. de Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genet. 1997; 17: 215-217Crossref PubMed Scopus (884) Google Scholar); (ii) the yeast frataxin (Yfh1p) is involved in the regulation of iron homeostasis (16Pandolfo M. Mitochondrion. 2002; 2: 87-93Crossref PubMed Scopus (28) Google Scholar, 17Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (831) Google Scholar) and is required for maturation of [Fe-S] cluster containing proteins (18Muhlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (287) Google Scholar), and its inactivation results in iron accumulation in mitochondria and is correlated to oxidative stress (18Muhlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (287) Google Scholar, 19Lange H. Muhlenhoff U. Denzel M. Kispal G. Lill R. J. Biol. 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Chem. 2003; 278: 31340-31351Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar); (iv) Yfh1p was shown to directly interact, within the cell, with proteins of the [Fe-S] cluster assembly machinery, for example the scaffold ISU1p and the cysteine desulfurase Nfs1p (18Muhlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (287) Google Scholar, 25Ramazzotti A. Vanmansart V. Foury F. FEBS Lett. 2004; 557: 215-220Crossref PubMed Scopus (112) Google Scholar, 26Gerber J. Muhlenhoff U. Lill R. EMBO Rep. 2003; 4: 906-911Crossref PubMed Scopus (314) Google Scholar), as well as with aconitase, a mitochondrial [Fe-S] enzyme (27Bulteau A.L. O'Neill H.A. Kennedy M.C. Ikeda-Saito M. Isaya G. Szweda L.I. Science. 2004; 305: 242-245Crossref PubMed Scopus (319) Google Scholar); and (v) its ability to deliver iron to ISU-type proteins and aconitase was demonstrated in vitro (27Bulteau A.L. O'Neill H.A. Kennedy M.C. Ikeda-Saito M. Isaya G. Szweda L.I. Science. 2004; 305: 242-245Crossref PubMed Scopus (319) Google Scholar, 28Yoon T. Cowan J.A. J. Biol. Chem. 2004; 279: 25943-25946Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 29Yoon T. Cowan J.A. J. Am. Chem. Soc. 2003; 125: 6078-6084Crossref PubMed Scopus (330) Google Scholar). On the other hand, a YFH1 deletion is not lethal in Saccharomyces cerevisiae, indicating that another iron donor might exist in mitochondria (17Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (831) Google Scholar, 30Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (348) Google Scholar). The bacterial ortholog of frataxin is the CyaY protein, which displays ∼25% sequence identity to human and yeast frataxin. The situation in bacteria appears to be more complex. Indeed, a knock-out of the cyaY gene alone confers no phenotype: no defect in iron content, no sensitivity to oxidative stress, and no auxotrophy (31Li D.S. Ohshima K. Jiralerspong S. Bojanowski M.W. Pandolfo M. FEBS Lett. 1999; 456: 13-16Crossref PubMed Scopus (69) Google Scholar). It is possible that the specific growth conditions wherein the cyaY gene expression plays a prominent role have not been defined yet. Interestingly, during the preparation of this manuscript, it was reported that a double knock-out in cyaY and yggX in Salmonella enterica resulted in defects in [Fe-S] cluster metabolism (32Vivas E. Skovran E. Downs D.M. J. Bacteriol. 2006; 188: 1175-1179Crossref PubMed Scopus (48) Google Scholar). Structural analysis of E. coli CyaY (x-ray structure at 1.4 Å resolution and NMR structure) and yeast and human frataxin revealed a common protein fold for all frataxin homologs (33Cho S.J. Lee M.G. Yang J.K. Lee J.Y. Song H.K. Suh S.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8932-8937Crossref PubMed Scopus (104) Google Scholar, 34Nair M. Adinolfi S. Pastore C. Kelly G. Temussi P. Pastore A. Structure (Camb.). 2004; 12: 2037-2048Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 35Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar, 36Lee M.G. Cho S.J. Yang J.K. Song H.K. Suh S.