BBAMCR-17401; No. of pages: 6; 4C: 2, 3 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria☆ F. Wayne Outten ⁎ University of South Carolina, Department of Chemistry and Biochemistry, 631 Sumter Street, Columbia, SC 29208, USA

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Article history: Received 17 September 2014 Received in revised form 31 October 2014 Accepted 3 November 2014 Available online xxxx Keywords: Suf Isc Iron–Sulfur Fe–S cluster Plasmodium B. subtilis

a b s t r a c t Fe–S clusters play critical roles in cellular function throughout all three kingdoms of life. Consequently, Fe–S cluster biogenesis systems are present in most organisms. The Suf (sulfur formation) system is the most ancient of the three characterized Fe–S cluster biogenesis pathways, which also include the Isc and Nif systems. Much of the first work on the Suf system took place in Gram-negative Proteobacteria used as model organisms. These early studies led to a wealth of biochemical, genetic, and physiological information on Suf function. From those studies we have learned that SufB functions as an Fe–S scaffold in conjunction with SufC (and in some cases SufD). SufS and SufE together mobilize sulfur for cluster assembly and SufA traffics the complete Fe–S cluster from SufB to target apo-proteins. However, recent progress on the Suf system in other organisms has opened up new avenues of research and new hypotheses about Suf function. This review focuses primarily on the most recent discoveries about the Suf pathway and where those new models may lead the field. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. © 2014 Published by Elsevier B.V.

1. Introduction Iron–sulfur (Fe–S) cluster metalloproteins play myriad roles in cell function, ranging from amino acid biosynthesis to transcriptional regulation. These diverse functions arise from the multiple types of Fe–S clusters assembled in vivo, ranging from relatively simple [2Fe–2S] clusters, found in some classes of ferredoxin, to complex, mixed-metal clusters, such as the [Mo–7Fe–9S] cluster (or FeMo cofactor) of nitrogenase [23,56]. Fe–S cluster function is also intimately associated with the protein frameworks to which they are bound. Protein environment can greatly alter Fe–S cluster reduction potentials and overall sensitivity to oxidation, providing a means to subtly tailor Fe–S cluster function to fit specific biochemical functions. Due to their versatility and the ready availability of ferrous iron and sulfide in the early Earth environment, Fe–S cluster metalloproteins have become intimately associated with key metabolic and regulatory pathways in most organisms. The addition of oxygen to the atmosphere exposed Fe–S clusters as a potential Achilles heel of cellular metabolism [58]. Solvent exposed Fe–S clusters can readily react with oxygen and its derivatives (H2O2 and O•2) [16,27,30,34]. Such oxidation events often lead to cluster disassembly and release of Fe2+, which in the cellular milieu can undergo further Fenton chemistry with H2O2 to produce highly toxic hydroxyl radicals.

☆ This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. ⁎ Tel.: +1 803 777 8151. E-mail address: [email protected].

Under this selective pressure, some Fe–S cluster proteins were replaced with less sensitive, often iron-free alternative enzymes. Despite their sensitivity to oxidation, the essential roles of many Fe–S cluster proteins have been maintained throughout evolution. As a supplemental strategy to replacing these key metalloproteins with other, less sensitive alternatives, many aerobic organisms have evolved with complex antioxidant systems that minimize the accumulation of reactive oxygen species in the cell [26,28]. In addition, the in vivo biogenesis systems for Fe–S clusters themselves have become a complex and interlocking network of highly regulated pathways that maintain adequate levels of Fe–S cluster assembly even under adverse conditions such as oxidative stress or iron starvation. The Fe–S cluster biogenesis systems of aerobic and facultative aerobic organisms have been selected for their robust activity and careful handling of assembly intermediates that allow them to keep pace with the increased Fe–S cluster demand that comes with an aerobic lifestyle. One such Fe–S cluster system, the Suf pathway, was likely present in a simple form in the earliest progenitors of modern species [7,72]. While Suf is still maintained in a simple form in many anaerobes, the pathway has progressively grown more complex throughout evolution [7,62,72, 76,77]. Over the past 15 years the Suf system has been the subject of in depth biochemical, genetic, and regulatory studies. Much of this accumulated knowledge of Suf function has been well reviewed in other publications, to which I point the reader for more in depth coverage of the Suf system [5,7,29,63,82]. Here I will focus primarily on recent progress on Suf in more diverse organisms beyond the Gram-negative model organisms, Erwinia chrysanthemi (recently renamed Dickeya dadantii) and Escherichia coli, in which it was first characterized [49,53,

