coli and purified.The purified protein was found to be mainly dimeric and did not contain any cofactor.Incubation with iron ions, cysteine and Synecho-cystisIscS led to the formation of on
Trang 1A dimer of the FeS cluster biosynthesis protein IscA from
cyanobacteria binds a [2Fe2S] cluster between two protomers
and transfers it to [2Fe2S] and [4Fe4S] apo proteins
Markus Wollenberg1, Carsten Berndt1, Eckhard Bill2, Jens D Schwenn1and Andreas Seidler1
1
Biochemie der Pflanzen, Fakulta¨t fu¨r Biologie, Ruhr-Universita¨t Bochum, Germany;2Max-Planck Institut fu¨r Strahlenchemie, Mu¨lheim, Germany
Two proteins with similarity to IscA are encoded in the
genome of the cyanobacterium Synechocystis PCC 6803
One of them, the product of slr1417 which accounts for
0.025% of the total soluble protein of Synechocystis was
over-expressed in E coli and purified.The purified protein
was found to be mainly dimeric and did not contain any
cofactor.Incubation with iron ions, cysteine and
Synecho-cystisIscS led to the formation of one [2Fe2S] cluster at an
IscA dimer as demonstrated (by the binding of about one
iron and one sulfide ion per IscA monomer) by UV/Vis,
EPR and Mo¨ssbauer spectroscopy.Mo¨ssbauer
spectro-scopy further indicated that the FeS cluster was bound by four
cysteine residues.Site-directed mutagenesis revealed that of
the five cysteine residues only C110 and C112 were involved
in cluster binding.It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers.The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min.Surprisingly, incubation
of cluster-containing IscA with apo adenosine 5¢-phospho-sulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster.This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units Keywords: assembly; cofactor; iron sulfur cluster; IscA; Synechocystis
Iron–sulfur proteins are widely distributed among the
organisms studied so far.Their main function is in electron
transfer but they also play roles in regulation and as sensors
[1].The assembly of FeS clusters can be achieved in vitro
from iron ions and sulfide in the presence of reductants
under exclusion of oxygen.However, because of their
toxiticity the concentrations of free Fe and sulfide in vivo is
extremely low.Therefore, a different mechanism is required
for cellular FeS cluster assembly.Recent work on the
assembly of the FeS cluster of nitrogenase and other
enzymes have revealed genes and proteins required for the
biological formation these clusters [2,3].In many bacteria
these proteins are encoded in an putative operon of at least
seven genes [4]
Sulfur is mobilized from cysteine by the action of the
enzyme IscS (or in the case of nitrogenase NifS), a cysteine
desulfurase [4,5].IscS (NifS) interacts with the iron-binding
protein IscU (NifU) where an iron–sulfur cluster is
assem-bled [6–9].Depending on the experimental conditions in vitro
one or two [2Fe2S] clusters can be assembled at IscU [10]
Apparently the two [2Fe2S] clusters can be rearranged to form one [4Fe4S] cluster.At least some eukaryotic IscU-like proteins (termed ISU) seem to assemble only [2Fe2S] clusters [11,12].This cluster has been shown to be transferred to a mitochondrial apo ferredoxin to generate
a holo ferredoxin carrying a [2Fe2S] cluster [12]
Other proteins which have been shown to be involved in FeS cluster biosynthesis are the two chaperones HscA and HscB.There are indications that HscA/B interact with IscU and it has been suggested that these proteins keep IscU in a conformation to facilitate FeS cluster assembly or the transfer of the cluster from IscU to the apo FeS protein [13,14].This would make IscU the key player in FeS cluster formation in apo FeS proteins.Although this is a possible mechanism for the majority of the organisms studied so far
it might not be the general pathway as there are some organisms which lack the typical IscU protein, for example some archea and the non-nitrogen fixing cyanobacteria Also some NifU proteins do not have similarity to IscU The NifU protein from Azotobacter vinelandii consists of three domains.The N-terminal domain is very similar in sequence and size to IscU, the central domain is similar to a protein carrying a [2Fe2S] cluster known as Bfd and the C-terminal domain is similar to NifU from some diazo-trophs such as Rhodobacter capsulatus [15].This C-terminal domain, the function of which is unknown, is also present as
a separate protein in other organisms that lack the typical IscU, for example in the cyanobacterium Synechocystis PCC 6803 [16,17]
Other proteins known to be important for FeS cluster synthesis are a [2Fe2S] ferredoxin of unknown function and
Correspondence to A.Seidler, Biochemie der Pflanzen, Fakulta¨t fu¨r
Biologie, Ruhr-Universita¨t Bochum, 44780 Bochum, Germany.
Fax: +49 234 321 43 22, Tel.: +49 234 322 45 49,
E-mail: Andreas.Seidler@ruhr-uni-bochum.de
Abbreviations: APS reductase, adenosine 5¢-phosphosulfate
reductase; Nif IscA, IscA protein encoded in the nif gene cluster;
orf, open reading frame.
