Iron–sulfur clusters (Fe–S) are amongst the most ancient and versatile inorganic cofactors in nature which are used by proteins for fundamental biological processes. Multiprotein machineries (NIF, ISC, SUF) exist for Fe–S cluster biogenesis which are mainly conserved from bacteria to human. SUF system (sufABCDSE operon) plays a general role in many bacteria under conditions of iron limitation or oxidative stress. In this mini-review, we will summarize the current understanding of the molecular mechanism of Fe–S biogenesis by SUF. The advances in our understanding of the molecular aspects of SUF originate from biochemical, biophysical and recent structural studies. Combined with recent in vivo experiments, the understanding of the Fe–S biogenesis mechanism considerably moved forward. Keywords Biosynthesis · Iron–sulfur cluster · Metallocenter assembly · Mechanism · SUF Introduction cofactors must be tightly regulated. Multiprotein machiner- ies exist for Fe–S cluster biogenesis which are mainly con- Iron–sulfur clusters (Fe–S) are amongst the most ancient and served from bacteria to human, although elaborate systems versatile inorganic cofactors in nature. They are used by pro- have diverged through evolution. teins for fundamental biological processes such as nitrogen Three distinct types of biosynthetic machinery have fixation, photosynthesis, respiration, DNA repair [ 1–4]. The emerged from bacteria, archaea and eukaryotic organelles, most common types of Fe–S are the 2Fe–2S and the cubane based on biochemical evidence and organization of genes in 2+ 4Fe–4S clusters that contain either ferrous (F e ) or ferric bacterial operon. Whereas the NIF system plays specialized 3+ 2− (Fe ) iron and sulfide (S ) . In most cases, thiolate from roles in the maturation of Fe–S proteins in nitrogen fixing cysteine coordinate iron ions of the cluster although there are organisms such as A. vinelandii [5, 6], the ISC machinery is increasing examples of nitrogen coordination—provided by the primary system for general Fe–S cluster biosynthesis in histidine or arginine residues—and oxygen coordination— bacteria . Moreover, along with additional components, from aspartate or tyrosine. Examples of coordination by the ISC system constitutes the eukaryotic mitochondrial exogenous ligands, such as water molecules, enzyme sub- machinery for Fe–S cluster biogenesis. Components in strates or cofactors have also been observed . Because of eukaryotes were discovered by a variety of genetic screens the toxicity of free iron and sulfur, the biogenesis of Fe–S performed on Saccharomyces cerevisiae based on Fe homeo- stasis, amino acid biosynthesis, ribosome biosynthesis and DNA repair . The third bacterial assembly system, termed The original version of this article was revised due to a SUF, plays a similar general role as ISC in many bacteria, retrospective Open Access order. but is operative only under conditions of iron limitation or oxidative stress . Not surprisingly, the bacterial SUF * Sandrine Ollagnier de Choudens email@example.com system also forms the basis of the Fe–S cluster biogenesis machinery in plant chloroplasts, an O -producing organelle Laboratoire de Chimie et Biologie des Métaux, Biocat, that is most likely inherited from the cyanobacterial ances- Université Grenoble Alpes, Grenoble, France tor of plastids [10, 11]. The SUF system also appears to be Laboratoire de Chimie et Biologie des Métaux, CNRS, the sole system for Fe–S cluster biogenesis in archaea and BioCat, UMR 5249, Grenoble, France cyanobacteria, as well as many Gram-positive, pathogenic CEA-Grenoble, DRF/BIG/CBM, Grenoble, France Vol.:(0123456789) 1 3 582 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 and thermophilic bacteria. Genomic analyses revealed that inhibitory effect of d -cycloserine is due to SufS inhibition the number and type of operons coding for these systems (it may inhibit other PLP enzymes) it is a promising start to vary from one microorganism to another. Some contain all identify drugs that target Suf function. Recent investigations systems, others two or only one, and some only contain a in S. aureus showed that SUF system is the target system for part of one system [9, 12, 13]. a polycyclic molecule named molecule 882 . In