BacterialIscUisawellfoldedandfunctionalsingledomain protein
Salvatore Adinolfi
1
, Francesca Rizzo
1
, Laura Masino
1
, Margie Nair
1
, Stephen R. Martin
1
, Annalisa Pastore
1
and Piero A. Temussi
1,2
1
National Institute of Medical Research, London, UK;
2
University of Naples Federico II, Napoli, Italy
Iron–sulfur clusters are widely represented in most organ-
isms, but the mechanism of their formation is not fully
understood. Of the two main proteins involved in cluster
formation, NifS/IscS and NifU/IscU, only the former has
been well studied from a structural point of view. Here we
report an extensive structural characterization of Escherichia
coli IscU. We show by a variety of physico-chemical tech-
niques that E. coli IscU construct can be expressed to high
purity as a monomeric protein, characterized by an ab fold
with high a-helix content. The high melting temperature and
the reversibility of the thermal unfolding curve (as measured
by CD spectroscopy) hint at awell ordered stable fold. The
excellent dispersion of cross peaks in the
1
H-
15
N correlation
spectrum is consistent with these observations. Monomeric
E. coli IscUis able to provide a scaffold for Iron–sulfur
cluster assembly, but has no direct interaction with either
Fe(II) or Fe(III) ions, suggesting the need of further partners
to achieve a stable interaction.
Keywords: Friedreich ataxia; iron–sulfur cluster; NMR;
thermal stability.
Metalloproteins hosting iron–sulfur clusters (isc) are
present in most organisms [1,2], and are involved in several
processes, including electron transport, generation of
organic radicals and regulatory processes. Although Iron–
sulfur clusters are widely diffuse in nature, the detailed steps
leading to their assembly are still mostly unknown. Owing
to the toxicity of iron and sulfide ions, it is probable that the
formation of Fe–S clusters is mediated by protein–protein
interactions. NifS and NifU, the specific proteins involved
in the building of Fe–S clusters were originally identified
within the nif operon of Azotobacter vinelandii [2], but have
counterparts in the isc family of other organisms. Most
genetic and biochemical studies hint at a mechanism for
prokaryotes in which IscS andIscU play a central role [3,4].
This mechanism however, is also preserved in eukaryotic
cells. IscS is analogous to NifS as it provides sulfane
equivalents to IscU via catalytic cysteine desulfurization [5].
IscU, like the previously characterized NifU [3] coordinates
a transient [2Fe)2S] cluster. Escherichia coli IscU is
homologous to the amino terminal domain of NifU with
which it shares three conserved cysteines and the binding of
a transient [2Fe-2S] cluster [6]. We undertook a systematic
study of the Isc proteins of E. coli. Considering the central
role of IscU in Fe–S cluster biosynthesis, suggested,
inter alia, by the fact that it is one of the most conserved
sequence motifs in nature and the fact that its three
dimensional structure has not yet been published, we
decided to start the study of bacterial IscU.
A further motivation for studying bacterialIscUis its
possible connection with CyaY, the bacterial orthologue of
frataxin, a small protein expressed at abnormally low levels
in Friedreich’s ataxia patients [7]. Consistent evidence shows
that Friedreich’s ataxia arises from disregulation of mito-
chondrial iron homeostasis, with concomitant oxidative
damage leading to neuronal death [8–13]. Accumulating
evidence suggests that frataxin is involved in iron meta-
bolism [14–20]. A possible function of CyaY in the complex
chain of events involved in Isc formation might be to supply
iron ions to IscU, as suggested by a recent report by Yoon &
Cowan [21] on the interaction between the corresponding
human orthologues. A detailed structural characterization
of IscU and, most of all, the nature of its interaction with
iron ions may help to clarify this function.