(Received 22 October 2002, revised 14 January 2003,
accepted 12 February 2003)
Trang 2a protein termed IscA.This protein was shown to be
important for FeS cluster synthesis in Escherichia coli
[18,19], and yeast [20–22]
In the cyanobacterium Synechocystis there are three
open reading frames (ORFs) that encode proteins with
sequence similarity to IscS, one to the C-terminal domain
of IscU and two to IscA [16,17].Because of the lack of
operon organization the assignment of other genes to FeS
cluster biosynthesis is difficult.There are ORFs which
encode for proteins with sequence similarities to ferredoxin
and HscA/B found in isc gene clusters in other organisms
In this study we characterized one of the two IscA
proteins from Synechocystis, IscA1, the product of orf
slr1417
Experimental procedures
Construction of the expression plasmid
The ORF slr1417 was amplified from chromosomal DNA
of Synechocystis PCC 6803 by PCR using the primers
PRiscA11 (5¢-GGAATTCCATATGAGCCAAGCCACC
GCTACC-3¢) and PRiscA12 (5¢-GATCTAAGCTTAAA
CCCCAAAGGATTTACC-3¢).The resulting 376-bp
frag-ment was cleaved with NdeI and HindIII and cloned into
the expression plasmid pRSET5a [23] cleaved with the same
enzymes creating the plasmid pISCA1.The transcription of
slr1417on this plasmid is under control of the strongF10
-promoter of the T7 phage
Site-directed mutagenesis of the cysteine residues
Site-directed mutagenesis was carried out by the method
described by Kunkel et al.[24] for the introduction of single
Cys-to-Ala mutations and using the Stratagene Multi
Site Mutagenesis Kit for introducing multiple mutations
The following mutagenic oligonucleotides MOslr1417-1
5¢-CCCCCACCCGTAgcgctAGATCTTTGCCCTG-3¢,
MO-slr1417-2 5¢-GTAGGACATGCCAGAggcGCCCCCTTG
ACG-3¢, MOslr1417-3 5¢-GCAAACTTTTCCGaTCGgc
MOslr1417-5 5¢-CCAAAGGATTTACCggcgCCACAGG
TTTG-3¢ (lower case type indicates the introduced
muta-tions, new cleavage sites Eco47III, KasI, PvuI, PvuII and
KasI, respectively, are shown in italic type) were used to
change the codons of the Cys residues to Ala.The presence
of the mutations were verified by digestion with the enzymes
cleaving the introduced restriction sites and the sequence of
the gene from one clone for each mutagenesis was confirmed
by DNA sequencing
Protein over-expression and purification
The expression plasmids for the expression of IscA1 and its
variants were transformed into E coli strain BL21(DE3)
containing the plasmid pLysS.Cells were grown in Luria–
Bertani (LB) medium containing 100 mgÆL)1ampicillin and
50 mgÆL)1chloramphenicol at 25C up to an D600¼ 0.6
Then expression was induced by addition of 0.5 mM
isopropyl thio-b-D-thiogalactoside.Six hours after
induc-tion the cells were harvested by centrifugainduc-tion at 6000 g and
4C for 10 min, resuspended in 50 mM Hepes/NaOH/
10 mMEDTA and stored at)70 C until further use.Cell lysis was achieved by thawing the cells and completed by two additional cycles of freezing and thawing.Then MgCl2 was added to a final concentration of 15 mM as well as
125 U Benzonase (Boehringer, Mannheim) per 10 mL buffer.After incubation for 1 h on ice cell debris was sedimented by centrifugation (15 000 g for 30 min at 4C) The sediment from 1 L cell culture was resuspended in
2 5 mL 50 mMHepes/NaOH/10 mMEDTA and sedimented
as before.Residual membrane fragments in the combined supernatants were sedimented by ultra centrifugation at
100 000 g and 4C for 1 h Removal of E coli DNA was carried out by adding streptomycin sulfate to a final concentration of 10 mM.After incubation on ice for 1 h the cell extract was cleared by centrifugation for 20 min at
20 000 g and 4C
In order to purify IscA1 solid ammonium sulfate was added to a final concentration of 1.23M.After incubation for 1 h at 0C the precipitate was removed by centri-fugation as above.More ammonium sulfate was added until a final concentration of 2.05M was reached.After another hour of incubation at 0C the precipitated proteins were sedimented by centrifugation at 20 000 g and 4C for 20 min.The sediment was dissolved in 5 mL
20 mM Hepes/NaOH pH 8.0 Residual amounts of ammonium sulfate were removed by dialysis against the same buffer
Further purification was achieved by hydrophobic interaction chromatography.A Butyl-Sepharose column (2.6· 15 cm, Pharmacia) connected to a Bio-CAD 700E workstation (PerSeptive Biosystems) was equilibrated with