http://dx.doi.org/10.1016/j.bbamcr.2014.11.001 0167-4889/© 2014 Published by Elsevier B.V.

Please cite this article as: F. Wayne Outten, Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbamcr.2014.11.001

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55,72]. Where necessary I will allude back to results from E. chrysanthemi and E. coli in order to compare and contrast that work to the newest discoveries in other organisms. 2. The Suf Fe–S cluster biogenesis system: An overview The core Suf system is composed of the SufB Fe–S cluster scaffold and the SufC ATPase (Fig. 1) [7,72]. SufB seems distinct from the IscU scaffold found in the well-characterized Isc pathway. SufB is unstable and prone to heterogeneous oligomer formation when expressed alone. Fe–S cluster assembly on SufB results in formation of a [4Fe–4S] cluster but, unlike IscU, no stable [2Fe–2S] intermediate is observed during SufB cluster assembly [9,38,79]. SufC is homologous to the nucleotide hydrolysis domains of the ATP-binding cassette (ABC) family of transporters that use ATP hydrolysis to drive transport across cellular membranes. Together the two proteins can form a SufB2 C 2 complex that is capable of forming and transferring Fe–S clusters to apo-acceptors [8,38,57,60]. The exact role of the SufC ATPase activity in this process is unknown but deletion of sufC or mutations in the ATP binding site abolish in vivo function of the Suf pathway [18,48,64,80]. The core Suf system can be augmented by a number of accessory proteins. SufD is a paralogue of SufB but does not appear to function

Fig. 1. Models of Suf function in multiple organisms and organelles. Solvent accessible structures are used for SufB (red), SufC (green), SufD (blue), SufE (orange), SufS (yellow), and SufU (magenta). Gray arrows with question marks indicate possible Fe–S cluster assembly/trafficking steps that have not been verified experimentally.

