Iron-Sulfur Cluster Assembly

CHARACTERIZATION OF IscA AND EVIDENCE FOR A SPECIFIC AND FUNCTIONAL COMPLEX WITH FERREDOXIN*

Sandrine Ollagnier-de-ChoudensDagger , Tony Mattioli§, Yasuhiro Takahashi, and Marc FontecaveDagger ||

From the Dagger  Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DBMS-CB, CEA/CNRS/Université Joseph Fourier, UMR 5047, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France, § Section de Bioénergétique, DBCM, Bat 532, CEA Saclay, 91191 Gif sur Yvette, France, and the  Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

Received for publication, April 2, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (iron-sulfur 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 (11-19).

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 IscA has a specific relationship with ferredoxin (Fdx),1 another member of the isc operon.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification-- E. coli C41(DE3)pET21-EcFdx and C41(DE3)pRKISC cells for expression of Fdx and the products of the isc operon, respectively, were grown as described by Nakamura et al. (21). Fdx purification was done as reported by Ta and Vickery (22). The iscA gene was cloned into BamHI-SalI sites of pQE-30 (Qiagen) and pMAL-c2 (New England BioLabs, Inc.) vectors to make pQiscA-55 and pMiscA-15 for production of IscA either with a histidine tag (IscAH) or fused with the maltose-binding protein (MBP-IscA), respectively. Primers used for iscA amplification were 5'-GGATCCTCGATTACACTGAGCGACAG-3' (forward primer; underlined bases indicate a BamHI site) and 5'-GTCGACTATCAAACGTGGAAGCTTTCG-3' (reverse primer; underlined bases indicate a SalI site). E. coli M15(pREP4) (Qiagen) and TB1 (New England BioLabs, Inc.) strains were transformed with pQiscA-55 and pMiscA-15, respectively. Expression was induced by addition of 1 mM (M15(pREP4)pQiscA-55) or 0.3 mM (TB1pMiscA-15) isopropyl-beta -D-thiogalactopyranoside (Eurogentec) to exponentially growing cells in terrific broth medium (Difco) at 37 °C for 3 h. M15(pREP4)pQiscA-55 cells (17 g) were sonicated in buffer A (20 mM sodium phosphate, pH 7.4, 50 mM NaCl) containing approximately 20 µg of DNase I, 10 mM MgCl2, and protease inhibitors (1 tablet, Roche Molecular Biochemicals), and after centrifugation (90 min, 45,000 rpm, 5 °C) soluble proteins (1.2 g) were used for further purification. IscAH was purified by chromatography first onto a Ni-NTA affinity column (Amersham Pharmacia Biotech) and then onto a Superdex-75 gel filtration column 16/60 (Amersham Pharmacia Biotech). TB1pMiscA-15 cells (16 g) were sonicated in buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol and centrifuged (90 min, 45,000 rpm, 5 °C). Soluble proteins (1.5 g) were loaded onto an amylose resin affinity column (New England BioLabs, Inc.). MBP-IscA purification and then separation of the MBP domain from the IscA protein were accomplished as described by the manufacturer. About 4 mg of pure IscA were obtained from 20 mg of MBP-IscA.

Iron and Sulfide Binding to IscA-- The protein was incubated for 3 h inside a glove box (Jacomex B553 (NMT)) with a 3-5-fold molar excess of both Na2S (Fluka) and Fe(NH4)2(SO4)2 (Aldrich) in the presence of 5 mM dithiothreitol in 0.1 M Tris-HCl, pH 8.0, and then desalted on Sephadex G-25 (80 ml, same buffer).

Ferredoxin Binding to IscA-- IscAH (1 mg) was allowed to bind anaerobically onto a Ni-NTA affinity column (1 ml). Crude extracts (70 mg) from C41(DE3)pRKISC E. coli cells were loaded onto the resulting IscA column, and the column was washed with buffer A. IscAH and protein(s) retained by the column were eluted with buffer A containing 500 mM imidazole. The resulting eluate was analyzed by SDS-polyacrylamide gel electrophoresis.