20 mM Hepes/NaOH pH 8.0 containing 760 mM ammo-nium sulfate and 1 mMdithiothreitol (buffer A).The flow rate was 5 mLÆmin)1 throughout chromatography.A protein solution containing 80 mg protein was adjusted
to 760 mMammonium sulfate and 1 mMdithiothreitol and loaded onto the column.The column was then washed with 140 mL buffer A.Elution of IscA was carried out with a gradient of 760 mM to 0 mM ammonium sulfate
in 20 mM Hepes/NaOH pH 8.0, 1 mM dithiothreitol in
420 mL
The pooled fractions containing IscA1 were dialysed against 10 mMsodium phosphate pH 6.5, 1 mM dithiothre-itol (buffer B), concentrated and further purified by hydroxy apatite chromatography.A hydroxy apatite col-umn (0.5· 5 cm, Pharmacia) was equilibrated with buffer B.The flow rate was 1 mLÆmin)1throughout the chroma-tography.The protein (2 mg) was loaded and pure IscA1 was eluted with buffer B while the contaminating proteins were kept bound to the column material.Pooled fractions containing IscA1 were concentrated and dialysed against
20 mMHepes/NaOH pH 8.0
SynechocystisIscS (Slr0387) was overexpressed in E coli and purified as described in [25] Synechocystis ferredoxin (Ssl0020) was overexpressed in E coli according to Barth
et al [26].Purification was carried out as described by Jaschkowitz and Seidler [25].Expression and purification of Catharanthus roseus adenosine 5¢-phosphosulfate (APS) reductase was carried out as described by Prior et al [27] The activity expressed as sulfite formed per mg protein and reaction time was usually about 7.5 l Æmg)1Æmin)1
Trang 3Incorporation of the FeS cluster into IscA1
and variants
Incorporation of the FeS cluster into IscA1 and variants
was achieved by incubating IscA1 (concentration range: 50–
200 lM) with five equivalentsL-cysteine and two equivalents
Fe(NH4)2(SO4)2 (sometimes Fe(III) ammonium citrate)
under anaerobic conditions in 20 mM Hepes/NaOH
pH 8.0 and 85 mM 2-mercaptoethanol.Other
concentra-tions of Fe2+and cysteine up to a 10-fold molar excess were
also used in some experiments but did not lead to a higher
Fe or sulfide content in IscA1.The reaction was started with
the addition of catalytic amounts of IscS.After 2 h of
incubation the reaction was stopped by gel filtration using
spin columns (0.5· 8 cm) filled with Sephadex G25 or a
PD 10 column (Pharmacia), both equilibrated with 20 mM
Hepes/NaOH pH 8.0 For Mo¨ssbauer samples metallic
57Fe was dissolved in H2SO4and titrated with ammonium
hydroxide to pH2
The stability of the cluster was investigated by
incuba-tion of holo IscA1 in 20 mM Hepes/NaOH pH 8.0 in the
presence and absence of oxygen and a reductant (5 mM
dithiothreitol or 85 mM 2-mercaptoethanol) at 25C
FeS cluster transfer from IscA1 to apo ferredoxin
Apo ferredoxin was obtained from holo ferredoxin as
described by Meyer et al [28].To prevent oxidation of the
sulfhydryl groups of ferredoxin 10 mM dithiothreitol was
added to all solutions.Concentration of apo ferredoxin was
determined by a Bradford assay with BSA for the
calibra-tion curve; the correccalibra-tion factor used was determined by
comparison of the holo ferredoxin concentration assayed
according to Bradford and using the extinction coefficient
(E423nm¼ 6400M )1).For the FeS cluster transfer reaction
2.5 nmol apo ferredoxin and 5 nmol holo IscA1 were
incubated in 100 lL 20 mMHepes/NaOH pH 8.0
contain-ing 5 mMdithiothreitol in argon atmosphere for 2 h unless
indicated otherwise.Analysis of the transfer reaction was
made by nondenaturing PAGE using 20% polyacrylamide
gels
FeS cluster transfer from IscA1 to APS reductase apo
protein fromCatharanthus roseus
The [4Fe4S] cluster of the APS reductase was removed by
treatment of the enzyme (15 lMin 100 mMHepes/NaOH
pH 8.0) with 0.3 mM K3Fe(CN)6 and 0.75 mM EDTA
with a concomitant loss of activity.The apo protein was
then purified by gel filtration using a PD10 column
(Pharmacia) and 100 mMHepes pH 8.0 as column buffer
Cluster transfer was obtained by incubation of 200 pmol
apo APS reductase with 800 pmol IscA1 carrying a
[2Fe2S] cluster in 300 lL 20 mM Hepes/NaOH pH 8.0/
5 mM dithiothreitol.In control experiments the same
quantity of apo APS reductase was incubated with IscA
without cluster and with 800 pmol Fe(NH4)2(SO4)2 and
800 pmol Na2S.After certain time intervals aliquots with
100–200 ng APS reductase were removed and diluted to
30 lL with 10 mM Tris/HCl pH 8.0 Then 70 lL of
