Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis\xe2\x80\xa0
Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich\xe2\x80\x99s ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homolog CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.
<!>Preparation of human recombinant proteins<!>Preparation of E. coli recombinant proteins<!>Titrations to favor protein complex formation<!>Cysteine desulfurase activity measurements<!>Fe-S Cluster Formation<!>Ratio of protein complex components for activity measurements<!>IscUec and CyaY stimulate the cysteine desulfurase activity of the human SD complex<!>Iron-dependent stimulation of cysteine desulfurase reaction for SD complexes<!>CyaY can substitute for FXN in Fe-S cluster synthesis by human SD complexes<!>CyaY and FXN have minor effects on the cysteine desulfurase activity of IscSec complexes<!>CyaY and FXN inhibit Fe-S assembly by IscSec complexes<!>DISCUSSION
<p>Iron-sulfur (Fe-S) clusters are protein cofactors that are essential for most living organisms. Fe-S clusters primarily function in electron transfer, but also participate in substrate binding and activation, protein stabilization, regulation of gene expression or enzyme activity, radical generation, and as a sulfur donor (1–3). These biological functions require the Fe-S cluster to vary from solvent accessible to completely buried, and to be accommodated in a diverse set of protein scaffolds (4). Despite this diversity, conserved metallochaperone proteins recognize and insert Fe-S clusters into apo Fe-S proteins through mechanisms that remain poorly understood. This assembly system must also control the reactivity of the building blocks for Fe-S clusters, ferrous iron and sulfide, to reduce toxicity and cell death. In humans, ferrous iron is prone to undergo Fenton chemistry: reaction with hydrogen peroxide to generate highly reactive hydroxyl radicals that damage proteins, lipids, and DNA, whereas sulfide is more toxic than cyanide, is a potent inhibitor of cytochrome c oxidase, and targets olfactory nerves, the eyes, and the brain (5). Defects in these metallochaperone systems have profound effects on iron metabolism, and result in mitochondrial dysfunction and human disease (6–8).</p><p>In prokaryotes, three different Fe-S assembly systems have been characterized: the nitrogen fixation (NIF), iron-sulfur cluster assembly (ISC), and mobilization of sulfur (SUF) pathways (9–13). Escherichia coli contains both ISC and SUF systems and appears to use the ISC pathway under 'normal' growth conditions and the SUF system under conditions of iron limitation or oxidative stress (14). The ISC operon is controlled by IscR, a Fe-S cluster binding transcriptional repressor, and contains genes for a cysteine desulfurase (IscS), scaffold protein (IscU), ferredoxin (Fdx), IscA, molecular chaperones (hscA and hscB), and IscX (also known as yfhj and ORF3). The scaffold protein IscU (in this manuscript the E. coli homolog is referred to as IscUec, or Uec) is the central protein in Fe-S cluster biosynthesis and is responsible for the assembly of [2Fe-2S] and [4Fe-4S] clusters (15). IscS (E. coli homolog referred to as IscSec or Sec) catalyzes the PLP-dependent breakdown of cysteine to alanine and provides the inorganic sulfur for Fe-S clusters (16, 17). Fdx likely provides electrons for cluster assembly and IscA may be an iron donor or alternate scaffold (18–20). The function of IscX is unknown, but it appears to compete with CyaY for binding to IscS (21–23). Once the cluster is formed, the chaperones hscA and hscB facilitate the transfer of intact Fe-S clusters to apo target proteins (24–26). A homolog of human frataxin (E. coli homolog referred to as CyaY or as Cec in protein complexes in this manuscript) is not part of the ISC operon, but has been shown to bind to the IscSec-IscUec (SecUec) complex (21, 27, 28) and inhibit Fe-S cluster assembly (29).