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 920-921Crossref PubMed Scopus (7) Google Scholar). NMR spectroscopic studies on CyaY showed that is able to bind iron in both Fe2+ and Fe3+ oxidation states through conserved Asp and Glu residues in the same region of the protein as in the case of human frataxin (34Nair M. Adinolfi S. Pastore C. Kelly G. Temussi P. Pastore A. Structure (Camb.). 2004; 12: 2037-2048Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It was shown that CyaY can accommodate 2 Fe2+/protein when incubated with Fe2+ anaerobically. Under these conditions the protein was shown to be a tetramer in solution. Upon ferrous iron oxidation CyaY is able to bind an initial group of 6 Fe3+/protein and up to 26 Fe3+/protein using a large excess of iron (35Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar, 37Bou-Abdallah F. Lewin A.C. Le Brun N.E. Moore G.R. Chasteen N.D. J. Biol. Chem. 2002; 277: 37064-37069Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The iron-loaded form of CyaY is present in solution as a mixture of monomer and oligomers, with only the latter containing iron (35Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar). Furthermore, iron-containing oligomers are resistant to iron chelation by EDTA or citrate, showing strong affinity of CyaY oligomers for Fe3+ (35Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar). So far, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported. In this work we demonstrate for the first time a specific interaction between E. coli CyaY and an ISC protein (IscS), which provides a link between CyaY and [Fe-S] cluster biosynthesis. Furthermore, we show a functional role for the iron-containing CyaY oligomers during [Fe-S] cluster assembly on the scaffold protein IscU in vitro. Materials—All of the chemicals were of reagent grade and obtained from Sigma-Aldrich or Fluka unless otherwise stated. 57Fe2O3 was converted into ferric chloride by dissolving it in hot concentrated (35%) hydrochloric acid of analytical grade (Carlo Erba) and repeatedly concentrated in water. Plasmid pET-22b-CyaY for production of recombinant E. coli CyaY was kindly provided by Prof. Se Won Suh (33Cho S.J. Lee M.G. Yang J.K. Lee J.Y. Song H.K. Suh S.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8932-8937Crossref PubMed Scopus (104) Google Scholar). Plasmid pTrcIscU for production of non-His-tagged recombinant E. coli IscU was kindly provided by Prof. Larry E. Vickery (38Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar), and plasmids pQE-IscU, pQE-IscS and pRKISC for production of E. coli IscU, IscS, and all proteins from the isc operon, respectively, were provided by Prof. Y. Takahashi (39Tokumoto U. Nomura S. Minami Y. Mihara H. Kato S. Kurihara T. Esaki N. Kanazawa H. Matsubara H. Takahashi Y. J. Biochem. (Tokyo). 2002; 131: 713-719Crossref PubMed Scopus (97) Google Scholar, 40Tokumoto U. Takahashi Y. J. Biochem. (Tokyo). 2001; 130: 63-71Crossref PubMed Scopus (215) Google Scholar, 41Nakamura M. Saeki K. Takahashi Y. J. Biochem. (Tokyo). 1999; 126: 10-18Crossref PubMed Scopus (169) Google Scholar). Plasmid pSL219 and pEB586 for production of non-His-tagged IscS and IscSC328S proteins were kindly provided by S. Leimkühler and E. Bouveret, respectively (42Gully D. Moinier D. Loiseau L. Bouveret E. FEBS Lett. 2003; 548: 90-96Crossref PubMed Scopus (95) Google Scholar). Expression and Purification of Proteins—Recombinant E. coli CyaY containing a C-terminal His6 tag was expressed and purified as previously described with some minor modifications (36Lee M.G. Cho S.J. Yang J.K. Song H.K. Suh S.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 920-921Crossref PubMed Scopus (7) Google Scholar). Following metal-chelate chromatography on nickel-nitrilotriacetic acid resin (Amersham Biosciences) a gel filtration on a HiLoad 16/60 Superdex 75 prep-grade column (Amersham Biosciences) was performed (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 3 mm DTT). 