100 mM Tris/HCl pH 8.0, 100 mM NaSO3, 500 mM
NaSO, 60 lM[35S]APS and 10 mM reduced glutathione
were added and incubated for 3 min at 30C.The reaction was stopped by adding 0.1 mL acetone and the amount of acid volatile sulfite was determined as described
by Schwenn and Schriek [29]
Spectroscopic methods Absorption spectra were recorded with a Beckman DU7400 diode array spectrophotometer.X-band EPR spectra were recorded with a Bruker ESP 300E spectrometer equipped with a helium flow cryostat (Oxford Instruments ESR 910),
an NMR Gaussmeter and a Hewlett Packard Frequency counter
Mo¨ssbauer data were recorded with a spectrometer of the alternating constant–acceleration type.The minimum experimental line width was 0.24Æmm s)1 (full width at half-height).The sample temperature was maintained constant either in an Oxford Instruments Variox or an Oxford Instruments Mo¨ssbauer-Spectromag cryostat.The latter is a split-pair superconducting magnet system for applied fields up to 8 T where the temperature of the sample can be varied in the range 1 5–250 K The field at the sample
is perpendicular to the c-beam.The 57Co/Rh source (1.8 GBq) was positioned at room temperature inside the gap of the magnet system at a zero-field position.Isomer shifts are quoted relative to iron metal at 300 K
MS The identity of the protein was confirmed by MALDI-TOF MS using a Voyager System DEPRO 6061 (PerSep-tive Biosystems).A saturated solution of sinapinic acid in 0.05% trifluoroacetic acid and 25% acetonitrile was used
as matrix
Determination of sulfide and iron Determination of sulfide and iron were carried out as described by Siegel [30] and Fish [31]
Gel electrophoresis and Western blotting SDS/PAGE was carried out according to Seidler [32] and the gels were stained with Coomassie brilliant blue.Non-denaturing gel electrophoresis was carried out according to Laemmli [33] except that SDS was omitted.The samples loaded on to the gel contained 20 mMdithiothreitol.The gels were subsequently stained with 0.1 mM Stains All (Sigma) in 30 mM Tris/HCl pH 8.8, 10% formamide and 25% isopropanol.Destaining was carried out after washing with water and illumination with white light on an overhead projector for 1–2 min
Results
In Synechocystis PCC 6803 there are two ORFs (slr1417, iscA1 and slr1565, iscA2) of which the deduced amino acid sequence showed homology to IscA.IscA proteins are characterized by the presence of three conserved cysteine residues of which two are in a CGCG (or in case of SynechocystisIscA2 CSCS) motive (Fig.1).Synechocystis IscA1 has two additional cysteine residues which are also
Trang 4present in two other proteins found in oxygenic
photosyn-thetic organisms, Arabidopsis thaliana and the red algae
Porphyra purpurea.According to a prediction using the
computer program TARGET P the Arabidopsis protein is
localized in the chloroplast.In Porphyra purpurea the
protein is encoded by the chloroplast genome
Western blotting with antibodies raised against the
purified product of ORF slr1417 revealed that this ORF
represents a true gene encoding a protein with an apparent
molecular mass of 13 kDa (Fig.2).Using the recombinant
purified protein as standard it was estimated that this IscA
protein represents 0.025% of the total soluble protein of
Synechocystis
Over-expression, purification and characterization
of IscA1 The ORF slr1417 was amplified by PCR from chromoso-mal DNA of Synechocystis PCC 6803 and cloned into the expression plasmid pRSET5a.One clone was sequenced and the sequence of the cloned DNA fragment was found to
be identical with the sequence deposited in the cyanobase Expression was carried out with the strain BL21(DE3)/ pLysS/pISCA1.Six hours after induction a protein with an apparent molecular mass of 13 kDa accumulated to 15%
of the soluble proteins of E coli (Fig.3).This molecular mass is very similar to the 12 929 Da calculated from the
Fig 1 Sequence alignment of IscA protein from various organisms The conserved cysteine residues are marked by asterisks.Accession numbers are Slr1417 (Synechocystis IscA1) NP_440066, Athal1 (Arabidopsis thaliana IscA1) AC007067.4, P_purp (Porphyra purpurea) NP_053827, Athal2 (Arabidopsis thaliana IscA2) AC005825.3, Athal3 (Arabidopsis thaliana IscA3), AC006921.5, A_vinIscA (Azotobacter vinelandii IscA) T44283, A_vinOrf6 (Azotobacter vinelandiiNifIscA) Q44540, Slr1565 (Synechocystis IscA2) NP_442892.