</p><p>A similar ISC Fe-S cluster assembly pathway functions in the matrix space of eukaryotic mitochondria (8, 30). The Fe-S assembly proteins are encoded by nuclear DNA and often contain a targeting sequence that is cleaved upon import into mitochondria. For the human pathway, the cysteine desulfurase NFS1 (59% identical to IscSec), which forms a eukaryotic-specific functional complex with ISD11 (31–33), the scaffold protein ISCU2 (also known as Isu2; 70% identical to IscUec), and FXN (20% identical to CyaY) form a NFS1-ISD11-ISCU2-FXN (SDUF) complex (34, 35) that is analogous to the IscSec-IscUec-CyaY (SecUecCec) complex. Recent in vitro studies indicate that FXN is an allosteric activator of Fe-S cluster assembly that increases the catalytic efficiency (kcat/KM) for the cysteine desulfurase and the rate of Fe-S cluster biosynthesis (34). Intriguingly, in vitro data indicate E. coli CyaY and human FXN have opposing regulator functions in Fe-S cluster biosynthesis (29, 34). Different physiological functions may also be supported by different in vivo phenotypes for E. coli and mouse model systems lacking frataxin (36, 37). However, both CyaY and FXN can at least partially complement strains of yeast lacking the frataxin homolog Yfh1 (38, 39), which implies similar functions.</p><p>To resolve this discrepancy and better understand the role of frataxin in Fe-S cluster biosynthesis, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous components were interchanged. Surprisingly, our results reveal that the cysteine desulfurase component, IscSec/NFS1-ISD11, rather than CyaY/FXN or IscUec/ISCU2 dictates the functional differences in how the frataxin homologs modulate Fe-S cluster assembly.</p><!><p>Human NFS1 (Δ1–55) and ISD11 were coexpressed in Escherichia coli strain BL21(DE3) as previously described (34, 40). Cells were lysed by French press in 50 mM Tris pH 8.0, 500 mM NaCl, and 5 mM imidiazole. The NFS1-ISD11 (SD) complex was purified as previously described (34) except that a cation exchange column was added between the Ni-NTA and S300 gel filtration chromatography steps. The yellow SD fractions from the Ni-NTA column were combined with 100 μM pyridoxal 5′-phosphate (PLP), 5 mM dithiothreitol (DTT), and 2 mM ethylenediaminetetracetic acid (EDTA), diluted 3-fold with 50 mM Tris pH 8.0, loaded onto a cation exchange column (16/14, POROS 50HS), and eluted with a linear gradient from 0–1000 mM NaCl. Human ISCU2 (Δ1–35) was purified as previously described (34) except the fractions for the cation exchange column were eluted with a linear gradient from 0 – 1000 mM NaCl in 50 mM Tris, pH 7.5 buffer and a S100 rather than a S300 gel filtration column was used. Human FXN (Δ1–55) was purified as previously described (34) except a S100 rather than a S300 column was used. The Bradford method (41) or extinction coefficients of 42670, 8250, and 26030 M−1cm−1 at 280 nm (42) were used to estimate the protein concentration of SD, ISCU2, and FXN, respectively.</p><!><p>A plasmid containing the IscSec gene (29) was transformed into BL21(DE3) E. coli cells and grown at 37 °C. Protein expression was induced with 0.5 mM IPTG when the OD600 was ~0.6. Cells were harvested 5 hours later and lysed by sonication in 50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidiazole, and 100 μM PLP. After centrifugation, the supernatant was loaded on a Ni-NTA column (16/13, GBiosciences) and eluted with a linear gradient from 5 to 250 mM imidazole. Yellow fractions were concentrated and further purified on a Sephacryl S300 column (26/60, GE Healthcare) equilibrated in 50 mM HEPES pH 8.0, 250 mM NaCl. Plasmids that contained either IscUec or CyaY genes (29) were transformed into BL21(DE3) cells and grown at 37 °C. Protein expression was induced with 0.5 mM IPTG when the OD600 was ~0.6. Cells were harvested 3.5 hours later and lysed by sonication in 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT. The supernatant was loaded onto a GST column and eluted with a linear gradient from 0–7 mM glutathione. The fractions that bound the column were concentrated and dialyzed into 50 mM Tris pH 8.0, 0.5 mM EDTA, and 1 mM DTT and then incubated at room temperature overnight with TEV protease. The tag cleavage product was loaded onto the GST column and the flow through was further purified on a S100 gel filtration column equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl. The Bradford method (41) or extinction coefficients of 41370, 11460, and 28990 M−1cm−1 at 280 nm (42) were used to estimate the protein concentration of IscSec, IscUec, and CyaY, respectively.</p><!