Using complementary biophysical and biochemical tech-
niques, we report here a structural characterization of this
protein and demonstrate that E. coli IscU can be obtained
as a recombinant well-folded protein. We demonstrate that
our construct can function as a scaffold for a transient Fe–S
cluster, but NMR chemical shift perturbation indicates that
E. coli IscU does not bind iron ions directly.
Materials and methods
Protein production
E. coli IscU was subcloned by PCR from bacterial genomic
DNA. The constructs were cloned into pET24d-derived
plasmid vectors (Novagen, Merck, Germany) as fusion
proteins with His-tagged glutathione S-transferase and a
cleavage site for tobacco etch virus protease which leaves,
after cleavage, only two additional amino acids (GlyAla) at
the protein N-terminus. The constructs were expressed in
E. coli strain BL21(DE3). For protein expression, the cells
were inoculated in Luria–Bertani medium with kanamycin
(30 mgÆL
)1
), induced for 3–4 h by addition of 0.5 m
M
isopropyl thio-b-
D
-galactoside. After the cultures reached
Correspondence to A. Pastore, National Institute of Medical Research,
The Ridgeway, London NW71AA, UK.
Fax: + 44 208 906 4477, Tel.: + 44 208 959 3666,
E-mail: apastor@nimr.mrc.ac.uk
Abbreviations: isc, iron–sulfur cluster.
(Received 19 February 2004, revised 22 March 2004,
accepted 24 March 2004)
Eur. J. Biochem. 271, 2093–2100 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04112.x
an attenuance of 0.6–0.8 at 600 nm, the cell pellets were
harvested and frozen. The frozen cells were thawed in a lysis
buffer (20 m
M
Tris/HCl, pH 8, 150 m
M
NaCl, 10 m
M
2-mercaptoethanol) and subsequently sonicated and centri-
fuged. The protein was purified by affinity chromatography
using Ni-nitrilotriacetic acid gel or a glutathione S-sepharose
column. Tobacco etch virus protease cleavage was then
obtained by incubating the protein overnight at 4 °C.
Further purification from glutathione S-transferase was
carried out by gel filtration chromatography on a G-75
column (Pharmacia).
15
N-labelled samples of IscU for
nuclear magnetic resonance studies were produced by
growing the bacteria in minimal medium using ammonium
sulfate as sole source of nitrogen.
The protein was desalted by dialysis against the final
buffer (either 20 m
M
Hepes/KOH pH 7.5 or 20 m
M
Tris/
HCl pH 7.5–8.0 with NaCl 50–150 m
M
and 20 m
M
2-mercaptoethanol) and concentrated with an Amicon
concentrator (model 8050; Amicon, Millipore, Billerica,
MA, USA). The purity of the recombinant protein was
checked by SDS/PAGE after each step of the purification
and by mass spectrometry of the final product. The
experimental mass of the IscU construct (13 977.9 Da), as
measured by electrospray mass spectrometry is in perfect
agreement with the expected value (13 976.7 Da). The
same protocol was used for the expression of E. coli IscS.
Probe of the oligomeric state of
E. coli
IscU in solution
Analytical gel filtration experiments were performed using a
prepacked HiLoad 10/30 Superdex 75 column (Pharmacia).
The column was equilibrated with Tris/HCl buffer (pH 8.0),
in the presence of 200 m
M
NaCl. Ovalbumin (43 kDa),
chymotrypsinogen A (25 kDa) and ribonuclease A
(13.7 kDa) were used as molecular standards for the mass
calibration. Samples of nonreconstituted IscU (1 mL in
20 m
M
Tris/HCl at pH 8.0, 150 m
M
NaCl and 10 m
M
2-mercaptoethanol) were loaded using a static loop
(1 mL) and were eluted with the same equilibrating buffer.
Sedimentation equilibrium experiments were carried out
using a Beckman XL-A analytical ultracentrifuge equipped
with UV absorption optics (Beckman Coulter Ltd, High
Wycombe, UK). The measurements were performed at
20 °C using speeds of 9000, 12 000 and 20 000 r.p.m. and
rotor An 60 Ti. Protein concentrations were in the range
10–40 l
M
. Data were recorded using different ionic strength
conditions (20 m
M
Tris/HCl at pH 8 with 50 m
M
or
150 m
M
NaCl and 10 m
M
2-mercaptoethanol). Each meas-
urement was repeated after 6 h to ensure that equilibrium
had been reached and that proteolysis was not occurring. In
all datasets, the absorbance of the depleted area at a final
speed of 40 000 r.p.m. provided an experimental value for
the baseline offset. The data were analysed with the
ORIGIN
XL
-
A
/
XL
1 package (Beckman) and fitted to the following
equation:
AðrÞ¼Aðr
0
Þexp Mðx
2
=2RTÞð1 À mqÞðr
2
À r
2
0
Þ
ÂÃ
where A(r) and A(r
0
) are the optical absorbances at radius r
and at the reference radius r
0
, respectively; M is the
molecular mass; x the angular velocity; R the gas constant;
T the absolute temperature; m the partial specific volume of
the solute and q is the density of the solvent. The equation
assumes the presence of asingle species at equilibrium. Data
fitting to this model yields an apparent average molecular
mass for all solutes in the cell.