Trang 5DNA sequence of IscA1.The protein was purified by
ammonium sulfate precipitation, hydrophobic interaction
and finally hydroxy apatite chromatography with a yield of
55 mg per litre E coli culture.The identity of the protein
was verified by MALDI-TOF MS.The purified protein had
a molecular mass of 12798 Da (data not shown) indicating
that the start methionine was removed by E coli after
protein translation
Incorporation of an FeS cluster into IscA1
IscA1 was incubated unaerobically with IscS, cysteine and
either Fe(II)(NH4)2(SO4)2 or iron(III) ammonium citrate
With both iron sources the sample turned brownish and this
colour remained even after gel filtration using a Sephadex
G25 column.The protein exhibited an absorption spectrum
with maxima at 330, 420 (with a shoulder at 470) and
580 nm (Fig.4) which is indicative for the formation of a
[2Fe2S] cluster.A small absorption maximum at 325 nm was also observed in the apo protein.This is due to a covalent modification in a minor fraction of the protein as this absorption was still present after dialysis for 24 h against 8M urea/5 mM dithiothreitol.MS revealed small peaks at M + 59 Da and M + 96 Da (M¼ 12 798 Da) which we have not been able to assign unambiguously.This modification might be localized close to the amino acid residues C110 and/or C112 as this absorption is missing in variants where one of these residues is replaced by alanine (see Fig.7)
Iron and sulfide content of IscA1 after reconstitution of the FeS cluster was determined to be 1.2 and 0.9, respect-ively, per protein monomer, irrespective of the concentration
of Fe and cysteine used (up to 10-fold molar excess).Gel filtration experiments revealed that IscA was present either
as a dimer ( 65%) or as a tetramer ( 35%) (Fig.5) The stability of the FeS cluster depended strongly on the experimental conditions.In the presence of oxygen and in the absence of a reductant the cluster had at 25C a half-life time
of 10 min.The stability increased when either oxygen was omitted or a reductant (dithiothreitol or 2-mercaptoethanol) was added (t1/2 180 min).The addition of a reductant to
an anaerobic solution of IscA1 containing the FeS cluster had no effect on the cluster stability
Spectroscopic characterization of the FeS cluster
in IscA1
In order to study the nature of the FeS cluster in IscA1 EPR experiments were carried out.The cluster was found to be EPR silent as only a minor g¼ 4.3 signal from ubiquitously and nonspecifically bound ferric ions was detected and there was none of the typical S¼ 1/2 spectra, indicating that the FeS cluster was in a diamagnetic state.Addition of dithionite to the protein solution did not lead to the appearance of an EPR signal, and neither addition of dithionite nor illumination of the sample with strong white light in the presence of FMN and EDTA resulted in any
Fig 4 Absorption spectrum of IscA1 with and without FeS cluster The protein concentration was 30 l M in 20 m M Hepes/NaOH pH 8.0 The spectra were recorded under Argon atmosphere.
Fig 2 Western blot of purified recombinant IscA1 and soluble protein
extract from Synechocystis Lanes 1, 2 and 3: 7.5, 15 and 30 ng purified
recombinant IscA1, respectively; lane 4: 60 lg soluble Synechocystis
proteins.
Fig 3 SDS/PAGE of IscA1 at various stages of its purification.
Lane 1, molecular mass standard; lane 2, crude extract from E coli
BL21(DE3)/pLysS/pISCA1; lane 3, IscA1 after ammonium sulfate
precipitation; lane 4, IscA1 after hydrophobic interaction
chroma-tography; lane 5, IscA1 after hydroxy apatite chromatography.
Trang 6change in the absorption spectrum which would indicate a
reduction of the FeS cluster
To get more information about the nature of the FeS
cluster Mo¨ssbauer spectroscopy was applied.The zero-field
spectrum obtained at 80 K could be fitted with two or three
superimposing symmetric Lorentzian line doublets, where
the main species had an isomer shift of d¼ 0.27 and a
quadrupole splitting DEQ¼ 0.57 mmÆs)1and contributed
with 86% to the spectrum (Fig.6 left panel, Table 1).The parameters are typical for [2Fe2S]2+clusters ligated by four cysteine residues.The doublet remained almost unchanged except for a small line broadening when an external field of
10 kG was applied at 80 K confirming the diamagnetic nature of the FeS cluster.A minor subspectrum with 12% relative intensity and isomer shift of d¼ 0.50 mmÆs)1was split into a hardly resolved magnetic six-line pattern in the applied field (data not shown).The high isomer shift which clearly excludes sulfur coordination of the iron sites and the magnetic behaviour strongly indicate the presence of some nonprotein bound (superparamagnetic) iron(III) aggregates that precipated during iron incubation.We mention that their amount was less in the better assembly assays of the various experiments.A third subspectrum that was observed only in some preparations has a high isomer shift of d¼ 1.3 mmÆs)1 and a quadrupole splitting of
DEQ¼ 1.3 mmÆs)1 which unambiguously indicate the presence of residual high-spin Fe(II) starting agent.The results of the iron and sulfur determination together with the spectroscopic data strongly indicate that a dimer of IscA can bind one [2Fe2S]2+cluster
Residues involved in ligation of the FeS cluster The symmetric Mo¨ssbauer subspectrum of the FeS cluster and the low isomer shift are typical of tetrahedral sulfur coordination for both iron sites and, hence, indicated all-cysteine ligation of the FeS cluster in IscA1.Because IscA has only three conserved cysteine residues it was possible that the [2Fe2S] cluster is bound in between two molecules
of IscA.However, it could not be excluded that the two additional cysteine residues play a role in FeS cluster binding in Synechocystis IscA1.To investigate which residue
is involved in cluster binding and assembly one, two or three cysteine residues were replaced by alanine using site-directed
Fig 6 Zero field Mo¨ssbauer spectra of IscA1 and the variant IscAC44A at 80 K The FeS cluster was assembled as described in Experimental procedures.The protein concentration was adjusted to 0.4 m M Crosses indicate the measured data points, [2Fe2S] 2+ , Fe(III) and Fe(II) the calculated subspectra.
