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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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-
-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.
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RESULTS |
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-
-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.
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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.
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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-
-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).
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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.
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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).
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DISCUSSION |
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.