CD and fluorescence studies
Far and near UV CD spectra were recorded on a Jasco
J-715 spectropolarimeter (Jasco UK Ltd, Great Dunmow,
Essex, UK) equipped with a cell holder thermostatted by
aPTC-348Peltiersystem.FarUVmeasurementswere
performed in 10 m
M
buffer at pH 7.5 using protein
concentrations of 7–35 l
M
from two independent protein
preparations. The spectra were recorded in fused silica
cuvettes of 1 mm path length. Ten scans were averaged and
the appropriate buffer baseline was subtracted. Spectral
decomposition for secondary structure predictions was
achieved by combining the CONTIN, SELCON and
CDSSTR methods [22].
Near UV spectra required a 2 mm path length cuvette
and a 490 l
M
protein concentration. Variations of the CD
signal were studied as a function of temperature over the
range 10 °Cto95°C using a heating rate of 1 °Cper
minute.
Fluorescence measurements were recorded on a SPEX
Fluoromax spectrometer (Glen Spectra Ltd, Middlesex,
UK) fitted with a thermostatically controlled jacketed cell
holder and interfaced with a Neslab RTE-111 water bath
(Thermo-Neslab, Portsmouth, UK). Fluorescence emission
spectra in the range 300–450 nm were recorded at 20 °C
with an excitation wavelength of 290 nm.
Nuclear magnetic resonance spectroscopy
NMR spectra at 25 °CwererecordedonVarianINOVA
spectrometers (Varian Ltd, Walton-on-Thames, UK) oper-
ating at 500, 600 and 800 MHz
1
H frequency. Typically,
0.3–0.5 m
M
unlabelled or
15
N uniformly labelled protein
samples were used. Water suppression was achieved by the
Watergate pulse-sequence [23]. The spectra were processed
and zero-filled to the next power of two using the
NMRPIPE
program [24]. Baseline correction was applied when neces-
sary. The spectra were analyzed using the
FELIX
(MSI) and
XEASY
programs [25].
Experimental
15
NT
1
and T
2
relaxation times and
heteronuclear
1
H-
15
N NOE values were measured on
uniformly labelled
15
N IscU 0.3 m
M
samples in 20 m
M
Tris/HCl at pH 7.0, 50 m
M
NaCl and 10 m
M
dithio-
threitol. The spectra were acquired at 11.7 T (500 MHz
proton frequency) using standard pulse sequences [26] and
analyzed using
NMRPIPE
/
NMRDRAW
or
XEASY
.ForT
1
and T
2
measurements, peak intensities were determined
for 109 amide resonances as a function of the relaxation
delay and the data were then fitted by least-squares fitting
to asingle exponential. Correlation times were calculated
from the T
1
/T
2
ratios according to the so-called model-
free approach [27]. Experimental
1
H-
15
N steady state
NOE values were determined from the peak intensity
ratios of amide resonances obtained by recording inter-
leaved 2D Watergate
1
H-
15
N HSQC spectra with and
without a saturation delay of 4 s anda repetition delay of
4.2 s [28].
2094 S. Adinolfi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
NMR titrations
Titration of IscU with Fe(II) was carried out by NMR,
typically starting with 0.4 m
M
protein in 20 m
M
Tris/HCl,
50 m
M
, NaCl at pH 7 and adding aliquots of ferrous
ammonium sulfate up to a ratio of 1 : 6.