4The abbreviation used is: DTT, dithiothreitol. After these two purification steps the protein was judged 99% pure by SDS-PAGE. The protein was concentrated by ultrafiltration using an YM10 membrane (Amicon) and was stored at –80 °C. Recombinant E. coli IscU (non-His-tagged), E. coli His-tagged IscU, E. coli His-tagged IscS (both N-terminal His-tagged), E. coli IscS (non-His-tagged), and E. coli IscSC328S (non-His-tagged) were expressed and purified as previously described (38Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 39Tokumoto U. Nomura S. Minami Y. Mihara H. Kato S. Kurihara T. Esaki N. Kanazawa H. Matsubara H. Takahashi Y. J. Biochem. (Tokyo). 2002; 131: 713-719Crossref PubMed Scopus (97) Google Scholar, 40Tokumoto U. Takahashi Y. J. Biochem. (Tokyo). 2001; 130: 63-71Crossref PubMed Scopus (215) Google Scholar, 42Gully D. Moinier D. Loiseau L. Bouveret E. FEBS Lett. 2003; 548: 90-96Crossref PubMed Scopus (95) Google Scholar, 43Leimkuhler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). For non-His-tagged IscU an additional gel filtration step on a HiLoad 16/60 Superdex 75 prep-grade column (Amersham Biosciences) in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 3 mm DTT was added to further purify the protein. Concentrated protein solutions were stored at –80 °C. Preparation of Cell-free Extracts—E. coli BL21(DE3)-RIL harboring plasmid pRKISC, E. coli BL21(DE3) harboring plasmid pTrcIscU and E. coli BL21(DE3) harboring plasmid pET-22b-CyaY were grown as previously described (36Lee M.G. Cho S.J. Yang J.K. Song H.K. Suh S.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 920-921Crossref PubMed Scopus (7) Google Scholar, 38Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 44Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (228) Google Scholar). Overexpression of proteins of the isc operon, of non-His-tagged IscU and of His-tagged CyaY was induced by addition of 0.5 mm isopropyl β-d-thiogalactopyranoside. After collecting the cells by centrifugation, the bacterial pellets were resuspended in buffer (100 mm Tris-HCl, pH 7.5, 50 mm NaCl), and cells were broken by sonication (10 × 10 s; amplitude 25%). The soluble protein fraction was obtained by centrifugation at 45000 rpm (Ti50.2 rotor), 4 °C for 90 min in a Beckman ultracentrifuge (Beckman Coulter, Inc.). The obtained supernatant (cell-free extract) was directly used for protein-protein interaction studies (see below). Protein-Protein Interactions between CyaY and Proteins of the isc Operon—His-tagged pure CyaY was loaded onto a gravity flow 0.2-ml nickel-loaded metal chelating Sepharose column (Amersham Biosciences). Cell-free extracts from a 2- or 3-liter culture of E. coli cells overexpressing the complete isc operon or non-His-tagged IscU were passed over this CyaY column. The column was washed with 30 column volumes of buffer (100 mm Tris-HCl, pH 7.5, 100 mm NaCl containing 0–20 mm imidazole), and bound proteins were eluted using buffer containing 0.5 m imidazole. These experiments were performed aerobically at 20 °C. Eluted proteins were analyzed by SDS-PAGE and identified by N-terminal sequencing. The same experiment was performed either aerobically or anaerobically with pure non-His-tagged IscU and non-His-tagged wild-type or mutated (C328S) IscS (between 0.5 and 1 equivalent with regard to CyaY). Iron Binding to CyaY—Purified CyaY (235 μm) was incubated with a 15-fold molar excess of either FeCl3 (aerobically) or Fe(NH4)2(SO4)2 (anaerobically) in 100 mm Tris-HCl, pH 7.5, 150 mm NaCl for 2 h at 4 °C. After incubation the mixtures were centrifuged for 10 min at 13,000 rpm in an Eppendorf table centrifuge (Eppendorf AG) and subsequently desalted on a NAP10 column (Amersham Biosciences). Aliquots of iron-loaded CyaY were stored at –80 °C. The iron binding properties of CyaY-Fe3+ and CyaY-Fe2+ were investigated by dilution with subsequent desalting, incubation with iron chelators like EDTA or citrate, and incubation with chemical reducing agents like DTT or dithionite. To study the dilution effects