Fe-S Cluster Transfer from IscA to Fdx-- Apoferredoxin was prepared as described by Nishio and Nakai (23). Typically, apoferredoxin was incubated with purified holoIscAH for 2 h at 25 °C in 0.1 M Tris-HCl, pH 8.0, 30 mM KCl.

Analysis-- Protein concentration (24), protein-bound iron (25), and labile sulfide (26) were determined as described previously.

Spectroscopy-- UV-visible spectra were recorded with a Cary 1 Bio (Varian) spectrophotometer. Resonance Raman spectra of frozen samples held at 15 K were recorded using a modified Jobin-Yvon T64000 monochromator equipped with a back-illuminated liquid N2-cooled charge couple device detector and the 496.5 nm line from an Ar+ laser (Coherent Innova 100). Spectral resolution was 4 cm-1.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of IscA-- pMiscA-15, which carries the iscA gene fused with the malE gene upstream, was used to transform E. coli TB1 cells. The malE gene encodes the MBP, which served to bind IscA selectively to an amylose affinity column. Another plasmid, pQiscA-55, containing a modified iscA gene to produce a protein with a His tag at the N terminus (IscAH), was also used. Induction with isopropyl-beta -D-thiogalactopyranoside resulted in overexpression of MBP-IscA and IscAH as shown by SDS-polyacrylamide gel electrophoresis of whole cells and sequencing of the N terminus (data not shown), respectively.

MBP-IscA was purified from soluble extracts by first binding to an amylose resin followed by elution with 10 mM maltose. Recovery of IscA was achieved by treatment of the eluted protein with Factor Xa, which cleaves the construct between MBP and IscA. A further purification was carried out during ion-exchange chromatography on an UnoQ column. The cleavage step is rather tedious, and a few milligrams of IscA (95% pure) could be obtained from 1.5 g of bacterial extracts.

IscAH was purified with a Ni-NTA column that specifically retains proteins containing a cluster of histidines at the N terminus. A gel filtration step using Superdex-75 then completed the purification. During that step, IscA eluted mainly in two peaks, the major one corresponding to a dimer and the minor one to a tetramer. Combining the two fractions containing IscA, 80 mg of IscAH could be obtained from 1.2 g of bacterial extracts in a more than 95% pure form as judged by gel electrophoresis (data not shown).

Biochemical and Spectroscopic Characterization of IscA and IscAH-- Purified solutions of IscA were metal-free and colorless (Fig. 1). However, incubation of IscA inside a glove box with a 3-5-fold molar excess of both ferrous sulfate and sodium sulfide in the presence of dithiothreitol followed by desalting on a Sephadex G-25 column within the glove box resulted in a brownish protein containing an iron-sulfur cluster as shown by iron and sulfide quantitation and UV-visible spectroscopy (Fig. 1). The protein (holoIscA) contained equivalent amounts of iron and sulfide but never more than two atoms of each per polypeptide chain (maximum, 1.7-1.8) and was EPR-silent. Its light absorption spectrum displayed bands at 330 and 420 nm (Fig. 1).


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Fig. 1.   UV-visible light absorption spectra of IscA in 0.1 M Tris-HCl, pH 8.0, 30 mM KCl. Isolated IscA (dashed line, 1.2 mg/ml) and holoIscA (solid lines, 1.2 mg/ml, 1.7 iron and sulfide atoms/polypeptide) before and after exposure to air for 10, 15, 20, 30, 45, 60, and 120 min.

The low temperature (15 K) resonance Raman spectrum in the Fe-S stretching region of holoIscA excited at 496.5 nm is shown in Fig. 2. Based on the observed frequencies and the relative intensity pattern of the 287 cm-1 band compared with the 333 cm-1 band, the holoIscA resonance Raman spectrum is indicative of a [2Fe-2S]2+ cluster. The spectrum is in agreement with those published for other ferredoxin [2Fe-2S]2+ clusters and model complexes excited at similar wavelengths (27-30). All the above spectroscopic properties were similarly observed with holoIscAH preparations.


