From the Laboratoire de Chimie et Biochimie des
Centres Rédox Biologiques, Département Résponse et
Dynamique Cellulaire-Chimie Biochimie,
CEA/CNRS/Université Joseph Fourier, UMR
5047, 17 Ave. des Martyrs, Grenoble 38054, cedex 09, France, the
§ Cell and Molecular Biology, Göteborg Universitet,
Box 462, Göteborg 40530, Sweden, the ¶ National Center for
Scientific Research, Demokritos, Institute of Materials Science, Ag.
Paraskevi, Attiki 15310, Greece, the
Department of Biological
Applications And Technologies, University of Ioannina, Ioannina 45110, Greece, and the ** Laboratoire de Chimie Bacterienne-CNRS,
IBSM, 31 Chemin Joseph Aiguier, Marseille 13402, cedex 20, France
Received for publication, January 10, 2003, and in revised form, March 7, 2003
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ABSTRACT |
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SufA is a component of the recently discovered
suf operon, which has been shown to play an important
function in bacteria during iron-sulfur cluster biosynthesis and
resistance to oxidative stress. The SufA protein from Erwinia
chrysanthemi, a Gram-negative plant pathogen, has been purified
to homogeneity and characterized. It is a homodimer with the ability to
assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by
Mössbauer spectroscopy. These clusters can be transferred to
apoproteins such as ferredoxin or biotin synthase during a reaction
that is not inhibited by bathophenanthroline, an iron chelator. Cluster
assembly in these proteins is much more efficient when iron and sulfur
are provided by holoSufA than by free iron sulfate and sodium sulfide.
We propose the function of SufA is that of a scaffold protein for
[Fe-S] cluster assembly and compare it to IscA, a member of the
isc operon also involved in cluster biosynthesis in both
prokaryotes and eukaryotes. Mechanistic and physiological implications
of these results are also discussed.
Iron-sulfur [Fe-S] proteins play important roles in electron
transfer, in redox and non-redox catalysis, in regulation, and as
sensors within all living organisms, prokaryotes and eukaryotes (1, 2).
The biosynthetic process by which defined proportions of iron and
sulfur atoms are mobilized from their storage sources and combined in a
controlled way to generate the various iron-sulfur cluster prosthetic
groups is still far from understood. It requires a complex protein
machinery that is only now becoming identified and characterized.
In the bacteria Escherichia coli and Azotobacter
vinelandii, from which most of the available information is
derived, this machinery has been found to be encoded by a highly
conserved cluster of at least seven genes,
iscRSUA-hscBA-fdx, also named the
ISC1 (for
iron-sulfur cluster) machinery (3,
4). Disruption of these genes generally results in both decreased
cluster content and activity of many important [Fe-S] enzymes such as
aconitase or succinate dehydrogenase, whereas overexpression of the
operon yields increased production of recombinant [Fe-S] proteins
(5-9). Homologs to the proteins of the isc operon from
E. coli have been found in the mitochondria of eukaryotes
and, in yeast, shown by genetic experiments to play crucial roles in
[Fe-S] cluster assembly (10-20).
IscS proteins are pyridoxal-phosphate (PLP)-dependent
cysteine desulfurases that catalyze the mobilization of sulfur atom from cysteine for incorporation into clusters (21, 22). IscU and IscA
proteins are able to assemble transient and labile [2Fe-2S] and
[4Fe-4S] clusters, as shown by Mössbauer and Raman resonance spectroscopy (23-31). These clusters can be rather efficiently transferred to apoferredoxins in vitro, and both IscU and
IscA were proposed to function as scaffold proteins for mediating
general [Fe-S] cluster assembly (23, 27, 30-32). HscA and HscB
proteins are molecular chaperones (33). They both interact with IscU, and a complex has been detected in two-hybrid experiments between HscA
and IscA (34-37). As a consequence they are supposed to be required
for optimizing conformations that facilitate [Fe-S] cluster assembly
and transfer from IscU/IscA to the target apoproteins. IscU but not
IscA also makes a complex with IscS from which it directly gets the
sulfur atoms required for [Fe-S] cluster synthesis (25, 38-40).
Finally, both IscU and IscA can form complexes with Fdx, the product of
another gene of the ISC machinery (23, 29, 30). Fdx is a [2Fe-2S]
ferredoxin, thus suggesting that electron transfer steps are part of
the cluster assembly process (41). Even though all of these proteins
have been isolated in pure form and extensively characterized, the
detailed mechanism by which they work together to incorporate an
[Fe-S] cluster into an apoprotein is not known and is the subject of
intense studies in several laboratories.
As part of our efforts to understand this important biological
reaction, we studied the properties of the IscA protein from E. coli (23). There are many reasons for this. First, it is important
to understand why most organisms contain both IscA and IscU proteins,
with apparently similar functions. It is generally assumed that IscU
proteins are the key players in the cluster assembly process, even
though there is no clear evidence for it. In support to this notion is
the observation that, in Saccharomyces cerevisiae, a
knockout of both iscU homologs, ISU1 and
ISU2, is lethal, whereas a knockout of both iscA
homologs, ISA1 and ISA2, only results in retarded
growth on non-fermentable carbon sources, accumulation of iron in
mitochondria, and marked decrease in the activities of mitochondrial
and cytosolic [Fe-S] enzymes (12, 15, 16, 42). On the other hand, it
was recently shown, using an in vitro [Fe-S] cluster
assembly assay, that extracts from S. cerevisiae
mitochondria depleted in ISA1 displayed severely decreased
activities during cluster assembly in biotin synthase, a [4Fe-4S]
enzyme, when compared with wild-type extracts (43). A second indication
for the importance of IscA-type proteins is the recent discovery of an
additional operon in bacteria and intracellular parasites, named
suf, also involved in iron and sulfur metabolism. The
suf operon contains six genes, sufABCDSE,
including an iscA homolog, sufA, and an
iscS homolog, sufS (44-46).
In contrast to S. cerevisiae, the genes of the
isc operon are necessary for optimal growth but are not
essential for the viability of E. coli cells. In an E. coli mutant, in which the entire isc operon has been
deleted, the activity of [Fe-S] proteins is only 2-10% of their
activity in wild-type cells (46). This is presumably due to the
presence of the suf genes as supported by the fact that
overexpression of suf operon restores the growth phenotype and activity of [Fe-S] proteins in the mutant cells lacking all components of the ISC machinery. Furthermore, lethality was observed when both the isc and suf operons were
inactivated (46).