><p>Cysteine desulfurase assays were used to determine reaction stoichiometries to favor protein complexes for different combinations of human and E. coli proteins. Cysteine desulfurase assays (see below for details) were initiated with 100 μM L-cysteine and included 0.5 μM of the cysteine desulfurase (SD or IscSec) and increasing amounts of the scaffold protein (ISCU2 or IscUec). Once the saturating number of equivalents of the scaffold protein were determined (Table 1), additional titrations were performed with increasing amounts of frataxin (FXN or CyaY).</p><!><p>Assay mixtures (800 μL) contained 0.5 μM cysteine desulfurase (SD or IscSec), 10 μM PLP, 2 mM DTT, 5 μM Fe(NH4)2(SO4)2, and increasing or saturating amounts (Table 1) of the scaffold protein (ISCU2 or IscUec) and frataxin (FXN or CyaY) in 50 mM Tris pH 8.0, and 250 mM NaCl (34, 40, 43). Samples were incubated in an anaerobic glovebox (10~14 °C) for 30 minutes. The cysteine desulfurase reactions were initiated by addition of L-cysteine (0.01 to 1 mM) at 37 °C and quenched after 10 minutes by the addition of 100 μl of 20 mM N,N-dimethyl-p-phenylenediamine in 7.2 N HCl and 100 μl of 30 mM FeCl3 in 1.2 N HCl, which also initiated the conversion of sulfide to methylene blue. After a twenty minute incubation at 37 °C, the absorption at 670 nm due to methylene blue formation was measured and compared with a Na2S standard curve to quantitate sulfide production. Cysteine desulfurase reactions were also performed for mixed species Fe-S assembly complexes with 0 to 80 equivalents of Fe(NH4)2(SO4)2 and 100 μM L-cysteine. Units are defined as μmol sulfide/μmol cysteine desulfurase per minute at 37 °C. Rates were fit to the Michaelis-Menten equation using KaleidaGraph (Synergy Software).</p><!><p>Assay mixtures contained 8 μM of the cysteine desulfurase (SD or IscSec), 5 mM DTT, 200 μM Fe(NH4)2(SO4)2, 100 μM L-cysteine, 50 mM Tris pH 8.0, and 250 mM NaCl in a total volume of 0.2 mL. In addition, the number of equivalents (relative to the cysteine desulfurase) for the scaffold protein (ISCU2 or IscUec) and frataxin (FXN or CyaY) required to saturate the activity (above; Table 1) were included in the assay mixture. The scaffold protein was incubated with 5 mM DTT in 50 mM Tris pH 8, 250 mM NaCl in an anaerobic glovebox for 30 minutes prior to mixing with the remaining assay components in an anaerobic cuvette and then incubated at 10 °C for 30 minutes in the cuvette holder. The reaction was initiated by injecting L-cysteine to a final concentration of 100 μM with a gas-tight syringe. Fe-S cluster formation was monitored at 456 nm at 10 °C and then the first 3000 seconds were fit as first-order kinetics using KaleidaGraph. The rate was converted to the activity using an extinction coefficient of 5.8 mM−1cm−1 at 456 nm for [2Fe-2S] cluster absorbance (15). Units are defined as the μmol of [2Fe-2S] cluster / μmol cysteine desulfurase per minute at 10 °C.</p><!><p>The objectives of this study were to provide insight into functional differences in frataxin-mediated regulation of Fe-S cluster biosynthesis for prokaryotes and eukaryotes by measuring cysteine desulfurase and Fe-S formation activities of mixed species protein complexes. The sulfur generated by the cysteine desulfurase reaction can be directly measured or can be used as a component of the Fe-S formation reaction. Titrations were performed to determine the relative ratios of the different components for maximum activity. Previous experiments revealed that adding 3 equiv of ISCU2 minimized the cysteine desulfurase activity of the NFS1-ISD11 (SD) complex (Table 1), likely through formation of a NFS1-ISD11-ISCU2 (SDU) species (34). Likewise, adding 3 equiv of FXN to SDU maximized the cysteine desulfurase activity by forming a NFS1-ISD11-ISCU2-FXN (SDUF) complex (34). A similar strategy was used here to determine and then compensate for weaker binding of E. coli proteins to the human assembly complex and human proteins to the E. coli assembly complex.