Báo cáo khoa học: Bacterial IscU is a well folded and functional single domain protein pdf

coli IscU is able to provide a scaffold for Iron–sulfur cluster assembly, but has no direct interaction with either FeII or FeIII ions, suggesting the need of further partners to achieve

Bacterial IscU is a well folded and functional single domain protein

Salvatore Adinolfi1, Francesca Rizzo1, Laura Masino1, Margie Nair1, Stephen R Martin1, Annalisa Pastore1 and Piero A Temussi1,2

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

coliIscU 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 a well ordered stable fold The excellent dispersion of cross peaks in the1H-15N correlation spectrum is consistent with these observations Monomeric

E coli IscU is 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 and IscU 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 bacterial IscU is 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 coliIscU does not bind iron ions directly

Materials and methods

Protein production

E coliIscU 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 colistrain 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 mM isopropyl thio-b- -galactoside After the cultures reached

Correspondence toA 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)

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 mM Tris/HCl, pH 8, 150 mM NaCl, 10 mM

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) 15N-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 mMHepes/KOH pH 7.5 or 20 mMTris/

HCl pH 7.5–8.0 with NaCl 50–150 mM and 20 mM

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 ofE 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 mM 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 mM Tris/HCl at pH 8.0, 150 mM NaCl and 10 mM

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 lM Data were recorded using different ionic strength

conditions (20 mM Tris/HCl at pH 8 with 50 mM or

150 mMNaCl and 10 mM2-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 theORIGIN

XL-A/XL1 package (Beckman) and fitted to the following

equation:

AðrÞ ¼ Aðr0Þexp Mðx 2=2RTÞð1  mqÞðr2 r2Þ

where A(r) and A(r0) are the optical absorbances at radius r

and at the reference radius r0, 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 a single 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

a PTC-348 Peltier system Far UV measurements were performed in 10 mM buffer at pH 7.5 using protein concentrations of 7–35 lM 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 lMprotein concentration Variations of the CD signal were studied as a function of temperature over the range 10°C to 95 °C using a heating rate of 1 °C per 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°C were recorded on Varian INOVA spectrometers (Varian Ltd, Walton-on-Thames, UK) oper-ating at 500, 600 and 800 MHz1H frequency Typically, 0.3–0.5 mM unlabelled or 15N 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 theNMRPIPE program [24] Baseline correction was applied when neces-sary The spectra were analyzed using theFELIX(MSI) and XEASYprograms [25]

Experimental 15N T1 and T2 relaxation times and heteronuclear 1H-15N NOE values were measured on uniformly labelled 15N IscU 0.3 mM samples in 20 mM Tris/HCl at pH 7.0, 50 mM NaCl and 10 mM 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 For T1 and T2 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 a single exponential Correlation times were calculated from the T1/T2 ratios according to the so-called model-free approach [27] Experimental 1H-15N steady state NOE values were determined from the peak intensity ratios of amide resonances obtained by recording inter-leaved 2D Watergate 1H-15N HSQC spectra with and without a saturation delay of 4 s and a repetition delay of 4.2 s [28]

NMR titrations

Titration of IscU with Fe(II) was carried out by NMR,

typically starting with 0.4 mMprotein in 20 mMTris/HCl,

50 mM, 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 mM L-cysteine to a reaction mixture

containing 76 lM 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]) A fivefold

excess (relative to IscU concentration) of freshly prepared

ferric ammonium citrate and 4 mM2-mercaptoethanol were

added to the mixture All experiments were performed in

20 mMTris/HCl at pH 7.5 and 200 mMNaCl 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 lMnonreconstituted IscU

was used as blank

Results

E coli IscU is a stable well folded protein

IscU could be purified to a homogeneous construct The

secondary structure of the protein and 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 Tmof 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 is a small thermal unfolding

transition occurring at low temperature However, the near

UV CD signal (285 nm) also gives a transition with a Tmof

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 1D1H 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 the1H and15N dimensions of the

1H-15N HSQC spectrum of uniformly15N labeled sample

of E coli IscU confirms that the protein is well 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 1H homonuclear NOESY of IscU

NMR relaxation measurements (T1, T2 and hetero-nuclear15N-[1H] NOE values) were recorded at 25°C and

600 MHz on an15N uniformly labeled sample of IscU to provide a measure of the local degree of flexibility (Fig 3) The mean values for T1 and T2 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 T1and T2values

is relatively homogenous without significant deviations from the mean values The experimental 15N-[1H] 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)2Ædmol)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.

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 is a monomeric protein The sample was characterized for its aggregation state, using three independent techniques The molecular mass of native

E coliIscU was first estimated by gel filtration The elution profile of E coli IscU presents a single 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 ) in 20 m 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-15N 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)and15N-[1H] heteronuclear NOE (C)measure-ments recorded at 25 °C and 600 MHz on a uniformly15N-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.

(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 lM 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 (sc)

of 9.8 ns In globular proteins, scis 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]

TheE 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 [Fe2S2]2+ cluster Typical experiments of Iron–sulfur cluster reconsti-tution were performed, as described in Agar et al [30]

A ten-fold excess ofL-cysteine (based on the concentration

of IscU monomer) was added in an argon glove box to a reaction mixture containing 100–400 lM IscU in the presence of 0.5–5.0 lM IscS, a 5-fold excess of ferric ammonium citrate (based on the concentration of IscU monomer), and 4 mM 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 apo 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 and a 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 [Fe2S2]2+ cluster in

A vinelandii IscU we recorded a UV-visible absorption spectrum (Fig 5) with characteristic bands at 320, 407 and

447 nm and a pronounced shoulder at 513 nm, character-istic of a [Fe2S2]2+cluster

E coli IscU does not bind iron ions independently

of Isc formation Although there is a general consensus that IscU is the cradle of the [Fe2S2]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 1H-15N 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.

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 IscU is well 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 1H-15N NMR 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 maritimawas 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)1H-15N HSQC spectrum of uniformly15N labeled sample of E 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 M L -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.

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 [Fe2S2]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