Genetic experiments using Erwinia chrysanthemi, a
Gram-negative plant pathogen, and E. coli, have recently
provided more detailed information on the suf genes. In both
species the suf operon was found to be under the
Fe-dependent repressor Fur, and, in E. coli at
least, it was also found to belong to the oxidative stress OxyR-dependent regulon (47-49). This suggests a specific
function of the SUF machinery during repair of [Fe-S] clusters as a
consequence of oxidative stress and a strong iron limitation. SufC
appears to be the most critical protein in this system, because
inactivation of sufC in E. chrysanthemi resulted
in (i) decreased Fe uptake, (ii) increased accumulation of free
intracellular iron levels, (iii) increased sensitivity to oxidative
stress, (iv) delay of the induction of the transcriptional activator
SoxS by SoxR, a homodimeric [2Fe-2S] redox-regulated transcription
factor, (v) decrease activities of enzymes containing oxygen labile
[Fe-S] clusters, under oxidative stress, and (vi) decreased virulence of E. chrysanthemi (44, 48, 50). Decreased iron uptake was shown to be a consequence of a decreased ability to obtain iron from
ferrisiderophores, such as chrysobactin or ferrioxamin, possibly because [Fe-S] clusters in ferrisiderophore reductases are not correctly assembled in this mutant (44, 48, 50).
With the exception of SufS, the Suf proteins have been very little
studied at the biochemical level so far (51-53). However, this system
provides a unique tool to understand mechanisms of [Fe-S] cluster
assembly, because it is simpler than the ISC system. Indeed, it does
not contain equivalents of the ferredoxin protein (Fdx) and of the
molecular chaperones (HscA and HscB). As discussed above we were
intrigued by the absence of an IscU-type protein, whereas a protein
(SufA) displaying significant sequence homology to IscA is part of the
machinery. This gives an opportunity to investigate the specific
function of an IscA-type protein. Fig. 1 shows the amino acid sequences
of SufA proteins from a variety of bacterial and archaeal sources.
Alignment with IscA proteins is possible demonstrating 52% identity
between SufA and IscA from the same organism, E. coli. In particular, the three invariant cysteines of IscA,
which have been shown by site-directed mutagenesis in the case of the
yeast protein to be essential for function and were proposed to be
involved in iron binding, are present in SufA proteins (15, 42). It is
thus very tempting to suppose that SufA has the potential to assemble
an [Fe-S] cluster and to serve in a way similar to IscA, as a
scaffold protein, for mediating [Fe-S] cluster assembly in target proteins.
With this protein the only other protein that SUF shares with ISC is a
cysteine desulfurase (SufS), which displays 23% identity to IscS. On
the other hand, the suf operon contains proteins that are
not present in cluster of ISC genes. The available data suggest that
SufB, SufC, and SufD, which are encoded by highly conserved genes
occurring in bacteria, archaea, plants, and parasites, work together to
form a multimeric ABC transporter complex with ATPase activity (50).
The role of the SufBCD complex is still unclear, but a possible
function would be to provide energy to the SUF machinery for [Fe-S]
cluster assembly. Additional characterization is needed to confirm the
presence and the role of the complex. Finally, nothing is known about
the function of SufE. In E. coli, a homolog of
sufE, referred as ygdK, lies just after the third cysteine desulfurase of that microorganism, named csd,
indicating a conservation of genetic organization between
sufS-sufE and csd-ygdK (48). Thus it is tempting
to suggest that SufE is also involved in [Fe-S] assembly. Similar
examples of this association are found in Pseudomonas
aeruginosa, Vibrio cholerae, and Haemophilus
influenzae (45).
Here we report, for the first time, the isolation and characterization
of SufA protein. We show that SufA from E. chrysanthemi can
assemble iron-sulfur clusters, which can be efficiently transferred to
target apoproteins.
Materials--
All chemicals were of reagent grade and obtained
from Sigma-Aldrich Chemical Co. 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.
5-Deaza-7,8-demethyl-10-methylisoalloxazine (DAF) was prepared
according to Ashton et al. (54).
S-Adenosylmethionine was from Roche Applied Science.
Pyridoxal 5'-phosphate was from Interchim. IscA, flavodoxin, and
flavodoxin reductase were available in our laboratory. Enzymes,
oligonucleotides, and culture media were purchased from New England
BioLabs, Oligo Express, and Difco, respectively. Pwo DNA
polymerase and the High Pure Plasmid Isolation kit, for plasmid DNA
purification, were from Roche Diagnostics (Mannheim, Germany).
Isolation of DNA fragments from agarose gels was performed using a
QIAquick gel extraction kit (Qiagen). DNA sequencing was performed by
Genome Express (France). Antibodies against VSV-G were obtained from
Roche Applied Science. E. coli MG1655 Cloning of the sufA Gene and Construction of the Overexpressing
Plasmids--
The sufA gene, encoding the SufA protein, was
amplified by PCR using genomic E. chrysanthemi 3937 DNA as a
template. Primers used for sufA amplification were
5'-CCGCATATGCAAACGCACGATGTAG-3' (forward primer, underlined
bases indicate a NdeI site) and
5'-GTTCTCGAGAAGCCCAAAACTTTCGCC-3' (reverse primer,
underlined bases indicate a XhoI site). PCR was run as
follows: genomic DNA was denatured for 10 min at 94 °C. The
Pwo DNA polymerase (0.5 unit), deoxynucleotide mix (0.2 mM each), and the primers (0.5 mM final
concentration) were added, and 30 cycles (30 s at 94 °C, 30 s
at 50 °C, then 30 s at 72 °C) were then performed,
followed by a final 10-min elongation step at 72 °C. The PCR product
was digested with NdeI and XhoI and then ligated
into a pET22b(+) vector (Novagen), digested with the same restriction
enzymes, for production of SufA with a histidine tag. The cloned gene
was then sequenced to ensure that no error was introduced during PCR
reaction. The plasmid was then named pET/SufA.
For the cross-linking experiment, a VSV-SufA encoding protein was
constructed as follow. Primers used for sufA amplification were
5'-CCCTACCATGGCTTACACTGATATCGAAATGAACCGCCTGGGTAAGATGCAAACGCACGATGTAGA-3' (forward primer, underlined bases indicate a NcoI site) and
5'-GCCTTCTCGAGTTAAAGCCCAAAACTTTCGC-3' (reverse primer,
underlined bases indicate a XhoI site). PCR was run as
follows: genomic DNA was denatured for 2 min at 94 °C. The
Pwo DNA polymerase (0.5 unit), deoxynucleotide mix (0.2 mM each), and the primers (0.5 mM final
concentration) were added and 30 cycles (30 s at 94 °C, 30 s at
60 °C, then 30 s at 72 °C) were then performed followed by a
final 10-min elongation step at 72 °C. The PCR product was digested
with NcoI and XhoI and then ligated into pBAD24
vector, digested with NcoI/SalI enzymes. The
cloned gene was then sequenced to ensure that no error was introduced
during PCR reaction. The plasmid was then named pA-VSV-SufA.