Fe–S cluster biogenesis
Typical experiments of Fe–S cluster biogenesis were carried
out by adding 4 m
ML
-cysteine to a reaction mixture
containing 76 l
M
IscU in the presence of different IscS
ratios, chosen to have formation kinetics compatible with
the spectroscopic measurements (the optimal ratio is
% 1 : 28 IscS/IscU according to Kispal et al.[4]).Afivefold
excess (relative to IscU concentration) of freshly prepared
ferric ammonium citrate and 4 m
M
2-mercaptoethanol were
added to the mixture. All experiments were performed in
20 m
M
Tris/HCl at pH 7.5 and 200 m
M
NaCl buffer using
a glove box under an argon atmosphere to obtain an
anaerobic environment. The Fe–S cluster formation was
followed with a UV-visible spectrophotometer Cary 50 Bio
(Varian Ltd, Walton-on-Thames, UK) recording spectra at
different times. A solution of 76 l
M
nonreconstituted IscU
wasusedasblank.
Results
E. coli
IscU isa stable wellfolded protein
IscU could be purified to a homogeneous construct. The
secondary structure of the proteinand its thermal stability
were first characterized by circular dichroism spectroscopy.
The far UV CD spectrum is that characteristic of a mixed
ab secondary structure content (Fig. 1A). Deconvolution of
the CD spectra yields the following percentages for secon-
dary structure elements: 40.8% of a-helix, 13.7% of
b-strands and 19.6% turns.
The thermal unfolding curve was % 95% reversible and
gave a T
m
of 71.5 ± 0.6 °C. As shown in Fig. 1B, the fit for
a simple two-step reversible transition is not perfect. This
is probably because there isa small thermal unfolding
transition occurring at low temperature. However, the near
UV CD signal (285 nm) also gives a transition with a T
m
of
71.5 °C without any detectable transition at lower tempera-
ture (data not shown).
Presence of a tertiary fold was checked both by one-
and two-dimensional NMR. The excellent chemical shift
dispersion in the 1D
1
H NMR spectrum of E. coli IscU
provides strong evidence for a stable globular fold
(Fig. 2A). The presence of characteristic ring-current
shifted peaks around 0 p.p.m. (e.g. the resonances at
0.02 p.p.m., 0.14 p.p.m. and 0.29 p.p.m.) in Fig. 2A
which arise from the spatial proximity of hydrophobic
residues to aromatic rings is also typical of proteins with
a well defined hydrophobic core. Likewise, the chemical
shift dispersion both in the
1
Hand
15
N dimensions of the
1
H-
15
N HSQC spectrum of uniformly
15
N labeled sample
of E. coli IscU confirms that the proteiniswell behaved
without relevant disordered regions (Fig. 2B). The num-
ber of observed backbone amide resonances (111), is
consistent with the expected ones (123). Figure 2C shows
the excellent quality of the
1
H homonuclear NOESY of
IscU.
NMR relaxation measurements (T
1
,T
2
and hetero-
nuclear
15
N-[
1
H] NOE values) were recorded at 25 °Cand
600 MHz on an
15
N uniformly labeled sample of IscU to
provide a measure of the local degree of flexibility (Fig. 3).
The mean values for T
1
and T
2
relaxation times are
608 ± 27 ms and 81 ± 6 ms, respectively (Fig. 3A,B).
Except for a few resonances (5), which are likely to
correspond to residues at the N- and C-termini and/or in
disordered loops, the distribution of both T
1
and T
2
values
is relatively homogenous without significant deviations
from the mean values. The experimental
15
N-[
1
H] NOEs
also range from 0.39 to 0.87 with an average of 0.68
Fig. 1. Circular dichroism spectrum and thermal unfolding of IscU.
(A) Far UV CD spectrum of E. coli IscU reported in terms of mean
residue mass ellipticity [h]/(degreeÆcm
)2
Ædmol
)1
). (B) Thermal dena-
turation curves of E. coli IscU. The spectra were recorded on a 10 l
M
protein in 10 m
M
buffer at pH 7.5.
