Bacterial IscU is a well folded and functional single domain 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 a well 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 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. 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 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 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 a single 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 and a 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 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 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 is a 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 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 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 is a 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 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 )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 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 [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 and a 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 is a general consensus that IscU is 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 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 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. References 1. Beinert, H. & Kiley, P. 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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