Overexpression and Purification of SufA--
For overproduction
of SufA, E. coli-competent BL21(DE3) strains were
transformed with pET/SufA vector, and 2 × 100 ml overnight cultures were used as an inoculum for 2 × 400 ml of LB medium (Difco) containing 100 µg/ml ampicillin. Cells were grown at 37 °C
to an A600 of 0.5, and expression was
induced with 0.5 mM of isopropyl- Aggregation State Analysis--
Fast protein liquid
chromatography gel filtration with an analytical Superdex-75 (Amersham
Biosciences) at a flow rate of 0.5 ml/min equilibrated with buffer A
was used for size determination. A gel filtration calibration kit
(calibration protein II, Roche Applied Science) was used as the
molecular weight standard.
Cell Fractionation Procedures--
E. chrysanthemi
A3559 strain was grown overnight in LB medium at 30 °C. When the
strain was transformed with plasmid pA-VSV-SufA, ampicillin was added
to the medium. A culture was used to inoculate fresh LB medium at
A600 0.35. After growth for 1 h,
L-arabinose 0.02% (w/v) was added and cultures were
incubated for 4 h at 30 °C. Culture was then divided in two
equal samples. On one hand, cells were pelleted and resuspended in 1.5 ml of Tris buffer (40 mM, pH 7.5) allowing for the
estimation of the amount of Suf proteins present in the cells. On the
other hand, spheroplasts were prepared (in 1.5 ml of Tris buffer) as
described by Bortoli-German et al. (55). After
centrifugation (10,000 rpm, 10 min, 4 °C) periplasmic fractions
containing supernatants were stored at 4 °C. Spheroplasts were
washed, resuspended in 1.5 ml of Tris buffer (40 mM, pH
7.5), and disrupted with pressure by French press treatment. After
centrifugation (15,000 rpm, 15 min, 4 °C), supernatants were
submitted to ultracentrifugation at 45,000 rpm during 1.5 h at
4 °C. The resulting supernatants corresponded to cytosol. Membranes
were resuspended in 1.5 ml of Tris buffer (40 mM, pH 7.5).
VSV-tagged SufA protein was detected using antibody raised against the
VSV-G epitope. For each location experiment, efficiency and reliability
of the cell fractionation procedure were checked using antibody raised
against Cel5, OutF, and MsrA.
Iron and Sulfide Binding to SufA--
The following procedure
was made anaerobically inside a glove box (Jacomex B553 (NMT)). ApoSufA
is obtained by irradiation with DAF in the presence of 10 mM EDTA. After 1 h of incubation, the colorless
protein was purified onto a Sephadex G-25 column equilibrated with
buffer C (0.1 M Tris-HCl, pH 8.0). ApoSufA (250-500 µM monomer) was then incubated in buffer C for 3-4 h
with a 3- to 4-fold molar excess of both Na2S (Fluka) and
either Fe(NH4)2(SO4)2 (Aldrich) or 57FeCl3 in the presence of 5 mM dithiothreitol (DTT). Then 2 mM EDTA was
added, and the solution was further incubated for 30 min. The protein
was desalted on Sephadex G-25 (80 ml, same buffer), and the colored
fractions were concentrated on Nanosep 10 (Amicon).
Preparation of Reconstituted Biotin Synthase
(recBioB)--
Reconstitution of apoprotein was achieved with
56Fe as described previously (56) and desalted over
Sephadex G-25 (equilibrated with buffer C) to remove adventitiously
bound iron after reconstitution.
[Fe-S] Cluster Transfer from HoloSufA to
Fdx--
Apoferredoxin was obtained from holoferredoxin as already
described (32). Typically, apoferredoxin (76 nmol) was incubated with
holoSufA (76 nmol) for 2 h at 25 °C in buffer D (0.1 M Tris-HCl, pH 8.0, 30 mM KCl). Transfer of the
[Fe-S] cluster from holoSufA to ferredoxin was monitored at time
intervals by EPR spectroscopy at liquid helium temperature, from the
ferredoxin-characteristic EPR signal obtained after reduction of the
cluster with 2 mM dithionite.
[Fe-S] Cluster Transfer from HoloSufA to ApoBioB--
ApoBioB
was obtained by irradiation of the as-isolated enzyme with DAF in the
presence of 10 mM EDTA and purified onto a Sephadex G-25
column equilibrated with buffer C. ApoBioB was then incubated in buffer
C with either a 2-fold molar excess of holoSufA or a 4-fold molar
excess of Fe2+ and S2 Biotin Synthase Activity--
The activity was assayed from the
amount of biotin formed from dethiobiotin. The standard reaction
mixture in a final volume of 50 µl of 0.1 M
Tris-HCl, pH 8, and 30 mM KCl contained 1.7 nmol of biotin
synthase monomer, 1 equivalent of pyridoxal 5-phosphate (PLP), 400 µM dethiobiotin, 1 mM DTT, 150 µM S-adenosylmethionine, 20 µM
flavodoxin, 4 µM flavodoxin reductase, 1 mM
NADPH, and 2 mM cysteine. The reaction was monitored at
37 °C. After 90 min of incubation, an aliquot was withdrawn and the
reaction stopped by the addition of 10% (v/v) 1 M
trichloroacetic acid. After centrifugation, the supernatant was
analyzed for biotin formation. Biotin was measured by a microbiological
method using Lactobacillus plantarum and a calibration curve
in each experiment. All the data presented in this report represent the
average of at least duplicate experiments.
UV-visible Spectroscopy--
UV-visible spectra were recorded
with a Cary 1 Bio (Varian) spectrophotometer.
Mössbauer
Spectroscopy--
57Fe-Mössbauer spectra were
recorded using 400-µl cuvettes containing 600-750 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.
Analysis--
Protein concentration (by monomer) was determined
by the method of Bradford (57). Protein-bound iron was determined under reducing conditions with bathophenanthroline disulfonate after acid
denaturation of the protein (58) and labile sulfide by the method of
Beinert (59).
SufA Proteins: Sequence Analysis--
Using the Blast search
algorithm against the protein data base with the sequence of SufA from
E. chrysanthemi we identified several proteins from
different organisms that displayed sequence homology to E. chrysanthemi SufA (Fig. 1).
These include SufA putative proteins as well as IscA proteins and ISAp
eukaryotic homologs. Like IscA, SufA proteins are characterized by the
presence of three conserved cysteines residues in the C-terminal
region, two of which are in a CGCG motif. A high sequence conservation was present around these cysteines as well as in some other important regions. SufA from E. chrysanthemi showed 59% identity with
SufA from E. coli and 42% with IscA from E. coli. In eukaryotes, in which genes are not organized within
operons, SufA and IscA homologs were also present (45). The proteins
from Homo sapiens and Arabidopsis thaliana
displayed 39 and 36% identity to SufA from E. chrysanthemi, respectively.