as an Fe–S scaffold. SufD interacts with SufC and SufB to form a SufBC2D complex (Fig. 1) [48,54,60]. The SufBC2D complex may replace SufB2C2 as the scaffold complex or it may be used for a distinct step in cluster assembly prior to formation of the final cluster on SufB2C2 (see next paragraph) [8,64]. SufA is a member of the A-type carrier (ATC) family of Fe–S cluster carrier proteins, which includes IscA and ErpA [77]. SufA can accept Fe–S clusters from the SufB scaffold and transfer them to downstream apo-proteins [9,20]. The ATCs are just one class of Fe–S cluster carrier proteins. Monothiol glutaredoxins and Nfu-like proteins have also been implicated in trafficking Fe–S clusters from scaffolds to target proteins [4,6,21,32,46,59,90]. In some systems the suf operon has its own dedicated transcriptional regulator while in other cases the suf pathway is controlled by multiple regulators that respond to stresses that disrupt Fe–S cluster metabolism [39,50,53,55,70,78,86,89]. Stepwise Fe–S cluster assembly requires the donation of sulfide and iron. While sodium sulfide can be used for in vitro Fe–S cluster reconstitution, most Fe–S cluster biogenesis systems that have been studied at the biochemical level contain a cysteine desulfurase enzyme (SufS in the Suf pathway) that uses a pyridoxal 5′-phosphate (PLP) cofactor to mobilize sulfur from L-cysteine (Fig. 1) [1,47,65,87,88]. This sulfur, bound to the cysteine desulfurase as a persulfide (R–S–SH) intermediate, is ultimately reduced and incorporated into the Fe–S cluster as sulfide (S2 −). This mechanism limits the release of toxic sulfide in the cytoplasm, mitochondria, or chloropolast. In vivo iron donation for Fe–S cluster assembly is quite murky but the Suf system seems to require both SufC ATPase activity as well as the presence of SufD in order for efficient iron acquisition in vivo (Fig. 1) [64]. These results suggest SufBC2D formation is required for iron incorporation into the SufB scaffold. Whether this effect is due to a direct role for SufC and SufD in iron mobilization or indirectly due to disruption of some other step of cluster assembly is not clearly understood. Clearly some organisms lack SufD but are still able to use SufBC for cluster assembly, suggesting there may be more than one route for iron acquisition or that organisms in certain ecological niches do not require specialized iron trafficking pathways (Fig. 1) [7]. One clear theme of the Suf system in Proteobacteria is that the stepwise Fe–S cluster assembly pathway is highly regulated by protein–protein interactions. The interaction of SufB with SufC is required to enhance the low basal ATPase activity of SufC in Thermotoga maritima [14,57] and similar effects have been observed when comparing E. coli SufC to SufB2C2 and SufBC2D (F.W. Outten and K.S. Thomas, unpublished data). In Proteobacteria and the chloroplast, SufS has low intrinsic cysteine desulfurase activity unless it is stimulated by the SufE partner protein that accepts persulfide from SufS via a conserved Cys residue [12,41,54,68,81,84]. Transfer of persulfide cycles the SufS desulfurase enzyme back to its resting state to allow initiation of another round of catalysis [52,54,68,81,84]. Recent studies have also suggested that SufE in E. coli may be able to allosterically enhance SufS activity by stimulating L-cysteine binding when the two proteins interact [71]. The concerted SufS–SufE cysteine desulfurase activity is further enhanced by the addition of the SufBC2D complex in E. coli [54]. The enhancement of SufS–SufE by the SufBC2D complex occurs because SufE physically interacts with SufB to transfer persulfide to the scaffold protein for cluster assembly [38]. The efficient transfer of persulfide to SufB allows SufE to accept another persulfide from SufS, thereby stimulating the overall production of persulfide from L-cysteine [38,68]. Since two key enzymes of the Suf pathway (SufC and SufS) are essentially inactive unless they interact with the correct partner proteins, this tight regulation controls the stepwise assembly of Fe–S clusters in vivo. One reason for the tight regulation of SufS activity could be to protect the reactive persulfide intermediate from perturbation by oxygen or reactive oxygen species. It was recently shown that E. coli SufS is more resistant to H2O2 mediated thiol oxidation than the E. coli IscS cysteine desulfurase [12]. The resistance of SufS is likely tied to the conformation of its active site Cys residue, which is more shielded when compared to the relatively exposed active site of IscS [11,17,40]. The

Please cite this article as: F. Wayne Outten, Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbamcr.2014.11.001

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low solvent exposure of the SufS active site likely explains why it becomes trapped in the persulfide intermediate state in the absence of its SufE sulfur transfer partner. Furthermore, SufE was able to stimulate and maintain SufS activity throughout exposure to levels of H2O2 that inactivate IscS [12]. Whether SufS is equally resistant to oxidative stress in other organisms remains to be confirmed.