Ó FEBS 2004 Characterization of E. coli IscU (Eur. J. Biochem. 271) 2095
for all residues excluding only two resonances, located
at 7.8 p.p.m. and 126.3 p.p.m. and at 8.0 p.p.m. and
124.8 p.p.m., which have negative values (Fig. 3C). Nega-
tive NOEs indicate highly flexible regions that, in globular
proteins, are usually observed for the amides at the N- and
C-termini. Positive values are typical of relatively rigid
regions [27]. We can therefore conclude that the IscU is
compact, without relevant differences of the local flexibility.
Finally, the environment of the unique tryptophan
(Trp76) of the IscU sequence was probed by fluorescence
measurements. The tryptophan fluorescence emission spec-
trum shows an emission band at 355 nm (data not
shown), suggesting that this residue is highly exposed to
the solvent.
E. coli
IscU isa monomeric protein
The sample was characterized for its aggregation state, using
three independent techniques. The molecular mass of native
E. coli IscU was first estimated by gel filtration. The elution
profile of E. coli IscU presents asingle peak at a molecular
mass corresponding to that of the monomer (13.9 kDa)
Fig. 2. Typical ID and 2D spectra of IscU. (A) 1D NMR spectrum of
non labeled IscU sample (0.5 m
M
)in20m
M
Tris/HCl at pH 7.0,
50 m
M
NaCl and 10 m
M
dithriothreitol. The spectrum was recorded at
25 °C and 800 MHz. (B)
1
H-
15
N HSQC spectrum of uniformly
15
N-labeled sample of E. coli IscU at 0.3 m
M
concentration recorded
at 25 °C and 600 MHz. (C) Amide region of partial 600 MHz
1
H
homonuclear NOESY of IscU.
Fig. 3. T
1
(A), T
2
(B) and
15
N-[
1
H] heteronuclear NOE (C) measure-
ments recorded at 25 °C and 600 MHz on a uniformly
15
N-labeled
sample of E. coli IscU at 0.3 m
M
concentration. The pulse sequence
used for the
15
N-[
1
H] heteronuclear NOE measurement is that pub-
lished by Farrow et al. [28]. In the absence of the sequential assign-
ment of the spectra, residue numbers are ordered according to their
resonances.
2096 S. Adinolfi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
(Fig. 4A). However, an additional peak, which could be
consistent with a dimer, appeared if no reducing agent was
used in the buffer.
Because the gel filtration profile strongly depends on the
protein shape, this result was confirmed in two different
concentration ranges by analytical ultracentrifugation, a
technique generally considered as the most accurate way to
detect oligomerization, and by estimating the correlation
time of the protein in solution from the NMR relaxation
measurements. The apparent molecular mass of IscU as
obtained by ultracentrifugation methods for 10–40 l
M
samples is 14.7 kDa, a value corresponding, within experi-
mental error, to the monomer molecular mass (Fig. 4B). No
significant differences were observed using different ionic
strength conditions.
The NMR relaxation measurements described in Fig. 3A
and B yielded a value for the rotational correlation time (s
c
)
of 9.8 ns. In globular proteins, s
c
is roughly proportional to
the molecular mass [29]. The value we obtain for IscU is
thus in excellent agreement with what is expected for
an % 14 kDa monomeric protein [29].
The
E. coli
IscU monomeric protein can host
a Fe–S cluster
To prove that the recombinant E. coli IscU can function as
a monomer, we checked whether it could promote the IscS-
mediated reconstitution of a reductively labile [Fe
2
S
2
]
2+
cluster. Typical experiments of Iron–sulfur cluster reconsti-
tution were performed, as described in Agar et al.[30].
A ten-fold excess of
L
-cysteine (based on the concentration
of IscU monomer) was added in an argon glove box to a
reaction mixture containing 100–400 l
M
IscU in the
presence of 0.5–5.0 l
M
IscS, a 5-fold excess of ferric
ammonium citrate (based on the concentration of IscU
monomer), and 4 m
M
2-mercaptoethanol. The IscU/IscS
ratio was used to vary the rate of cluster formation. A
simple, yet efficacious, way to characterize an Iron–sulfur
cluster is through its UV-visible absorption spectrum [30].