Cellular Location of SufA in E. chrysanthemi--
Previous
analyses revealed that SufB, SufC, and SufD proteins are located in the
cytosol of E. chrysanthemi. Therefore, we were interested in
analyzing the cellular location of SufA. A strain of E. chrysanthemi was transformed with a chimeric gene encoding a
VSV-SufA-tagged protein, which can be detected by reaction with an
antibody against the VSV region. Cell fractionation techniques revealed
that SufA was indeed located exclusively in the cytosol (Fig.
2).
Expression and Purification of SufA--
A SufA-(His)6
protein from E. chrysanthemi was overproduced in BL21(DE3)
E. coli cells. Analysis by SDS-electrophoresis gel of whole
cell extracts revealed a Coomassie Blue-stained band at around 15,000 Da (inset in Fig. 3,
lane 2). This molecular mass is in agreement with the mass
calculated from the DNA sequence (14,700 Da). After extraction, a high
yield of soluble SufA was obtained, even though a significant amount of
protein remained under unsoluble forms. Soluble SufA was purified with
a Ni-NTA column that specifically retains proteins containing a cluster of histidines. A gel filtration step using Superdex 75 was used to
determine the oligomerization state of SufA (Fig. 3). During that step,
SufA eluted in a major peak corresponding to a dimer, but a minor
amount of aggregated forms of larger size could also be observed.
Combining the two Superdex-75 fractions containing SufA, 47 mg of SufA
could be obtained from 180 mg of bacterial extracts, in a more than
95% pure form as judged by gel electrophoresis (inset in
Fig. 3, lanes 7 and 8). Edman degradation
analysis revealed a MQTHD sequence that corresponded to the predicted N
terminus sequence of SufA.
Biochemical and Spectroscopic Characterization of
SufA--
Concentrated purified solutions of SufA were slightly
pink-colored with a UV-visible spectrum that indicated the presence of
iron-sulfur clusters. Iron and sulfide analysis confirmed the presence
of stoichiometric amounts of iron and sulfide but with only 0.06-0.08
iron and sulfide atom per polypeptide chain. This small amount of
cluster could be removed by irradiation in the presence of deazaflavin
(DAF) and EDTA, a method used to prepare apoSufA, the apoprotein form.
Incubation of apoSufA inside an anaerobic glove box with a 5-fold molar
excess of both ferrous sulfate and sodium sulfide, in the presence of
dithiothreitol (DTT), followed by desalting on a Sephadex G-25 column,
resulted in an EPR-silent brownish protein (holoSufA) containing a
maximum amount of 1.7-2.1 atoms of iron and sulfide per polypeptide
chain. ApoSufA was unable to chelate iron in the absence of sodium
sulfide. Like the apo form, holoSufA eluted in two peaks during
Superdex-75 chromatography, the major one corresponding to a dimer and
the minor one to a trimer. The light absorption spectrum of
holoSufA displayed bands at 330, 420, and 460 nm, indicating the
presence of an iron-sulfur center (Fig.
4). This center was stable in the presence of 2 mM EDTA during 30 min under anaerobiosis.
The clusters were unstable both in aerated solutions or during
anaerobic reduction. In the presence of oxygen we observed a bleaching
of the protein solution, and the cluster had a half-life time of about
20 min at 25 °C. Addition of DTT did not stabilize the cluster under
aerobic conditions. In contrast, the light absorption spectrum did not
change when the protein was left overnight in the anaerobic glove box
(data not shown). Treatment of SufA with strong reducing agents (2 mM dithionite or illuminated DAF) under anaerobic
conditions followed by Sephadex G-25 chromatography also led to a
metal-free protein. We were unable to detect an EPR-active form under
either oxidative or reductive conditions.
We used Mössbauer spectroscopy to characterize the iron sites in
holoSufA preparations after reconstitution with 57Fe and
sulfide and treatment with 2 mM EDTA and desalting on
Sephadex G-25. Different conditions gave preparations with lower
proportions of defined clusters. One of these, treated with larger
concentrations of EDTA (10 mM), proved useful for analysis
of the Mössbauer data, because it contained a larger proportion
of [2Fe-2S] clusters and no more [4Fe-4S] clusters. Similar
results were obtained whether sulfur was provided by sodium sulfide or
by the SufS-dependent cysteine desulfurization reaction
(data not shown). Fig. 5 (A and B) shows the 77 K and 4.2 K Mössbauer spectra,
respectively, from a sample treated anaerobically with 2 mM
EDTA (sample 1: 0.74 mM final concentration; 1.7 iron/monomer), and Fig. 5 (C and D) shows the 77 K and 4.2 K spectra from a sample treated with 10 mM EDTA
(sample 2: 0.68 mM; 1.1 iron/monomer).
The 4.2 K spectrum of sample 2 (Fig. 5D) consists of two
major doublets, I and II. The parameters of I (
In the presence of 2 mM EDTA the spectra are different. We
first discuss the 77 K spectrum (Fig. 5A). The peak at
From an analysis of spectra in Figs. 5A and 5B we
estimate that 11% of the iron belongs to [2Fe-2S]2+
clusters and 40% to [4Fe-4S]2+ clusters. 30% of the
iron belongs to high spin mononuclear ferrous iron. A closer
examination of the spectrum in Fig. 5B suggests that the
remaining ~20% of the iron gives rise to a broad background extending from Iron-Sulfur Cluster Transfer--
The presence of iron sulfur
clusters in holoSufA led us to analyze the ability of these clusters to
be transferred to an acceptor apoprotein. To investigate this, two
[Fe-S] proteins were chosen as model substrate proteins: ferredoxin
(Fdx) and biotin synthase (BioB). In this study we present the results
obtained with proteins from E. coli, which display
very strong homologies to those from E. chrysanthemi (Fdx: 90% identity; BioB: 84% identity). Fdx is a [2Fe-2S] protein. The corresponding apo form of Fdx, apoFdx, was
incubated with a stoichiometric amount of holoSufA under anaerobic conditions, and the reaction was monitored by UV-visible spectroscopy because holoferredoxin displays characteristic light absorption bands
at 415 and 460 nm. Furthermore, the presence of the cluster in
ferredoxin can be confirmed, after reduction with an excess of
dithionite, both from its EPR signal, characteristic for a reduced
[2Fe-2S]+ cluster, and its UV-visible spectrum with a new
absorption band at 550 nm. From these assays we determined that 80% of
iron and sulfide initially present in holoSufA was recovered as a
[2Fe-2S] cluster in Fdx, after a 40-min reaction (data not shown). In
comparison, formation of the cluster by incubation of apoFdx with a
2-fold molar excess of iron and sulfide was highly inefficient with a yield of 5% after a 3-h reaction.