Fig 5 Gel filtration analysis of IscA containing a [2Fe2S] cluster.
Reconstituted IscA1 was loaded onto a Superose 12 HR 10/30 column
(Pharmacia) pre-equilibrated with a buffer containing 4 m M KH 2 PO 4 ,
16 m M Na 2 HPO 4 , 115 m M NaCl, 1 m M dithiothreitol.The flow rate
was 0.7 mLÆmin)1.The elution was monitored by absorption at
280 nm.Inset: calibration of the column using molecules with known
molecular masses.The following molecules were used: bovine c-globin
(158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa),
vitamin B12 (1.35 kDa).
Trang 7mutagenesis.The protein variants were over-expressed and
purified as the wild-type protein
Mutagenesis of residues C34 and C75, the less well
conserved residues, did not abolish the ability of IscA1 to
bind a [2Fe2S] cluster (Fig.7) However, for unknown
reasons the extent of cluster binding by the C75A variant
was found to be lower in all cluster insertion experiments.In
contrast, the extent of cluster formation in the variants
C34A and C34/75A was usually slightly higher than in the
unmodified protein (Fig.7).The Mo¨ssbauer spectrum of
the variant C34/75A could be fitted without any
d¼ 0.50 mmÆs)1contribution which also indicates a more
complete formation of the FeS cluster than in the
unmodi-fied protein (Table 1)
The variant C44A was also able to bind a [2Fe2S] cluster
The UV/Vis spectrum of this variant showed absorption
maxima at 330, 420 and 580 nm like the unmodified protein
However, the absorption of this variant in the visible region
was significantly lower and the peaks are less well resolved
The Mo¨ssbauer spectrum (Fig.6, right panel) could be fitted
with three doublets at d¼ 0.27, 0.50 and 1.30 (Table 1) The
contribution of the doublet originating from the [2Fe2S]2+ cluster (d¼ 0.27) was 39.4% which is considerably lower than in the unmodified protein.The doublet at d¼ 1.30 clearly originated from Fe(II).This result has two possible causes: either the FeS cluster was less stable or the assembly
of the cluster at C44A was impaired.However, in the presence of oxygen the cluster was found to have the same half-life time as that of the unmodified protein in 20 mM Hepes/NaOH pH 8.0, and was completely stable under reducing conditions.In the absence of oxygen and any reductant the FeS cluster was found to be slightly less stable than in the modified protein (t1/2 80 min compared to
180 min in the unmodified protein).When the cluster formation at IscA1 and the variant C44A was compared it appeared that the cluster formation was retarded (data not shown).This demonstrates that the cluster formation at the variant C44A was impaired
The variant C34/44/75A in which the three cysteine residues at positions 34, 44 and 75 were replaced by alanine was also able to bind a FeS cluster.The absorption spectrum was very similar to that of the variant C44A (data not shown).Together with the results obtained with the variants containing single mutations this indicated that none
of the three residues was involved in cluster binding
In variants where one of the two cysteine residues in the conserved CGCG motive (C110 or C112) were replaced by alanine no cluster formation was obtained (Fig.7).These data confirmed the above drawn conclusion that the [2Fe2S] cluster is bound by a dimer of IscA.Both protomers provide two ligands, C110 and C112
FeS cluster transfer to apo ferredoxin
As IscA was shown to be involved in FeS cluster assembly the potential transfer of the unstable FeS cluster to apo ferredoxin was studied.FeS cluster-containing IscA1 was incubated anaerobically with apo ferredoxin for 1 h.During this time the colour of the solution changed from brownish
to brown-red, a colour typical for holo ferredoxin.The mixture was analysed by nondenaturing PAGE where holo and apo forms of both proteins showed different mobility (Fig.8A) During incubation of 40 nmol IscA (20 nmol IscA dimer) with 20 nmol apo ferredoxin 80% of the apo ferredoxin was transformed into holo ferredoxin.In addi-tion, the absorption spectrum was typical for ferredoxin and different from the spectrum of FeS cluster-containing IscA1 (Fig.8C)
The kinetics of the transfer reaction was analysed Already after 2 min of incubation a significant portion ( 20%) of ferredoxin contained the FeS cluster, 50% of the centres were transferred after about 10 min (Fig.8b) Since C44 is totally conserved in all IscA proteins and since it has been shown for the yeast protein that this residue
is essential for the function of IscA in vivo [20,21] we tested the possibility of whether this residue plays a role in the cluster transfer.The variant C44A was reconstituted with the FeS cluster and subsequently incubated with apo ferredoxin.Samples analysed between 2 and 60 min after mixing of the two proteins did not reveal any difference in cluster transfer kinetics (data not shown).The same results were obtained with the variant C34/75A where the two additional cysteine residues were replaced by alanine
Fig 7 Absorption spectrum of IscA1 and the variants with single
cys-teine-to-alanine substitutions after assembly of the FeS cluster The
protein concentration was 30 l M in 20 m M Hepes/NaOH pH 8.0 The
spectra were recorded under argon atmosphere.