3. Recent advances in our understanding of sulfur donation for Fe–S cluster biogenesis SufS is the cysteine desulfurase in most Suf systems, with the notable exception of some Archaea that live in sulfide-rich environments and lack a SufS homologue [7]. SufS, and other close homologues such as CsdA, require a partner protein to accept the persulfide. In Proteobacteria, Cyanobacteria, and the chloropolast of some higher plants, the SufS partner protein is SufE while in Firmicutes such as Bacillus subtilis, the partner protein is SufU (Fig. 1) [2,41,52,54,67]. These partner proteins all share a common structural fold and a conserved Cys residue that accepts the persulfide sulfur from the active-site Cys residue of the cysteine desulfurase [19,35,62]. B. subtilis SufU diverges structurally from the SufE-like proteins in that it has two additional Cys residues that are poised near the sulfur acceptor site (Cys41 in B. subtilis SufU). Mutation of SufU Asp43 to an Ala results in purification of small amounts of Fe–S cluster, presumably bound to the 3 Cys residues [3]. The ability of SufU to bind small amounts of Fe–S cluster led to its designation as an Fe–S scaffold protein for the Suf system in Firmicutes [3,62]. This assignment is consistent with the extensive homology between SufU and the IscU Fe–S cluster scaffold of the Isc Fe–S cluster biogenesis system in Proteobacteria. However, the B. subtilis Suf system also contains SufB, raising the question of why two distinct scaffolds are present in this pathway. Furthermore, amino acid alignments do reveal small but key differences between IscU and SufU. SufU family members contain an 18–21 residue insert between the second and third Cys residue. SufU has also replaced a key His residue (H105 of IscU) used for cluster binding or stabilization with a Lys residue. Functionally, IscU does not enhance its cognate desulfurase IscS to the same levels as SufU enhances SufS, suggesting there are different roles for the two proteins [2,12,33,67]. In addition to low level Fe–S cluster binding, the three Cys residues of SufU together with the D43 residue, form a binding site for Zn2 + (Fig. 2) [36]. Recent work has analyzed the role of the Zn 2 + in the function of B. subtilis SufU [69]. The Ka of SufU for Zn2+ was measured via a competition assay with the high affinity chelator TPEN and shown to be 1017 M−1 making it one of the tightest zinc-binding proteins

Fig. 2. Structure of B. subtilis SufU (magenta) bound to Zn2+ (teal sphere). The four ligands to the zinc ion are shown in stick representation and labeled by residue. The structure is from PDB 2AZH.