Biotin synthase (BioB), the product of the bioB gene, can be
prepared in two different active forms. Form I, named recBioB, contains
one catalytically essential [4Fe-4S] cluster per polypeptide chain
and requires pyridoxal phosphate (PLP), cysteine, a source of
electrons, and S-adenosylmethionine for activity (60, 61). Form II contains an additional [2Fe-2S] cluster and does not require PLP and cysteine (62, 63). However, it is important to note that
in vitro and for unknown reasons both forms do not allow the
production of more than 1 mol of biotin per mol of enzyme (61, 62, 64).
In the experiment discussed below we studied the formation of the
[4Fe-4S] cluster of BioB. ApoBioB, the enzyme form lacking the
cluster(s), was incubated with 2 molar excess of holoSufA, providing
only a 4 molar excess of iron and sulfide, in the presence of DTT.
Cluster transfer from SufA to BioB was then monitored by assaying the
solution for biotin synthase activity. Because PLP was required in this
assay, it is likely that under these reaction conditions only recBioB
(form I) was generated. Fig. 6 shows the
time-dependent formation of active recBioB and demonstrates
that the reaction proceeds efficiently, because the maximal biotin
synthase activity (0.6 mol of biotin/mol of BioB monomer) was obtained
after 20-30 min of incubation. In comparison, very low activity
(0.10-0.12 biotin/BioB monomer) was observed when apoBioB was
incubated with 4 molar excesses of iron and sulfide either with or
without apoSufA (Fig. 6). Fig. 6 also shows the results obtained using
holoIscA from E. coli as an [Fe-S] cluster donor, during
cluster transfer to apoBioB. These data show that SufA and IscA are
equally efficient for activation of apoBioB. HoloIscA was prepared as
previously discussed (23). As a control for the specificity of the
reaction, we found that iron-sulfur clusters from the [4Fe-4S]
Iron-sulfur cluster assembly can proceed either via a concerted and
direct transfer of the cluster from holoSufA to the target apoprotein
or via an indirect transfer mechanism with first a release and
decomposition of the cluster in the solution, then a chelation of free
iron and sulfide by the acceptor apoprotein, and finally assembly of
the new cluster. To understand the mechanism by which the cluster
transfer reaction occurs, we studied the effects of a strong free iron
chelator on that reaction. Bathophenanthroline sulfonate (BPS) proved
suitable, because it does not inhibit BioB activity and does not
chelate out iron from SufA or IscA at concentrations below 150 µM (data not shown). BPS was expected to be an inhibitor of the cluster transfer reaction only in the case of an indirect transfer mechanism, which implies that iron becomes transiently accessible.
ApoBioB and holoSufA (or holoIscA) were incubated anaerobically at
18 °C with increasing concentrations of BPS, and biotin synthase
activity was measured after a 30-min reaction. As shown in Fig.
7, BPS did not inhibit the iron-sulfur
cluster transfer from holoSufA (or holoIscA) to apoBioB, because the
reaction proceeded equally well in the absence or in the presence of
150 µM of the iron chelator. On the contrary,
reconstitution of the cluster of biotin synthase during incubation of
apoBioB with 4 equivalents of iron and sulfide was inhibited by
increasing concentrations of BPS.
Cluster transfer was also observed with comparable rates in the absence
of DTT if apoBioB was first treated with 50 mM DTT anaerobically and then desalted. This shows that DTT was not required during the [Fe-S] transfer process but rather for reduction of disulfide bridges present in the aerobic preparations of apoBioB.
On the basis of its strong amino acid sequence similarity with
IscA (Fig. 1), a protein of the isc operon shown to be able to assemble an iron-sulfur center, we made the hypothesis that SufA,
one of the components of the SUF machinery, would also have the
potential to bind clusters. This is now unambiguously shown here for
the first time with a purified preparation of the homodimeric recombinant SufA protein from E. chrysanthemi. Iron and
sulfide analysis of both as-isolated and reconstituted proteins as well as UV-visible and Mössbauer spectroscopy are consistent with the
presence of iron-sulfur clusters in this protein.
There are many similarities between IscA and SufA proteins. These
similarities are discussed in the following paragraphs.
First, both purified proteins are found essentially in the homodimeric
form with some tendency to aggregate into larger polymers (Fig. 3).
Second, IscA and SufA proteins have in common three fully conserved
cysteines (Cys-51, Cys-115, and Cys-117 for SufA from E. chrysanthemi, Fig. 1). A similar pattern of two cysteine residues separated by one amino acid CXC has been reported for an
aldehyde oxidoreductase from Desulfovibrio gigas. In
this case X-ray crystallography has shown that these cysteines are
bound to different irons in the same [2Fe-2S] cluster (66). In the
case of ISA1p and ISA2p from the yeast S. cerevisiae, these
cysteines have been proposed to function as binding residues for iron,
because individual amino acid substitutions for each of the three
cysteine residues yielded the same phenotypes as gene knockouts (15,
42). However, as yet the corresponding mutated proteins have not
been purified and characterized. On the contrary, in the case of ISA1p
from Schizosaccharomyces pombe, the corresponding mutated
proteins were isolated in pure form and found to be able to coordinate a cluster under the in vitro reconstitution conditions
employed for the wild-type protein (29). These clusters displayed
UV-visible and Mössbauer spectroscopic properties quite
comparable to those of the wild-type protein. Even though the stability
of the clusters of the mutant proteins was found to be greatly reduced
with regard to that of the wild-type protein, these results did not
provide an unambiguous confirmation that these three conserved
cysteines indeed are iron-binding residues. Nevertheless, at the
present stage, we favor such an hypothesis and speculate that they play an iron-binding function in SufA, but this must be further investigated by site-directed mutagenesis. It is important to note that not only is
there no other cysteine in SufA, but also two additional fully
conserved residues, Asp-71 and Asp-97, are possible candidates as
iron-binding residues and thus should be considered in future site-directed mutagenesis studies. At least one of these residues might
provide the fourth ligand to a cluster bound by three cysteines.
Third, the clusters in SufA from E. chrysanthemi are
air-sensitive and are rapidly decomposed during exposure to air. This probably explains why the protein was isolated mainly in the apoprotein form after chromatographic purification. Furthermore, the protein also
loses its cluster during anaerobic reduction with dithionite. This
oxidative and reductive lability of SufA clusters was also observed in
the case of IscA from E. coli or Azotobacter
vinelandii and ISA1p from S. pombe (23, 26, 29). It
should be noted that, in both SufA and IscA, iron is tightly bound to
the protein only in the presence of sulfide. These proteins are not
able to, although in some cases only very weakly, chelate
ferrous iron alone.