</p><p>First, protein reaction ratios were determined for the human SD complexes by measuring cysteine desulfurase activities with increasing amounts of the scaffold protein, either ISCU2 or IscUec, and frataxin, either FXN or CyaY. The cysteine desulfurase activity of SD decreased with addition of either ISCU2 or IscUec and minimized after the addition of ~2 equiv/SD (Figure 1A). Three equiv of ISCU2 or IscUec were therefore included with SD for kinetic experiments of the SDU or NFS1-ISD11-IscUec (SDUec) complexes (Table 1). The binding of frataxin homologs to the SDU complex increased the cysteine desulfurase activity that maximized after the addition of either 3 equiv of FXN or 80 equiv of CyaY (Figure 1B). Similarly, the binding of frataxin homologs to the SDUec complex increased the cysteine desulfurase activity that maximized after either 200 equiv of FXN or 30 equiv of CyaY (Figure 1C).</p><p>Second, protein reaction ratios were determined for IscSec using similar titrations. The cysteine desulfurase activity of IscSec slightly decreased with addition of IscUec and slightly increased with addition of ISCU2 (Figure 1D). This perturbation in IscSec activity maximized after the addition of 3–5 equiv of IscUec or ISCU2. To favor complex formation, five equiv of IscUec or ISCU2 were therefore added to IscSec (Table 1) for all kinetic experiments of IscSec-IscUec (SecUec) and IscSec-ISCU2 (SecU) complexes, respectively. Titrations of up to 50 equiv of CyaY or FXN did not significantly change the cysteine desulfurase activity of either the SecUec or SecU complex (data not shown). Therefore, protein complex stoichiometries of 1:5:10 were used for the SecUecCec and SecUCec complexes based on the number of equivalents of CyaY previously determined to inhibit Fe-S cluster formation (29). Similarly, protein complex stoichiometries of 1:5:15 were arbitrarily chosen for the Michaelis-Menten kinetics (below) of the SecUecF and SecUF complexes (Table 1).</p><!><p>Saturating amounts of ISCU2 or IscUec and FXN or CyaY determined above were added to the SD complex and the rates of the cysteine desulfurase reaction were measured as a function of the L-cysteine concentration. The human SD complex exhibited a kcat/KM of 660 M−1s−1 for the cysteine desulfurase reaction (Table 2), which is slightly higher (primarily due to a lower KM for L-cysteine) than previously reported values of ~100 M−1s−1 (34, 40). The addition of ISCU2 or IscUec and formation of the SDU or SDUec complex slightly decreased the kcat (Figure 2 and Table 2). The binding of FXN to form the SDUF and SDUecF complexes stimulated the kcat for the cysteine desulfurase reaction by ~7-fold relative to the SDU and SDUec complexes (Figure 2). Similarly, CyaY binding and formation of the SDUCec and SDUecCec complexes resulted in a 4–6 fold increase in the kcat relative to SDU and SDUec (Figure 2). These experiments show that IscUec can functionally substitute for ISCU2 and that CyaY can replace FXN as an activator of the cysteine desulfurase reaction for the human Fe-S assembly complex.</p><!><p>Cysteine desulfurase activities were determined for SD complexes as a function of added iron. From 0 to 80 equiv of Fe(II) were added to the SDUF, SDUCec, SDUecF, and SDUecCec complexes and the cysteine desulfurase activity was measured (Figure 3A). The activity maximized after about 1, 1, and 5 equiv for the SDUF, SDUCec, and SDUecF complexes, respectively. In contrast, the SDUecCec complex did not exhibit a significant Fe-based activation. In addition, the SD complexes were incubated with 10 equiv of ferrous iron and the Michaelis-Menten parameters for the cysteine desulfurase activity were determined (Table 2). Ferrous iron had a large effect on the kcat of the SDUF complex, as previously reported (34), a moderate effect on the SDUCec and SDUecF complexes, and no effect on the SDUecCec complex (Figure 3B).</p><!><p>The rate of Fe-S cluster assembly for the SDU, SDUec, SDUF, SDUCec, SDUecF, and SDUecCec complexes were determined by monitoring the increase in absorbance at 456 nm (15) at 10 °C and converting this change in absorbance to an activity measurement (μmol [2Fe-2S] / μmol of complex per minute). In the absence of FXN or CyaY, the SDU and SDUec complexes exhibited very low or no activity and were similar to a control reaction in which sulfide was substituted for cysteine (Table 2 and Figure 4). The addition of FXN greatly stimulated the Fe-S assembly activity of SDU and SDUec. In contrast, addition of CyaY resulted in a very modest or no increase in Fe-S assembly activity of the SDU and SDUec complexes, respectively. A CyaY-based stimulation was more readily observed when the Fe-S cluster assembly assays were repeated at room temperature (Figure 4A inset). Thus, E. coli CyaY can substitute for human FXN in the human Fe-S assembly complex and strongly stimulate the cysteine desulfurase activity. CyaY also weakly stimulates the Fe-S cluster formation reaction.