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currently characterized. The enhancement of SufS activity by SufU requires Zn2 + to be bound to SufU and the apo-form of SufU was found to be structurally altered compared to the zinc holo-form [69]. In contrast to IscU, exhaustive in vitro attempts to reconstitute stoichiometric amounts of Fe–S clusters on zinc-bound or apo-SufU consistently show little to no cluster formation on SufU [3,69]. Since the Zn2+ binding site is the same as the presumptive cluster binding site, the high affinity of SufU for zinc would seem to rule out any cluster binding so long as zinc is present. In fact, it was shown by EXAFS that the Zn2+ site is not perturbed during attempted in vitro Fe–S cluster reconstitution, confirming that zinc cannot be easily released to allow cluster binding to SufU [69]. Since the zinc appears to be required for structural stability of SufU, the apo-form of SufU is also not a good Fe–S scaffold in vitro. Based on these results, the current model of SufU function is that it acts as a sulfur transfer partner for SufS but is not a bona fide scaffold protein [69]. 4. A role for the Suf system in the cytosol of some eukaryotes Initial characterization of the subcellular localization of the Suf proteins indicated that Suf was restricted to the cytosol of Bacteria and Archaea, to the plastids of red algae and higher plants [80,83,85], and to the plastid-derived apicoplast of some Plasmodium species (see Section 5) (Fig. 1). The one notable exception to that observation was the AtSufE1 protein of Arabidopsis thaliana. A. thaliana contains three proteins with SufE-like domains. AtSufE1 contains an N-terminal SufE domain paired with a C-terminal BolA domain. AtSufE1 appears to be dual localized to both plastids and mitochondria and interacts with both the plastid-localized AtSufS and with AtIscS localized to the mitochondria [81,84]. Targeted deletion of the gene encoding AtSufE1 is embryonic lethal [81]. A. thaliana plastids also contain AtSufE2, which activates the cysteine desulfurase activity of AtSufS 40-fold but appears to be differentially expressed in flower and pollen tissues. A third SufE plastid homologue, AtSufE3, contains a SufE domain and a domain similar to bacterial quinolinate synthase (NadA). AtSufE3 activates AtSufS activity by 70-fold and forms a [4Fe–4S] cluster via the NadA domain, suggesting that the SufE sulfur transfer domain in this protein is dedicated to maintaining the Fe–S cluster used by the NadA domain [44]. The reason for the complexity of SufE functions in A. thaliana is unclear but demonstrates that the SufE sulfur transfer domain may play versatile roles in sulfur trafficking through fusion to other protein domains. Recent work has indicated that an unusual chimeric protein containing SufC and SufB fused together as Bh-SufCB is localized to the cytoplasm in the anaerobic parasite Blastocystis (Fig. 1) [76]. The sufC and sufB domains of the chimeric protein are most similar to homologues in anaerobic or thermophilic Archaea and Bacteria, especially the Methanomicrobiales, suggesting lateral gene transfer to Blastocystis from a related microbe. Bh-SufCB was able to perform Fe–S cluster biogenesis in a transgenic E. coli strain lacking the endogenous sufCD and iscUA genes [76]. Purified Bh-SufCB dimer was biochemically similar to the SufB 2C 2 complex in that it was able to enhance the cysteine desulfurase activity of E. coli SufS and SufE and displayed ATPase activity. The Bh-SufCB dimer could also be reconstituted with sub-stoichiometric amounts of [4Fe–4S] cluster in vitro [76]. The exact role of Bh-SufCB in Blastocystis cytoplasm is unclear. As a eukaryote, Blastocystis already contains the Cia1, Cia2, MMS19, Nbp35, Nar1, and Tah18 components of the cytoplasmic iron–sulfur cluster assembly (CIA) machinery, which is distinct from Suf [75]. It also is puzzling that even though Bh-SufCB can enhance E. coli SufS and SufE activity, there are no homologues of SufS and SufE in Blastocystis. Therefore it is not clear how Bh-SufCB would obtain an Fe–S cluster in the cytosol unless it interacts with the CIA machinery. Blastocystis does possess a mitochondrion-related organelle (MRO) with the standard Isc Fe–S cluster biogenesis system, which presumably carries out the bulk

Please cite this article as: F. Wayne Outten, Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbamcr.2014.11.001