Fourth, the SufA protein can bind close to 2 iron and sulfur atoms per
polypeptide chain mainly assembled within both [2Fe-2S]2+
and [4Fe-4S]2+ clusters, with typical Mössbauer
parameters: Fifth, both SufA and IscA are able, at least in vitro, to
efficiently transfer its iron and sulfide atoms to apoferredoxin (23).
The high transfer yield suggests that ferredoxin can take iron and
sulfur not only from [2Fe-2S] and [4Fe-4S] clusters preassembled in
SufA but also from unspecifically bound iron-sulfide species. These
indeed represent a significant proportion of total iron and sulfur in
holoSufA, as shown by Mössbauer spectroscopy. The formation of
the [2Fe-2S] cluster of ferredoxin is greatly accelerated when iron
and sulfur are preassembled in SufA (or IscA) when compared as
when they are provided in the form of free iron and sulfide salt. This
is consistent with the working hypothesis that SufA indeed plays the
role of a scaffold protein for assembly of [Fe-S] clusters, providing
a mechanism for bringing together elemental sulfur and iron into
transient assembled clusters. This function would not be a specific
property of the IscA-SufA proteins because IscU (bacteria), ISU
(yeast), and NifU (nitrogen-fixing bacteria) proteins were also
suggested to play this role (23, 27, 30-32, 67, 68). Because there is
no homolog of these proteins in the suf operon and because
the other components of that operon are unlikely to bind an [Fe-S]
cluster, it is likely that SufA plays the specific and unique function
of a scaffold protein during assembly of [Fe-S] clusters that is
dependent on the SUF machinery.
It can be speculated that, depending on the scaffold protein, IscA,
IscU, or SufA, different clusters, [2Fe-2S] or [4Fe-4S], might be
assembled in target proteins. So far this seems rather unlikely
considering the ability of these proteins to transiently assemble both
types of clusters (see above). Furthermore, this hypothesis is not
supported by the phenotypes of the knockout bacterial and yeast strains
where reduced activities of both [4Fe-4S] and [2Fe-2S] proteins
were observed (5, 7, 8, 69). In this study we show that SufA not only
serves as a [2Fe-2S] cluster donor to ferredoxin, as discussed above,
but also as a [4Fe-4S] donor for biotin synthase, BioB (Fig. 6). In
this case also cluster formation was achieved at a greater rate when
iron and sulfur atoms were provided by holoSufA as compared with free iron and sulfide. Furthermore, we show here that both holoSufA and
holoIscA can provide their clusters to BioB. Thus a single protein can
deliver both types of clusters to different proteins and, furthermore,
a given target can get its clusters from different scaffold proteins.
These in vitro results thus seem to rule out the notion that
the [Fe-S] cluster assembly process depends on specific combinations
of cluster donors and acceptors.
Besides the lack of U-type proteins, there are two major differences
between the ISC and the SUF systems. First, the SUF system lacks
molecular chaperones, corresponding to those of the ISC system:
DnaK/HscA and DnaJ/HscB in bacteria or Hsp70/SSQ1 and Hsc20/JAC1 in
yeast. On the other hand, the SUF system contains three cytoplasmic
proteins, SufB, SufC, and SufD, forming a complex and working together
as an ABC/ATPase machinery, suggesting a requirement for ATP
during SUF-dependent cluster synthesis and that SufBCD
provides energy to this pathway (50). Whether SufBCD also has a
molecular chaperone function is a possibility that needs to be studied.
The second difference is the absence in the SUF system of an electron
transfer protein such as ferredoxin. In yeast, mitochondrial ferredoxin
and ferredoxin reductase are essential components of the [Fe-S]
cluster assembling machinery, their deletion being lethal (14, 70, 71).
Furthermore, requirement for a potent redox system in this biosynthetic
pathway is not surprising because electrons might be required for
reduction and release of iron from storage or transport sources,
generation of sulfide, assembly of the cluster itself, and release of
the [Fe-S] cluster from its transient association with a scaffold protein. Thus either the SUF system does not require electrons and
operates by a different mechanism or it uses another reductase system
that needs to be identified.
We believe that SUF is an interesting system to investigate because it
uses a limited number of components for assembly of [Fe-S] clusters
and might represent the minimal combination for that process. In Scheme
1 we propose a hypothetical
SUF-dependent mechanism for [Fe-S] cluster biosynthesis.
In this mechanism SufA would serve as a unique scaffold protein
assembling iron and sulfur atoms into transient clusters. SufS,
possibly in association with SufE, would serve to generate sulfur from
cysteine. The source of iron is still unknown. Even though, in the
experiments reported here, SufBCD was not required for cluster transfer
reactions from SufA, it is tentatively included in Scheme 1. Genetic
studies have indeed shown the importance of this protein complex during [Fe-S] cluster assembly (46, 48, 50). Whether SufBCD has an effect on
cluster assembly in SufA or on cluster transfer in vitro
needs to be further studied. It might be relevant to note that the
cellular location of the SufBCD proteins is still a matter of
controversy, because SufC was found in the membrane in E. coli using microscopy-based technology and in the cytosol in both
E. coli and E. chrysanthemi using conventional
cell fractionation techniques (45,
48).2 Assuming that all Suf
proteins work together, the fact that SufA was found to be located in
the cytosol in the present study supports the later view. In Scheme 1
no electron source is involved, but one cannot exclude, as discussed
above, that a reductase not encoded by the suf operon
participates in the process. Mycoplasma sp., which
are considered to be the most primitive parasitic cells, might tell us
that it is even possible to do with less. These organisms contain in
their genomes only three proteins of the ISC machinery, IscU, IscS, and
HscB, and no bacterial-like ferredoxin reductase/ferredoxin system
(45).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
suf was described
previously (50).
-D-thiogalactopyranoside (Eurogentec) for
4 h at 37 °C. The bacterial pellet (7 g/800-ml culture) was
resuspended in 50 ml of buffer A (100 mM Tris-HCl, pH 7.5, 50 mM NaCl) and treated twice with a French press for
disruption. The cell lysate was centrifuged at high speed for 30 min,
at 4 °C. The supernatant (180 mg of soluble proteins) was loaded
onto a 5-ml Hi-trap column (Amersham Biosciences), charged with nickel,
and equilibrated with buffer A. Pure protein (47 mg) was eluted by a
25-ml gradient from 0.04 to 0.5 M imidazole with buffer B
(buffer A plus 1 M imidazole) and was washed twice with 10 ml of buffer A onto a BIOMAX-5K device (Millipore) to remove imidazole.