Samples of ÔapoÕ IscU, i.e. prior to cluster assembly, do not
have a visible chromophore but become red on anaerobic
treatment with catalytic amounts of IscS in the presence of
excess
L
-cysteine anda stoichiometric amount of ferric
ammonium citrate. The spectrum should contain charac-
teristic bands centered at % 320, 410 and 456 nm and a
pronounced shoulder at 510 nm [31]. Similarly to what is
observed for the assembly of a [Fe
2
S
2
]
2+
cluster in
A. vinelandii IscU we recorded a UV-visible absorption
spectrum (Fig. 5) with characteristic bands at 320, 407 and
447 nm anda pronounced shoulder at 513 nm, character-
istic of a [Fe
2
S
2
]
2+
cluster.
E. coli
IscU does not bind iron ions independently
of Isc formation
Although there isa general consensus that IscUis the
cradle of the [Fe
2
S
2
]
2+
cluster, there is disagreement on
the possibility of a direct interaction with iron ions. To
compound this debate, we tested the possible interaction
of IscU with iron ions by
1
H-
15
N correlation NMR
spectra. NMR is the ideal technique to detect even weak
interactions, because it operates at millimolar concentra-
tions, and can map local perturbations of electronic
density to specific protein sites. If a diamagnetic molecule
binds to a protein, we expect to detect chemical shift
perturbation of selected cross peaks in the spectrum that
correspond to the protons affected by the binding (e.g.
[32]). When paramagnetic species are present in solution
but not bound, a general and unspecific broadening of
the resonances mediated by the solvent is observed.
Binding of a paramagnetic species leads instead to major
Fig. 4. Probing the aggregation state of IscU. (Top) Calibration curve
for apparent molecular mass determination of E. coli IscU in native
conditions by gel filtration chromatography. A HiLoad 10/30 Super-
dex 75 column equilibrated with Tris/HCl buffer (pH 7.5), 100 m
M
NaCl was used, with ovalbumin (A; 43 kDa), chymotrypsinogen A (B;
25 kDa) and ribonuclease A (C; 13.7 kDa) as molecular standards for
the mass calibration. (Bottom) Sedimentation equilibrium distribution
of IscU measured using analytical ultracentrifugation. The data were
recorded at 20 °C and 20 000 r.p.m. Protein concentration was 20 l
M
in 20 m
M
Tris/HCl a 50 m
M
at pH 8.0, 50 m
M
NaCl and 10 m
M
2-mercaptoethanol. Lower panel: Experimental absorbance at 280 nm
as a function of the radial position and data fitting to the equation
reported in Materials and methods. Upper panel: Distribution of dif-
ferences between experimental and calculated values. The apparent
molecular mass for this experiment is 14 000 Da.
Ó FEBS 2004 Characterization of E. coli IscU (Eur. J. Biochem. 271) 2097
chemical shifts and/or disappearance of selected reso-
nances [33].
The spectra shown in Fig. 6B,C correspond to additions
of Fe(II) at iron/IscU ratios of 2 : 1 and 5 : 1, respectively.
Addition of Fe(II) up to a ratio of 5 : 1 has no detectable
effect on the chemical shifts and on the lineshapes of the
resonances, implying no direct binding. When the sample
was partially or completely oxidized by atmospheric oxygen
only diffuse broadening was observed.
The possibility that iron binding could be hindered by the
presence of sulfane sulfur [S(0)] on IscU, as suggested by
Nuth et al. [34], was ruled out by electrospray mass
spectrometry. As mentioned previously, the experimental
mass of the IscU construct (13 977.9 Da) agrees to the
Dalton with the expected value (13 976.7 Da) and does not
support the presence of additional sulfur atoms.
Discussion
Gathering detailed structural information on IscU-like
proteins has been limited by intrinsic folding properties.
The only structure reported so far is that of IscU from
Haemophilus influenzae (PDB ID: 1Q48), but this struc-
ture is not yet described in a paper. Recently, it was
possible to establish the secondary structure of IscU from
Thermatoga maritima but its tertiary structure could not
be determined because, according to these authors, the
protein behaves as a flexible molten globule-like state [35].