</p><!><p>Purified recombinant IscSec exhibited a kcat of 7.5 min−1 (Figure 5A and Table 2), which is similar to the previously reported value of 8.5 min−1 (44). Addition of IscUec diminished the kcat for the cysteine desulfurase reaction by a factor of 1.6, whereas a more moderate decrease in kcat was observed upon ISCU2 addition (Figure 5A). Previously, IscUec was shown to either have no effect on the activity of IscSec (45) or stimulate the activity 6-fold (46) using different experimental conditions and assays. The kcat for SecUec and SecU were increased slightly upon addition of either CyaY or FXN (Figure 5A and Table 2). The IscSec cysteine desulfurase activity was slightly decreased by added ferrous iron, consistent with a previous report (47), that appeared to occur within the first equiv of added iron for the SecUecF and SecUecCec complexes and 10 equiv of iron for the SecUF complex (Figure 5B). Steady state kinetics also revealed the kcat slightly decreased upon addition of 10 equiv of ferrous iron (Figure 5A). The slight decrease in cysteine desulfurase activity upon addition of iron (Figure 5) is opposite of the general Fe-based activation observed for SD complexes (Figure 3). Overall, these results indicate that binding of frataxin homologs and ferrous iron have minor effects on the cysteine desulfurase activity of IscS complexes.</p><!><p>The Fe-S cluster assembly rate for the SecUec and SecU complexes were similar to each other (Figure 6) and significantly higher than analogous SDUec and SDU complexes with NFS1-ISD11 (Figure 4). The addition of 10 equiv of CyaY to the SecUec and SecU complexes completely inhibited Fe-S cluster formation (Figure 6A and 6B), consistent with previous results (29). At 2 equiv, CyaY is a slightly more effective inhibitor of the native SecUec complex than the mixed species SecU complex. The addition of FXN to the SecUec and SecU complexes (Figure 6C and 6D) also lowered the rate and possibly the amount of cluster bound; however, FXN did not completely eliminate Fe-S cluster formation. Thus, bacterial IscS assembly complexes are inhibited in Fe-S cluster formation by the inclusion of either frataxin ortholog.</p><!><p>The physiological function of frataxin has been intensively investigated since it was associated with the neurodegenerative disease Friedreich's ataxia (FRDA; reviewed in (48, 49)). A decrease in FXN levels in yeast and mouse model systems results in a phenotype that includes loss of Fe-S cluster enzyme activity, accumulation of iron in mitochondria, and susceptibility to oxidative stress (50–52). In addition, FXN levels are correlated with the age of disease onset for FRDA patients (53, 54), and FXN missense mutations were identified that show FRDA-like phenotypes in mouse (52) and yeast (55) model systems. A role for frataxin as an Fe chaperone and/or donor in Fe-S cluster biosynthesis is often cited due to the frataxin depletion phenotype (50–52), the ability of frataxin to bind Fe (56–63), and the acceleration of Fe-S cluster formation by Fe-bound FXN in vitro (58). Recent in vitro data as well as data presented here reveals that human FXN binds to the SDU complex and activates both the cysteine desulfurase and Fe-S formation reactions (34). Moreover, assays using FXN missense mutations exhibit defects in binding and activation that appear to correlate with the severity of clinical progression for FRDA patients (64, 65). The physiological function of CyaY is less clear. In contrast to FXN in eukaryotes, deletion or overexpression of CyaY does not affect cellular growth, iron content, and survival after exposure to H2O2 (36, 66). Part of the complication for these types of studies is the presence of both the ISC and SUF Fe-S assembly systems in E. coli. If the physiological role of CyaY were to inhibit Fe-S cluster formation by the ISC system, then one would expect overexpression of CyaY in the absence of the SUF system to give a loss of Fe-S cluster phenotype. To the best of our knowledge this experiment has not been reported. Unfortunately, the complicated phenotype for human FXN depletion, the lack of a phenotype for CyaY depletion, and the opposing in vitro functions in Fe-S cluster assembly have contributed to the general confusion over the physiological role of this protein.