SufA protein was then aliquoted and stored at
80 °C.
. At time intervals (5, 10, 20, 30, and 60 min) biotin synthase activity was measured by
addition of all components required for the activity as described
below. HoloIscA from E. coli, prepared as previously
discussed (23), was also used in place of holoSufA.
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Fig. 1.
Sequence alignment of SufA-like proteins from
various organisms. The conserved cysteines are in
boldface, and conserved amino acids are marked by
asterisks. Ec, E. coli; St,
Salmonella typhimurium; Erc, E. chrysanthemi; Yp, Yersinia pestis;
Pa, P. aeruginosa; Av, A. vinelandii; Rs, Ralstonia solanacearum;
Hi, H. influenzae; Nm, Neisseria
meningitidis; Vc, V. cholerae;
At, Arabidopsis thaliana; and Hs,
Homo sapiens.
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Fig. 2.
SufA is located in the cytosol.
Immunoblot analysis of crude extracts (lanes 1), periplasmic
(lanes 2), cytoplasmic (lanes 3), and membrane
(lanes 4) fractions prepared from E. chrysanthemi
A3559 strain carrying pA-VSV-SufA. VSV-SufA was detected using
anti-VSV-G antibody. Given on the right side are the
proteins under study. OutF, Cel5, and
MsrA were used as markers for membrane, periplasmic, and
cytosolic compartments, respectively.
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Fig. 3.
Aggregation state of SufA. SufA (500 µg) after purification on a Ni-NTA column was loaded on an analytical
Superdex 75 column and eluted with 50 mM Tris-HCl, 5 mM DTT, pH 7.5. Inset: SDS-electrophoresis gel
of fractions during SufA purification. 1, molecular markers
(kDa): 175, 83, 62.5, 47.5, 32.5, 25, and 15; 2, soluble
extracts; 3, French press pellet; 4, French press
supernatant; 5, Ni-NTA column flow-through; 6,
Ni-NTA column washing (50 mM imidazole); 7,
Ni-NTA column eluate (200 mM imidazole); and 8,
Superdex 75 column (major peak).
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Fig. 4.
UV-visible absorption spectrum of
reconstituted SufA protein (100 µM,
1.7 iron/polypeptide chain) in 0.1 M Tris-HCl, 50 mM KCl, pH 8.
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Fig. 5.
Zero field Mössbauer spectra of
holoSufA from sample 1 (730 µM, 1.7 Fe/monomer) at 77 K (A) and 4.2 K (B)
and from sample 2 (680 µM, 1.1 Fe/monomer) at 77 K (C) and 4.2 K
(D). Solid and dashed lines
represent theoretical simulations using the parameters quoted in the
text. In B we show the theoretical contribution of the
doublets belonging to the [2Fe-2S]2+ (solid
line) and [4Fe-4S]2+ (dotted lines)
clusters, respectively.
= 0.27 mm/s and
EQ = 0.59 mm/s) are consistent with a
[2Fe-2S]2+ cluster. The parameters of II (
= 0.71 mm/s and
EQ = 3.28 mm/s) are typical for
Fe2+ mononuclear species in tetrahedral environment
comprising sulfur atoms. A closer examination of the 4.2 K spectrum
reveals that these major doublets are superimposed on a broad
background. This background (probably representing paramagnetic
iron-sulfide impurities) hinders us from quantitation of the [2Fe-2S]
and mononuclear ferrous forms from the 4.2 K spectrum. At 77 K,
however, the spectrum (Fig. 5C) consists solely of doublets.
From this spectrum we estimate that 26% of the iron belongs to
[2Fe-2S] clusters and 13% to mononuclear tetrahedral ferrous
species. 61% of the iron belongs to a broad doublet (dotted
line in Fig. 5C) with a mean isomer shift of 0.49 mm/s
and a quadruple splitting of 0.91 mm/s, which apparently arises from
the sites giving rise to the anomalous background at the 4.2 K spectrum.
1
mm/s and the broad peak at ~+2.5 mm/s can be assigned to mononuclear Fe2+(S = 2) sites. We have simulated these peaks
assuming two sites; site 1 with parameters
= 0.71 mm/s and
EQ = 3.20 mm/s and site 2 with
= 1.20 mm/s and
EQ = 2.80 mm/s. Site 1 contributes 13% to the spectrum and is attributed to
Fe2+(S = 2) in tetrahedral coordination comprising
sulfur atoms as in sample 2. Site 2 represents 17% of total iron, and
its parameters are consistent with Fe2+(S = 2) in
octahedral environment with a coordination of five or six N/O donors.
The rest of the iron gives rise to a broad asymmetric doublet, confined
in the region of 0-1 mm/s. We have simulated this part of the spectrum
assuming two doublets. One doublet has parameters similar to the
parameters of the [2Fe-2S]2+ cluster observed in Fig.
5D. Its presence is justified from the 4.2 K spectrum (see
below). The other doublet (dotted line in Fig.
5A) has apparent parameters
= 0.49 mm/s and
EQ = 0.92 mm/s. Unlike sample 2, at 4.2 K, in
Fig. 5B we still observe diamagnetic doublets in the central
part of the spectrum. We simulated this part assuming three doublets.
One doublet has parameters similar to the parameters of the
[2Fe-2S]2+ cluster observed in Fig. 5D of
sample 2. The other two doublets (dotted lines in Fig.
5B) have apparent parameters
1 = 0.46 mm/s,
EQ1 = 1.26 mm/s;
2 = 0.48 mm/s,
EQ2 = 0.82 mm/s. On average, the values
of the isomer shift (~0.47 mm/s) and quadruple splitting (1.04 mm/s)
are consistent with a [4Fe-4S]2+ cluster.
3 to +3 mm/s. This species is attributed to
paramagnetic iron-sulfide impurities.
protein of the anaerobic ribonucleotide reductase (65) and from the
[2Fe-2S] ferredoxin from E. coli (41) could not be
transferred to apoBioB.
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Fig. 6.
Kinetic of the [Fe-S] cluster transfer from
holoSufA/IscA to apoBioB. ApoBioB was incubated with either a
2-fold molar excess of holoSufA (X) or holoIscA ( ) or a
4-fold molar excess of Fe2+ and S2
with or
without apoSufA (
). At different times, 5, 10, 20, and 30 min, the
biotin synthase activity was measured by addition of the standard assay
mix (see "Experimental Procedures") to the protein solution.
RecBioB (
) was used as a positive control.
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Fig. 7.