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of de novo cluster assembly in that organism [76]. Most eukaryotes lack any Suf homologues or have Suf localized to plastid organelles and therefore rely on the CIA system to carry out Fe–S cluster biogenesis functions in the cytosol. Since the chimera protein is mildly upregulated in response to oxygen, acquisition of Bh-SufCB may provide some advantage to Blastocystis under oxygen stress [76]. 5. The importance of Suf in human pathogens: tuberculosis and malaria Mycobacterium tuberculosis is the causative agent of TB in humans. This organism contains a sufRBDCSUT operon but lacks the full isc Fe–S cluster biogenesis system [24]. The “sufR” in M. tuberculosis is an uncharacterized member of the ArsR superfamily of transcriptional regulators and is not to be confused with the well-characterized SufR regulator of the cyanobacterial suf genes [78]. The sufT gene encodes a protein of unknown function although it is homologous to eukaryotic cytosolic and plastid Fe–S cluster biogenesis components (Cia2 and HCF101, respectively) [66,75]. Attempts to delete the suf genes from M. smegmatis were unsuccessful, suggesting that the operon is essential in Mycobacteria [24]. Yeast two-hybrid assays using the M. tuberculosis genes revealed that SufB interacts with SufC and SufD in vivo. Interestingly, the sufB gene is the site of an intein insertion sequence. Inteins are proteins embedded in-frame in the coding sequence of another gene. Inteins typically catalyze their own posttranslational protein splicing to remove them from the host polypeptide [51]. It was shown that unspliced SufB is non-functional because it fails to associate with SufC or SufD by yeast two-hybrid assay [25]. These preliminary studies indicate that the Suf pathway and/or intein splicing may be good targets for novel antibiotic therapy to treat drug-resistant TB. Future studies are required to elucidate the biochemical functions of the novel Suf proteins present in Mycobacteria. Malarial parasites, such as P. falciparum, contain a non-photosynthetic plastid organelle known as the apicoplast. The apicoplast is responsible for a number of biochemical pathways, such as type II fatty acid synthesis, lipoate synthesis, and isoprenoid synthesis. Many of the pathways in the apicoplast require one or more Fe–S cluster metalloenzymes. P. falciparum contains the sufABCDSE system with all of the genes encoded in the nuclear genome except for sufB, which is encoded in the apicoplast genome. The SufC, SufS, and SufE proteins were shown to be localized to the apicoplast in vivo [18,37]. A dominant negative point mutation in SufC (K140A) was toxic due to a defect in the production of isoprenoids by the apicoplast, which could be rescued by the addition of isopentenyl pyrophosphate to the growth media [18]. A similar point mutation (K140R) in the E. coli SufC protein was previously shown to disrupt in vivo iron donation for Fe–S cluster assembly on the SufB scaffold protein [64]. The isoprenoid defect caused by expression of SufC(K140A) in P. falciparum resulted from the loss of the apicoplast organelle from that transgenic strain. The failure to synthesize isoprenoids appeared to be a downstream consequence arising from the loss of the apicoplast rather than an upstream cause of apicoplast loss. Based on these results it was hypothesized that apicoplast maintenance relies on the Suf-mediated maturation of one or more Fe–S cluster proteins in the apicoplast of P. falciparum [18]. The Suf system has also been studied in the P. berghei murine malaria model system. In that organism the nuclear-encoded sufC, sufD, sufE, and sufS genes could not be deleted using genetic approaches, suggesting they are essential for viability during the blood infection stages of Plasmodium growth [22]. The same study confirmed that SufA, SufC, SufD, SufE, and SufS were all localized to the apicoplast in P. berghei (Fig. 1). The sufA gene could be successfully deleted in P. berghei and the loss of sufA did not alter the progression of the parasite through its full life cycle, from the mosquito vector to erythrocytic growth in the mouse host [22]. The lack of a ΔsufA phenotype is superficially similar to what has been observed in