</p><p>Here we used a function replacement approach to understand the factors that mediate the dramatically different in vitro responses to frataxin homolog binding. More specifically, in vitro cysteine desulfurase and Fe-S cluster assembly activities were evaluated for protein complexes in which components from the recombinant human Fe-S assembly system: the NFS1-ISD11 cysteine desulfurase complex, the scaffold protein ISCU2, and FXN, and the recombinant E. coli system composed of the IscS cysteine desulfurase, the scaffold protein IscUec, and CyaY, were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is not controlled by the frataxin homolog but rather dictated by the cysteine desulfurase.</p><p>Interestingly, even though IscSec and NFS1 are almost 60% identical, IscSec exhibited a kcat/KM for the cysteine desulfurase activity that is 10–70 fold higher than that of the SD complex and was largely unchanged by the addition of IscUec and CyaY (34, 40). In contrast, the low activity of the SD complex could be rescued by the addition of ISCU2 and FXN; the SDUF complex had a kcat/KM that is about double that of IscSec alone or as a SecUec or SecUecCec complex. The SecUec and SDU complexes also exhibit striking differences in their ability to facilitate Fe-S cluster formation. The SecUec complex is highly active in the Fe-S cluster assembly assay, whereas the SDU complex exhibits little ability to mediate this reaction in the absence of frataxin. These differences were not due to the identity of the scaffold protein: interchanging the IscUec and ISCU2 proteins did not substantially alter the higher rate of the complexes containing Sec or the substantially inhibited rates of complexes containing SD (Figures 4, 6). Moreover, addition of CyaY or FXN to these reactions inhibited Fe-S assembly by Sec-containing complexes and activated Fe-S assembly by SD-containing complexes (Figures 4, 6), albeit inhibition of the E. coli complex by CyaY was more efficient than by FXN and activation of the human complex by FXN was more efficient than by CyaY.</p><p>Our data therefore suggest that intrinsic features of IscSec and human SD cysteine desulfurase components control both the basal activity as well as the effect of binding the frataxin homologs CyaY and FXN. In both cases, binding of the effector molecule switches the activity, either from "off" to "on" or from "on" to "off". In this sense, NFS1 behaves less like its ortholog IscS and more like its paralogs, such as SufS, which are characterized by modest activity that can be increased or activated by accessory proteins (67–69). Evolutionary innovations in cysteine desulfurases from eukaryotes that control the basal activity and intrinsic features of frataxin homolog regulation are not known; binding of ISD11 and conserved amino acid changes in eukaryotes near the active site could play important roles in this process. Precise molecular details that regulate these features are currently under investigation.</p><p>Our data are consistent with FXN and CyaY binding playing a role in modulating Fe-S cluster formation by trapping an "on" conformation of SDU or inducing an "off" conformation of SecUec (Figure 7). This model is consistent with the fact that SDU forms a stable complex with FXN (34, 35) and SecUec forms a stable complex with CyaY (21, 27, 28). The fact that FXN and CyaY can functionally replace each other in vitro, albeit at lowered efficiencies, is consistent with the formation of a complex with a common structure and with the ability of both of these proteins to suppress defects caused by deletion of YFH1, which encodes the S. cerevisiae frataxin homolog (38, 39). These modifications to the cysteine desulfurase may function as part of a protein-level regulatory mechanism. Future experiments will be aimed at providing additional mechanistic insight into the differences between the human SD complex and IscSec and how these differences control Fe-S cluster assembly.</p>