Table 1 Mo¨ssbauer parameters used for fitting of the signals obtained
from IscA and its variants.
Fe species d [mmÆs)1] DE Q [mmÆs)1] % of total signal
IscA1
IscA1-C34/75A
IscA1-C44A
Trang 8FeS cluster transfer to APS reductase apo protein
fromCatharanthus roseus
In order to investigate the assembly of [4Fe4S] cluster by
IscA1 the FeS cluster of APS reductase was removed by
treatment with K3Fe(CN)6and EDTA with a concomitant
loss of enzyme activity.Incubation of the apo protein with
two equivalents holo IscA dimer resulted in the restoration
of 40% of the initial activity (Fig.9).No reactivation was
observed when apo IscA or iron and sulfide ions were added
to the apo APS reductase.This demonstrates that the
[4Fe4S] cluster in APS reductase can be assembled from
[2Fe2S] precursors at IscA
Discussion
A number of proteins have been shown to be involved in the
assembly of FeS clusters although the exact function of
these proteins remained unclear.In this work we studied the
properties of the prominent form of Synechocystis IscA, the
product of orf slr1417, in vitro.A dimer of IscA1 was able to
bind a [2Fe2S] cluster.This cluster is in the Fe(III)Fe(III)
state as demonstrated by the absence of any EPR signal and
by Mo¨ssbauer spectroscopy.The oxidation state of the FeS
cluster was unrelated to the oxidation state of the iron ions
used for the assembly.This oxidation state is the only stable
state we have observed.All attempts to reduce (or oxidize)
this cluster were unsuccessful
This is in agreement with findings by
Ollagnier-de-Choudens et al.[34] and Krebs et al.[35] who also
observed an assembly of an FeS cluster at IscA from E coli and A vinelandii, respectively.In contrast with A vinelandii NifIscA we could not find experimental conditions which allowed the assembly of a [4Fe4S] cluster at IscA1 from Synechocystis
The question of which residues are involved in cluster binding was addressed by site-directed mutagenesis.Muta-genesis of C34, C44 and C75 had no effect on cluster
Fig 8 FeS cluster transfer from IscA1 to apo ferredoxin (A) Apo
ferredoxin was incubated with IscA1 carrying a FeS cluster.After 1 h
an aliquot containing 5 lg ferredoxin was removed and analysed by
nondenaturing PAGE.The gel was stained with Stains All.Lane 1,
5 lg holo ferredoxin; lane 2, 5 lg apo ferredoxin; lane 3, 10 lg holo
IscA1; lane 4, 5 lg ferredoxin after 1 h of incubation with IscA
car-rying a [2Fe2S] cluster.For unknown reasons apo ferredoxin was
stained poorly by Coomassie brilliant blue.Therefore, Stains All was
used which stained apo and holo ferredoxin equally well.However,
IscA was stained poorly by this dye.(B) Kinetics of the FeS cluster
transfer from IscA to ferredoxin.Apo ferredoxin (7.5 nmol) was mixed
anaerobically with 15 nmol holo IscA1 in 0.3 mL 20 m M Hepes/
NaOH pH 8.0/5 m M dithiothreitol.Aliquots of 40 lL were removed
after 2, 5, 10, 20, 30 and 60 min and the transfer was stopped by
addition of 20 nmol K 3 Fe(CN) 6 and 50 nmol EDTA which led to an
immediate destruction of the FeS cluster at IscA.The samples were
then frozen in liquid nitrogen and stored until they were analysed by
nondenaturing PAGE.From each aliquot removed after a certain time
5 lg ferredoxin was loaded onto a nondenaturing polyacrylamide gel.
(C) Absorption spectrum of ferredoxin after FeS cluster transfer from
IscA1.One hour after holo IscA1 and apo ferredoxin were mixed the
reductant was removed by gel filtration using a Sephadex G25 column.
In a control experiment apo ferredoxin was omitted.After incubation
for 45 min at 25 C in the presence oxygen absorption spectra were
recorded from both samples.The spectrum of the control sample
without ferredoxin was subtracted from the spectrum of the sample
were the FeS cluster was transferred from IscA to apo ferredoxin (solid
line).This spectrum and the spectrum of purified holo ferredoxin
(dotted line) were normalized at 278 nm.