Bathophenanthroline does not inhibit
activation of biotin synthase by holoSufA/IscA. ApoBioB was
incubated with a 2-fold molar excess of either holoSufA (X)
or holoIscA ( ) or a 4-fold molar excess of Fe2+ and
S2
(
) and increasing concentrations of
bathophenanthroline. After 30 min of incubation at 18 °C, biotin
synthase activity was measured as discussed under "Experimental
Procedures."
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
= 0.27 mm/s,
EQ = 0.59 mm/s and
= 0.47 mm/s,
EQ = 1.04 mm/s, respectively. Whether the [2Fe-2S] clusters are intermediate
species on the way to the [4Fe-4S] ones or they are degraded products
of labile [4Fe-4S] clusters remains to be shown. Furthermore, all
preparations of holoSufA, even those extensively treated with EDTA,
contained significant amounts of adventitiously bound iron, both high
spin Fe2+ mononuclear species and magnetic polymeric
Fe-sulfides, as shown by Mössbauer spectroscopy (Fig. 5). Thus,
despite great efforts to optimize the in vitro
reconstitution process, we failed to set up the conditions for an
efficient generation of clusters in SufA. IscA proteins from various
origins have been shown to chelate the same amount of iron and sulfur
atoms per polypeptide chain during reconstitution of the apo forms (23,
26, 29). On the other hand, contradictory results have been reported in the literature concerning the type of clusters present in IscA proteins
(23, 26, 29). Our analysis of the protein from E. coli by
Raman resonance spectroscopy led to the conclusion of the presence of
[2Fe-2S] clusters (23). However, this spectroscopy is not as
efficient for detection of [4Fe-4S] clusters in proteins, and we do
not completely exclude the possibility that [4Fe-4S] clusters are
also present in E. coli IscA. Mössbauer spectroscopy, which is the ideal method to discriminate between [2Fe-2S] and [4Fe-4S] clusters, has not been applied to this particular protein. Mössbauer spectroscopy has been used to investigate the clusters in two cases: ISA1p from S. pombe and IscA from A. vinelandii (26, 29). In the first case, only [2Fe-2S] clusters
(one per monomer) could be detected with typical Mössbauer
parameters (
= 0.27 mm.s
1 and
EQ = 0.56 mm.s
1) (29). On the
contrary, in the case of IscA from A. vinelandii, a detailed
study of the metal centers generated during reconstitution with free
iron and sulfide provided by enzymatic cysteine desulfurization revealed that the protein can assemble both [2Fe-2S] and [4Fe-4S] clusters, with Mössbauer parameters comparable to those of the clusters of SufA (
= 0.26 mm.s
1,
EQ = 0.55 mm.s
1 for [2Fe-2S]
and
= 0.44-0.46 mm.s
1,
EQ = 1.04-1.25 mm.s
1 for
[4Fe-4S]) (26). The [2Fe-2S] clusters have been unambiguously shown
to be transient species during the cluster assembly process, but the
final [4Fe-4S] clusters were shown to be degraded under prolonged
incubation into polymeric Fe-sulfides, confirming their significant
instability. This instability of the metal centers is probably at the
origin of the discrepancies described above. On the other hand, this
instability might also be an intrinsic functional property of these
proteins in relation to their role as sources of clusters during
[Fe-S] assembly in apoproteins (see below). Nevertheless, a more
definitive characterization of the iron centers in both IscA and SufA
require a better control of the reconstitution conditions that need to
be redefined.
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Scheme 1.
A current working model for [Fe-S]
cluster biosynthesis by suf operon.
Of note, a final aspect of our work involves the mechanism of cluster transfer from holoSufA to BioB. Our results suggest that this transfer is not a two-step process, with first a release of iron and sulfur atoms from holoSufA in solution followed by their uptake by the apoprotein target. A concerted mechanism is more consistent with the inefficiency of BioB reconstitution with free iron and sulfide as compared with holoSufA and the absence of inhibitory effects of bathophenanthroline, a strong ferrous chelator, on the transfer process. In contrast, the inefficient cluster formation by free iron and sulfur is greatly inhibited by this chelator. The hypothesis of a concerted transfer process is reasonable, considering the need for scaffold proteins for assembly of [Fe-S] clusters in cells. In other words, why should a cluster be transiently pre-assembled into SufA (or IscA) on the way to its final destination if this cluster would be disassembled before transfer to the target protein? We are interested in understanding the molecular details of the cluster transfer reaction, but this requires a better control of cluster assembly in SufA, which is currently under study.
Cluster assembly in biotin synthase Bio2p, the yeast equivalent of
BioB, has also been investigated in yeast. Using detergent extracts
from S. cerevisiae mitochondria, Mülhenhoff et
al. (43) have shown that cysteine, NADH, ATP, and DTT greatly
stimulated formation of clusters. Cysteine is the substrate of cysteine
desulfurase and is the precursor of sulfide, whereas NADH is the
electron source, in agreement with the requirement for ferredoxin
reductase/ferredoxin electron transfer chain. The requirement for ATP
is consistent with the fact that chaperones are involved in cluster
formation. The role of DTT has been discussed, but the possibilities
that Bio2p could be overoxidized in the mitochondrial extracts and that
DTT could be required for reducing disulfide bridges, as a prerequisite
for enzyme activity, were not considered. Our results show that DTT is
not required anymore during cluster transfer from SufA to BioB if BioB
has been pretreated with DTT first and then desalted. The most
interesting result in this study of Bio2p activation, in the context of
our work on IscA and SufA proteins, is that a deficiency in ISA1 caused
a strong decline in [Fe-S] assembly. In contrast, deletion of NFU1
resulted in only moderate defects. This, together again with the fact
that there are no U proteins (NifU or IscU) in the suf
operon, supports our hypothesis of A-type proteins (IscA and SufA)
playing a major role during [Fe-S] cluster biosynthesis, probably as
a scaffold protein for transient [Fe-S] cluster assembly and delivery
to target proteins.
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FOOTNOTES |
---|
* This work was supported by the French Greek Plato Collaboration Program of the Ministry of Foreign Affaires.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.:
33-4-38-78-91-03; Fax: 33-4-38-78-91-24; E-mail:
mfontecave@cea.fr.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M300285200
2 S. Ollagnier-de Choudens, L. Nachin, Y. Sanakis, L. Loiseau, F. Barras, and M. Fontecave, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: ISC, iron-sulfur cluster; DTT, dithiothreitol; BioB, biotin synthase; Fdx, ferredoxin; [Fe-S], iron-sulfur cluster; PLP, pyridoxal 5-phosphate; BPS, bathophenanthroline disulfonate; DAF, deazaflavin; VSV-G, vesicular somatidis virus-glycoprotein; Ni-NTA, nickel-nitrilotriacetic acid.
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