the iron-loaded protein was passed over a NAP10 column, which resulted in a dilution of 1:1.5. After 1 h of incubation the diluted protein was again passed over a NAP 10 column, which again resulted in a dilution of 1:1.5. These steps were repeated three times and the iron content before and after each dilution step was determined. To study the effects of iron chelators, iron-loaded CyaY (80 μm in iron) was incubated with a 60-fold molar excess (over iron) of either EDTA or citrate for 1 h. After desalting the iron content was determined and compared with that before chelator treatment. The effect of chemical reducing agents on the iron-loaded CyaY was investigated by incubation of CyaY (300 μm in iron) with a 10–20-fold molar excess of either DTT or dithionite for 1 h. After incubation the mixtures were desalted, and the iron content was determined and compared with that before the treatment. Oligomerization State of Iron-loaded CyaY—The oligomerization state of CyaY loaded with either Fe3+ or Fe2+ was determined by gel filtration on an analytical Superdex 75 HR 10/30 (Amersham Biosciences) and on a Tricorn high performance Superdex 200 10/300 GL column (Amersham Biosciences) in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl at a flow rate of 0.5 ml/min under aerobic conditions. Separation of Different Oligomerization States of CyaY-Fe3+—Different oligomeric forms of Fe3+-loaded CyaY were separated by preparative gel filtration on a HiLoad 16/60 Superdex 200 prep-grade column (Amersham Biosciences) in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl at a flow rate of 0.8 ml/min. Fractions of different oligomeric forms were collected, combined, and concentrated. The integrity of the separated forms after concentration was verified by reinjection onto a Tricorn high performance Superdex 200 10/300 GL column. Reduction of CyaY-Fe3+ by Biological Reducing Agents—CyaY-Fe3+ (150 μm in iron) was incubated with 2 mm l-cysteine or reduced glutathione in a total volume of 100 μl, and changes in the UV-visible absorption spectrum were followed spectrophotometrically. To verify the reduction of Fe3+ to Fe2+ the Fe2+-chelator ferrozine (750 μm) was added to the mixtures, and formation of a Fe2+-ferrozine complex was followed by UV-visible spectroscopy. Reconstitution of Iron-Sulfur Clusters on IscU—Cluster reconstitution was performed in an anaerobic chamber (Jacomex B553 (NMT)). Purified IscU was incubated with 5 mm DTT for 30 min. The DTT was removed from the solution by passage of the protein over a Micro-Biospin 6 Chromatography Column (Bio-Rad). Subsequently His-tagged IscU was incubated with catalytic amounts of His-tagged IscS and an excess of l-cysteine for 2 h. Following this preincubation CyaY-Fe3+ was added to the mixtures. Final concentrations were 50 μm IscU, 1 μm IscS, 2 mm l-cysteine, and 150 μm iron (within CyaY-Fe3+) in a total volume of 100 μl of 100 mm Tris-HCl, pH 8.0, 50 mm KCl. Formation of iron-sulfur clusters on IscU was followed by UV-visible spectroscopy using a Cary 1 Bio (Varian) spectrophotometer. The yield of [2Fe-2S] clusters formed on IscU was determined using an extinction coefficient ϵ = 5.8 mm–1 cm–1 (8Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar). A plot of the yield (%) of [Fe-S] as a function of time was fitted to a rate equation for a first order process, and the observed rate constant (kobs) was determined. Preparation of Mössbauer Samples—For Mössbauer measurements reconstituted IscU was prepared as described above in a total volume of 5 ml. After cluster reconstitution, the mixture was desalted by passage over a NAP25 column (Amersham Biosciences) and concentrated by ultrafiltration using a YM10 membrane (Amicon) to a final volume of 400 μl. The protein solution was transferred into a Mössbauer cup and frozen in liquid nitrogen. The final concentration of IscU in the sample was 527 μm. Mössbauer Spectroscopy—57Fe-Mössbauer spectra were recorded using 400-μl cuvettes containing 527 μm protein. Spectra were recorded on a spectrometer operating in constant acceleration mode using an Oxford cryostat that allowed temperatures from 1.5 to 300 K and a 57Co source in rhodium. Isomer shifts are reported relative to metallic iron at room temperature. Iron Determination—The iron content of proteins was determined as previously described (45Beinert H. Methods Enzymol. 