Trang 9binding and stability.However, when C44 or C75 were
replaced by alanine the cluster assembly seems to be
partially impaired.The Mo¨ssbauer spectrum of the variant
C44A showed the same doublet at d¼ 0.27 mmÆs)1as the
unmodified protein.The additional doublet at
d¼ 0.50Æmm s)1 was attributed to mesoscopic iron(III)
aggregates which were copurified with the protein upon gel
filtration.Treatment of FeS cluster-containing IscA1 with
ferri cyanide converted the iron species with 0.27 mmÆs)1
completely into the d¼ 0.50 mmÆs)1 form indicating a
destruction of the FeS cluster (data not shown).The
equivalent residue in Saccharomyces serevisiae IscA (178 in
the Saccharomyces ISA1 sequence) was found to be essential
for the function of this protein [20,21] In vivo it might have a
role in the assembly or stabilization of the FeS cluster.Krebs
et al.[35] suggested that two of the three fully conserved
cysteine residues are involved in cluster binding whereas the
third one provides an electron during cluster assembly for
the reduction of the cysteine persulfide at NifS/IscS.We
were unable to verify this hypothesis as we obtained a
reduced cluster assembly under the conditions used in Krebs
et al.[35] (no reductant, 8 mMcysteine).Further studies are
required to clarify the role of residue C44
Replacement of C110 and C112 led to a complete loss of
cluster-binding ability of IscA.Since the Mo¨ssbauer data
indicated an all-cysteine ligation of the cluster and the
variant C34/44/75A was still able to bind a FeS cluster we
concluded that the [2Fe2S] cluster is bound by C110 and
C112 in between the two protomers of the IscA dimer
The FeS cluster assembled at IscA could be transferred to
apo ferredoxin by incubation of the two proteins in the
presence of a reductant.This transfer was rapid because
already after 10 min 50% of the cluster was transferred.As
apo ferredoxin is also easily reconstituted by sulfide and iron
ions (data not shown) it was necessary to confirm the stability of the FeS cluster at IscA1 in the time course of the transfer experiment.However, in the presence of a reductant
no release of iron or sulfide was observed during the time course of the experiment
The mechanism of the cluster transfer is not known so far.However, all attempts to demonstrate a protein–protein interaction in between IscA and apo ferredoxin have failed (M.W and A.S., unpublished data) Since cluster transfer is also very efficient from Synechocystis IscA1 to apo FeS proteins from Catharanthus and Bacillus subtilis (C.Berndt, M.Wollenberg, E.Bill, A.Seidler and J.-D.Schwenn, unpublished data) it is possible that there is no specific docking site.In addition, IscA might provide FeS clusters for several or all FeS proteins in Synechocystis and other cyanobacteria which are quite numerous and it is difficult to believe that all apo FeS proteins have a specific docking site for holo IscA.It is possible that the presentation of thiol groups with a certain geometry leads to a replacement of two of the cysteine side chains provided by one IscA protomer.In a subsequent step the two cysteine side chains from the other IscA protomer might then be replaced by cysteine side chains from the FeS protein
It is somewhat surprising that the [2Fe2S] cluster containing IscA was able to reconstitute the [4Fe4S] cluster
at the apo APS reductase and at the phosphoadenosine 5¢-phosphosulfate reductase from Bacillus subtilis (C.Berndt, M.Wollenberg, E.Bill, A.Seidler and J.-D.Schwenn, unpublished data).This implies that either two IscA1 dimers react with one molecule of APS reductase at the same time
or, more likely, that the APS reductase binds transiently a [2Fe2S] cluster.So far only IscU [10],NifIscA [35] and FNR from E coli [36] have been reported to be able to bind both
a [2Fe2S] or a [4Fe4S] cluster at the same or overlapping sites.Since free iron and sulfide ions could not be incorpor-ated in apo APS reductase as shown by control experiments
in the absence of IscA we concluded that the FeS cluster is directly transferred from IscA.NifIscA from A vinelandii is able to assemble a [4Fe4S] cluster which might be transferred to nitrogenase apo protein which as holo protein contains a [4Fe4S] cluster.We concluded therefore that SynechocystisIscA assembles only [2Fe2S] clusters in order
to provide [2Fe2S] units for [2Fe2S] and [4Fe4S] apo proteins whereasNifIscA might supply [2Fe2S] or [4Fe4S] units for the polynuclear centres of nitrogenase.Further experiments are underway to study the assembly of the [4Fe4S] clusters by Synechocystis IscA
Acknowledgements
We thank D.Kessler for helpful discussions and U.Kokelj for excellent technical assistance.B.Lagoutte is gratefully acknowledged for the plasmid for the expression of Synechocystis ferredoxin and R.Scho¨pfer for the plasmid pRSET5a.This work was supported by the Deutsche Forschungsgemeinschaft and the Ministry of Sciences and Research of the German federal state of Nordrhein-Westfalen (Bennigsen-Foerder Program to A.S).
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