Evidence for secondary and tertiary structure seems
absent in the human and yeast homologues as stated in
Mansy et al. [36]. In the present work we have shown that
our construct of E. coli IscUiswell folded. The high
melting temperature and the reversibility of the thermal
unfolding curve (as measured by CD) hint at a well
ordered stable fold. This view is confirmed by the
excellent dispersion of cross peaks in the
1
H-
15
NNMR
correlation spectrum, by the quality of the homonuclear
NOESY spectrum (Fig. 2C and data not shown) and by
relaxation data. Altogether our data do not support a
flexible molten globule-like state for E. coli IscU.
Until now the only monomeric IscUs identified have been
the human protein [37] and that from Haemophilus influ-
enzae, whereas homologues from other organisms gener-
ally have been described as dimers, e.g. the IscU from
T. maritima was shown to form a homodimer [35]. Here we
present conclusive evidence that the E. coli orthologue also
behaves as a monomeric protein. Monomeric IscU is
Fig. 6. Probing for Fe(II) binding of IscU by NMR. (A)
1
H-
15
NHSQC
spectrum of uniformly
15
NlabeledsampleofE. coli IscU; (B) as for
sample A, after addition of Fe(II) in a Fe(II)/IscU ratio of 2 : 1;
(C) same as sample A with a ratio Fe(II)/IscU of 5 : 1 after partial
oxidation by atmospheric oxygen.
Fig. 5. UV-visible absorption spectrum of reconstituted IscU. The
spectrum of reconstituted IscU containing a [Fe
2
S
2
]
2+
cluster was
recorded 45 mins after adding 4 m
ML
-cysteine to a reaction mixture
containing 76 l
M
IscU in presence of IscS in the ratio 1 : 28 IscS/IscU,
a fivefold excess of freshly prepared ferric ammonium citrate (relative
to IscU concentration) and 4 m
M
2-mercaptoethanol.
2098 S. Adinolfi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
functional as an iron cluster assembly protein as,
when reacted with iron ions and IscS-produced active
sulfane, it showed the typical UV spectrum of a transient
labile [Fe
2
S
2
]
2+
cluster typical of the reconstituted protein
[30].
Another important result from our study is that E. coli
IscU does not interact directly with iron ions, independently
of Isc formation. The use of NMR is probably conclusive in
this respect because this technique can reveal even very
weak interactions. The generally accepted mechanism for
biological Iron–sulfur cluster assembly is based on the
hypothesis that persulfides catalytically formed on IscS
can be transferred to IscU for cluster assembly through
association of the two proteins [30]. The alternative
mechanism, proposed by Nuth et al. [34], based on initial
binding of iron by IscU, is not consistent with the reluctance
of IscU to accept iron ions indicated by our NMR data.
Accordingly, the mechanism of Fe–S cluster assembly based
on initial binding of iron followed by delivery of sulfur
equivalents, proposed for the T. maritima IscU [34], does
not seem applicable to E. coli IscU.
Within this frame, the role of CyaY in bacteria may well
be that of an iron chaperone which passes the iron to IscU,
as first suggested by Yoon & Cowan [21], and more recently
supported by independent line of experimental evidence
both based on in vivo and in organello studies [37,38]. This
process might however, require preformation of a (tran-
sient?) ternary complex with IscU/IscS and occur cooper-
atively only when a source of sulfur is also available.
Further studies to describe the molecular details of these
multiple interactions will be needed to understand this
complex phenomenon.
Acknowledgement
The project was funded by Seek A Miracle/MDA and the Friedreich’s
Ataxia Research Alliance (FARA) foundations.
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Supplementary material
The following material is available from http://blackwell
publishing.com/products/journals/suppmat/EJB/EJB4112/
EJB4112sm.htm
Fig. S1. Near UV thermal unfolding curve.
Fig. S2. 2D NOESY of IscU.
2100 S. Adinolfi et al.(Eur. J. Biochem. 271) Ó FEBS 2004
. Bacterial IscU is a well folded and functional single domain protein
Salvatore Adinolfi
1
, Francesca Rizzo
1
, Laura Masino
1
, Margie Nair
1
,. glutathione S-transferase and a
cleavage site for tobacco etch virus protease which leaves,
after cleavage, only two additional amino acids (GlyAla) at
the protein