1978; 54: 435-445Crossref PubMed Scopus (111) Google Scholar). Determination of Protein Concentration—The Bio-Rad protein assay was used according to the manufacturer's instructions using bovine serum albumin as a standard (46Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217547) Google Scholar). CyaY Interacts with IscS—Protein-protein interactions can provide a useful hint for identifying unknown protein functions. Because the CyaY protein is proposed to be a potential iron donor protein for [Fe-S] cluster biosynthesis, observable interactions of the protein with proteins involved in this process (ISC proteins) would provide an important hint toward CyaY function. To study the possible interactions of CyaY with proteins of the isc operon, an affinity chromatography approach was chosen. The His6-tagged CyaY, expressed and purified as already described (36Lee M.G. Cho S.J. Yang J.K. Song H.K. Suh S.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 920-921Crossref PubMed Scopus (7) Google Scholar), was loaded on a gravity flow Ni2+-charged metal chelating column, and in a first experiment, an E. coli cell-free extract (∼400 mg) prepared from a 3-liter culture of BL21(DE3)-RIL cells overexpressing the whole isc operon was passed over the CyaY-loaded column. Because all of the cellular proteins were not His-tagged, proteins retained on the column and eluted together with CyaY are those that interact with CyaY specifically. Bound CyaY and potential partners were then eluted in the presence of 0.5 m imidazole. Fig. 1 (lane 2) shows the analysis of eluted proteins by SDS-PAGE (15%). In addition to the band corresponding to CyaY (12200 Da), other bands are visible on the gel. N-terminal sequencing of the most intense bands revealed that the upper one, more intense, corresponds to IscS (∼45,000 Da), the lower one (∼14,000 Da), less intense, to IscU and that the intermediate ones (∼25000 Da) did not correspond to proteins of the isc operon (Fig. 1, lane 2). Binding of these proteins was due to high histidine contents favoring direct interaction with the nickel-loaded chelating Sepharose column. Apart from IscS and IscU, no other proteins from the isc operon were detected in significant amounts by SDS-PAGE. In a control experiment, performed either aerobically or anaerobically, using pure non-His-tagged IscS in place of the extracts, we observed that IscS eluted with CyaY in the 0.5 m imidazole fraction (Fig. 1, lane 3). Under identical conditions, no IscS could bind to the column in the absence of CyaY (data not shown). This demonstrated a specific interaction between CyaY and IscS. Because IscU had been previously shown to form a tight complex with IscS (47Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar, 48Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), we then addressed the question whether the presence of a small amount of IscU from the extracts on the gel (lane 2) was an indirect consequence of its binding to IscS rather than caused by direct binding to CyaY. To verify this hypothesis the same experiment as described above was repeated by passing extracts from a 2-liter culture of BL21(DE3) cells overexpressing only the IscU protein over a CyaY-loaded nickel-loaded chelating Sepharose column. Under these conditions IscU was again observed in small amounts in the imidazole fraction as shown by gel electrophoresis (Fig. 1, lane 4) and N-terminal sequencing. More surprising was the presence of a much higher amount of IscS in
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