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Fig. 2.   Low temperature (15 K) resonance Raman spectrum of holoIscA. The spectrum was recorded using 496.5 nm excitation from an Ar+ laser with 50 milliwatts of laser power at the sample (2 mM, 1.7 iron and sulfide atoms/polypeptide). Raman contributions from the buffer (i.e. mainly from the "ice band" at 230 cm-1) have been substrated.

The cluster in IscA and IscAH proved to be sensitive to oxygen. Whereas the spectrum in Fig. 1 did not change overnight in the glove box, admission of air to purified solutions resulted in slow bleaching and decay of the UV-visible bands (Fig. 1) with a half-life of 40 min. Such an aerated solution did not contain iron and sulfide after desalting onto a Sephadex G-25 column.

A Specific IscA-Ferredoxin Complex-- All the experiments described below had been carried out in the anaerobic glove box so that no loss of the cluster could occur during the time frame of the experiments. HoloIscAH was fixed to a Ni-NTA column, and this material served to identify which of the products of the isc operon had a strong affinity for IscA. Soluble extracts from isopropyl-beta -D-thiogalactopyranoside-induced E. coli C41(DE3)pRKISC cells expressing the whole operon were thus loaded onto the IscA affinity column. Then, elution with 500 mM imidazole resulted in a protein solution that contained, in addition to IscAH, only one protein as shown by SDS-polyacrylamide gel electrophoresis (Fig. 3) that proved to be Fdx by N terminus sequencing. A pure preparation of Fdx migrated at the same position on the gel. Pure Fdx solution was also used in place of extracts. In that case, more Fdx was found to be associated with IscAH with a stoichiometry close to 1:1.


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Fig. 3.   Binding of ferredoxin to IscAH. HoloIscAH (lanes 5 and 7) or apoIscAH (lane 6) was fixed onto a Ni-NTA column and used to identify IscA-binding proteins in E. coli C41(DE3)pRKISC extracts (lanes 5 and 6). In one experiment extracts were replaced by pure Fdx (lane 7). After elution with imidazole buffer, protein solutions (15-20 µg of protein) were analyzed by 15% SDS-polyacrylamide gel electrophoresis. Lane 1, molecular mass markers (97, 66, 45, 30, 20.1, and 14.4 kDa); lane 2, pure holoIscAH (8 µg); lane 3, pure holoFdx (8 µg); lane 4, extracts (70 µg).

In contrast, no protein could bind to an affinity column prepared with apoIscAH (Fig. 3), indicating that binding of Fdx to IscA was not unspecific and furthermore that the affinity of Fdx for IscA absolutely depended on the presence of the cluster in IscA.

Cluster Transfer from holoIscA to Apoferredoxin-- When apoFdx was incubated with a stoichiometric amount of holoIscAH (1.7 iron and sulfide atoms/polypeptide chain) under anaerobic conditions the solution slowly turned from brownish to red. Fig. 4 shows the formation of holoFdx as monitored by the appearance of characteristic well resolved light absorption bands at 415 and 459 nm (22). Further confirmation that Fdx has acquired a correctly assembled [2Fe-2S] cluster can be obtained from the appearance of a light absorption band at 550 nm (Fig. 4, inset) and an EPR signal (data not shown), both characteristic of a reduced [2Fe-2S]+ cluster, after anaerobic reduction with dithionite. From the intensity of both the band at 459 nm and the EPR signal we determined that ~70% of iron and sulfide initially present in holoIscAH was recovered as a [2Fe-2S] cluster in Fdx.


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Fig. 4.   Iron-sulfur transfer from holoIscA to apoferredoxin. Top, apoFdx (400 µg) was incubated with holoIscA (400 µg, 1.7 iron and sulfide atoms/polypeptide) in 0.1 M Tris-HCl, pH 8.0, 30 mM KCl for 30, 60, and 120 min under anaerobic conditions (solid lines) or after a 120-min reaction in the presence of air (dotted line). Arrows indicate absorbance at 415 and 459 nm. Bottom, light absorption spectrum after a 120-min reaction (20 nmol of Fdx) before (solid line) and after (dotted line) reduction with 1 mM dithionite.