E. coli [53]. However, in E. coli it has been shown that other ATC proteins (such as IscA) likely compensate for the loss of SufA and a severe phenotype is only observed if the genes are deleted in combination [43,73]. It is not clear how this would work in Plasmodium since the IscA1 and IscA2 proteins are likely localized to the mitochondria rather than the apicoplast. Possibly the NifU-domain containing protein, NFUapi, which is also apicoplast localized, compensates for the loss of sufA [21]. The distantly related NfuA protein of E. coli has been shown to act as an Fe–S cluster transfer protein, with some similarities to SufA and other ATCs, and functions under the same stress conditions as the Suf pathway [4,59]. Deletion of the gene encoding NFUapi in P. berghei caused only a mild decrease in the release of liver stage merozites from cultured hepatoma cells with no other measurable effects on the Plasmodium life cycle [21]. Hopefully future work will characterize a mutant with deletions of both sufA and nfu. The importance of the Suf system for apicoplast maintenance and viability in Plasmodium suggests that it could be a valid target for antibiotic therapy to inhibit blood stage and liver stage malaria parasites. Recently it was demonstrated that D-cycloserine could inhibit the in vitro cysteine desulfurase activity of P. falciparum SufS and SufE with a modest IC50 of 29 μM [10]. D-cylcoserine binds to the PLP cofactor of members of the aspartate amino-transferase superfamily (of which cysteine desulfurases are members). Upon binding, D-cycloserine forms a 3-hydroxyisoxazole-pyridoxamine adduct with PLP, causing inhibition of the target enzyme [15,42]. D-cycloserine is already in clinical use as a second line drug against the human pathogen M. tuberculosis [13]. D-cycloserine was also able to inhibit the blood stage growth of P. falciparum, with IC50 values from 60 to 70 μM depending on the exact infection cycle [10]. Although it has not been conclusively shown that the growth inhibitory effect of D-cycloserine is tied to SufS inhibition (as opposed to inhibition of other PLP enzymes), it is a promising start to identifying drugs that target Suf function. 6. Unanswered questions about Suf function The most critical gaps in our knowledge of Suf function are the biochemical details of in vivo iron donation. While SufC and SufD have been implicated in this process in E. coli, the situation is likely different in organisms that lack SufD. The discovery that SufBC 2 D binds FADH2 has raised the possibility that reducing equivalents from the cellular flavin pool may be used to mobilize ferric iron (by reduction to the ferrous form) during cluster biogenesis [79]. So far this question has not been adequately addressed in vivo and remains an intriguing hypothesis. In the Proteobacteria, the Suf system is primarily a stress-response Fe–S cluster biogenesis pathway that must acquire iron under conditions where iron homeostasis is disrupted (such as oxidative stress, iron starvation, or upon exposure to toxic metals) [31, 45,48,49,53,55,61,74]. The mechanism of iron acquisition by the stress-response systems may be different than the pathway utilized by organisms, such as B. subtilis, where Suf is the main Fe–S cluster biogenesis system. In addition to the question of iron donation, the role of Suf accessory proteins SufC, SufD, SufT, and SufU must be characterized to gain a full understanding of Suf function. Elucidation of their biochemical roles is especially important for comparing and contrasting Suf systems in different organisms in order to establish hypotheses for the complex phylogenetic distribution of the suf genes. Likewise, further characterization of the regulatory pathways, ranging from transcriptional to post-translational, that control Suf expression will be critical for placing Suf function in the context of the broader physiology of each organism and organelle. Finally, the recent discovery of complex subcellular distribution of Suf homologues in eukaryotes has opened up new possibilities for alternate Suf functions in those organisms, especially as it concerns SufE-mediated sulfur trafficking and the possibility of non-scaffold roles for SufBC in the Blastocystis cytoplasm.

Please cite this article as: F. Wayne Outten, Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbamcr.2014.11.001

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Acknowledgments This work and the studies conducted by the author were supported by National Institutes of Health grant GM81706 and by grants from the University of South Carolina to F.W.O. References [1] J.N. Agar, L. Zheng, V.L. Cash, D.R. Dean, M.K. Johnson, Role of the IscU protein in iron–sulfur cluster biosynthesis: IscS-mediated assembly of a 2Fe–2S cluster in IscU, J. Am. Chem. Soc. 122 (2000) 2136–2137. [2] A.G. Albrecht, F. Peuckert, H. Landmann, M. Miethke, A. Seubert, M.A. Marahiel, Mechanistic characterization of sulfur transfer from cysteine desulfurase SufS to the iron–sulfur scaffold SufU in Bacillus subtilis, FEBS Lett. 585 (2011) 465–470. [3] A.G. Albrecht, D.J. Netz, M. Miethke, A.J. Pierik, O. Burghaus, F. Peuckert, R. Lill, M.A. Marahiel, SufU is an essential iron–sulfur cluster scaffold protein in Bacillus subtilis, J. Bacteriol. 192 (2010) 1643–1651. [4] S. Angelini, C. Gerez, S. Ollagnier-de Choudens, Y. Sanakis, M. Fontecave, F. Barras, B. 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Please cite this article as: F. Wayne Outten, Recent advances in the Suf Fe–S cluster biogenesis pathway: Beyond the Proteobacteria, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbamcr.2014.11.001