In contrast, when apoFdx reacted with the same molar excess of iron (ferric or ferrous) and sulfide in the presence of dithiothreitol but in the absence of IscA no holoferredoxin could be formed after a 2-h reaction (not shown). The same result was obtained when holoIscA was incubated with apoFdx in air (Fig. 4) under conditions of spontaneous loss of the cluster of IscA (see above).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been recently shown that E. coli IscA greatly improves the efficiency of the maturation of overexpressed ferredoxins within E. coli cells (7). 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 (17-19). Although not lethal, deletion of isa1 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 mutated isa 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, consistent with the observation that the IscA polypeptide could not bind more than two iron and two sulfur equivalents. The resonance Raman spectrum of holoIscA is indicative of a [2Fe-2S]2+ cluster. The observed frequencies of the Agt and B3ut modes of the cluster at 287 and 333 cm-1, respectively, predominantly Fe-S(Cys) stretching modes, all fall within the range of those observed for naturally occurring clusters with complete cysteine ligation (280-290 and 325-340 cm-1, respectively) (27-30). However, the IscA sequence displays only 3 cysteines that are all fully conserved within the IscA family and are thus likely to be ligands to the cluster. Ligation by only 3 cysteines and one non-cysteinyl ligand is not inconsistent with the resonance Raman spectrum and might be the cause of the lability of the cluster. More experiments are needed to characterize the iron coordination chemistry in IscA. Nevertheless, the observed instability of the Fe-S cluster bound to IscA is consistent with IscA serving as a site for synthesis of intermediate Fe-S species, which would then be transferred to acceptor polypeptides as previously suggested (see above).

This hypothesis is supported by the finding that holoIscA specifically forms a tight 1:1 complex with ferredoxin, the product of the fdx gene present in the isc operon, as shown by the ability of an IscA column to extract Fdx exclusively from E. coli extracts expressing the whole operon. Several observations suggest that the IscA-Fdx complex has a functional relevance. First, formation of the complex absolutely depends on the presence of an Fe-S cluster in IscA raising the question of the contribution of the cluster itself to the IscA-Fdx association. Second, holoFdx can be formed in vitro by transfer of iron and sulfide from holoIscA to the apoFdx polypeptide. The fact that apoIscA and holoFdx, the products of the transfer reaction, do not form a complex anymore opens the interesting possibility that one molecule of IscA might contribute to the formation of several molecules of holoFdx. The catalytic role of IscA in formation of holoFdx is striking because almost no reaction occurs when apoFdx is incubated with free iron and sulfide in the absence of IscA. More experiments are required to understand this fascinating cluster transfer at the molecular and structural level. In particular, whether the transfer concerns the Fe2S2 core or instead involves degradation and reassembling of the binuclear entity into the apopolypeptide target is an interesting question that remains to be studied. The fact that no holoFdx could be formed in the presence of IscA undergoing an air-dependent loss of its clusters would be in favor of the first hypothesis. There are a number of other intriguing questions that deserve detailed studies: (i) which species directly supply iron and sulfur to IscA?, and (ii) is cluster transfer from IscA to apoFdx accelerated by other proteins?

From these results we propose that the specific IscA-mediated delivery of the Fe-S cluster to Fdx could be a key step in the biosynthesis of iron-sulfur proteins in the cell. 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). The same phenotype was observed with yeast cells depleted in the essential Fdx reductase, named Arh1p (32). These studies suggest that the complex process of biosynthesis of Fe-S clusters absolutely depends on a functional Fdx reductase-Fdx system, which probably participates in an essential electron transfer step. Whether holoFdx is functional alone or in association with holoIscA is an interesting question. Consequently, if the function of IscA is indeed to specifically control Fdx integrity and activity, as suggested by the present study, then IscA plays an important physiological role in particular for maturation of Fe-S proteins.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 33-4-76-88-91-03; Fax: 33-4-76-88-91-24; E-mail: mfontecave@cea.fr.

Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M102902200

    ABBREVIATIONS

The abbreviations used are: Fdx, ferredoxin; MBP, maltose-binding protein; NTA, nitrilotriacetic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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