particu- For all systems, the basic process of Fe–S biogenesis lar, when SuB, SufC and SufD are pulldown with molecule requires donation of iron (ferric or ferrous) and sulfide as 882, a direct interaction between molecule 882 and SufC bridging ligand for iron ions. Sulfide is provided by cysteine is observed (KD 2 µM). In agreement with this result, a desulfurase enzyme that uses l -cysteine as stable and safe strain deficient in the maturation of Fe–S biogenesis (ΔsufT, source of sulfur, whereas origin of iron is still unclear ΔnfU) displays an increased sensitivity to molecule 882 than (Fig. 1). The two components, iron and sulfide, first combine the wild-type. All these studies prove that SUF system is a on a protein that serves as a “scaffold” for cluster assembly good target for an antibiotherapy and may guide the develop- (Step 1, Fig. 1). Due to the lability of the scaffold bound- ment of new antimicrobials. cluster it can be transferred to appropriate apoform of metal- loprotein either directly or using a series of carriers proteins that mediate trafficking and targeting of the mature Fe–S The SUF biogenesis system proteins (Step 2, Fig. 1). The SUF system is the most ancient of the currently identi- Fe–S biogenesis and health fied system of biogenesis [9 ]. As mentioned before, in some organisms, the SUF system is the only system present, and In humans, a number of genetic diseases are associated with therefore, is essential for viability. In others, SUF operates in dysfunction of the ISC system, showing the importance to parallel with ISC and NIF [22, 23]. Lack of a functional suf study at a molecular level Fe-S cluster biogenesis process to operon is neutral for E. coli under normal growth conditions better arrest these diseases [14, 15]. The SUF system dis- [9, 12, 24]. In contrast, under oxidative stress, deletion of the plays also an important scientific interest in health. Indeed, suf genes made E. coli unable to produce functional forms of the SUF machinery is not conserved in humans and it is the enzymes containing oxygen-labile Fe–S clusters . The only Fe–S biogenesis pathway in some pathogens such as same observation was obtained when cells were exposed to Staphylococcus aureus (SufSBCDUTA, and Nfu) [16, 17], 2,2′-dipyridyl, an iron chelator . These observations led Mycobacterium tuberculosis (SufRBDCSUT) , parasites to the conclusion that suf operon is functional under oxi- Plasmodium (SufABCDSE) and Toxoplasma, making SUF dative stress and iron limitation. Further genetic analyses an attractive pathogen-specific drug target. Recently, it was demonstrated that suf operon operates under stresses owing demonstrated that d -cycloserine could inhibit in vitro the to regulators such as apo-IscR (oxidative stress and iron cysteine desulfurase activity of P. falciparum SufSE (IC deprivation), Fur/RhyB (iron limitation) and OxyR (oxi- of 20 µM) . d -Cycloserine binds to the PLP cofactor and dative stress) [25–28]. The suf operon contains two (SufB, forms a 3-hydroxyisoxazole-pyridoxamine adduct with PLP SufC) to more than six genes (SufA, SufB, SufC, SufD, causing inhibition of the enzyme. d -Cycloserine is in clinical SufS, SufE, SufU) organized as single polycistronic tran- use as a second line drug against M. tuberculosis  and scriptional units, showing that the role of SUF has evolved was shown to inhibit the blood stage growth of P. falciparum through evolution (Fig. 2). A recent phylogenetic analy- . Although it was not conclusively shown that the growth sis of the SUF pathway suggests that diversification into Fig. 1 Simplified Fe–S assem- bly mechanism Apo chaperones Cysteine S-S-H Scaﬀold desulfurase Carriers Holo 2+ - Fe e CysAla Fe-S proteins Step 1: Fe-SAssembly Step 2: Fe-STransfer 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 583 Fig. 2 Evolution of suf operon. Selected examples of suf oper- Methanocaldococcusvulcanius ons among Archae and bacteria. SufC SufB Genes for sufA, sufB, sufC, Metallosphaeracuprina sufD, sufS and sufU are color- SufC SufB SufD coded to reflect their homology in different organisms. Adapted from  Prochlorococcusmarinus SufB SufC SufD SufS Bacillus subilis SufC SufD SufS SufU SufB SynechococcusPC7002 SufR SufB SufC SufD SufS Escherichia coli SufA SufB SufC SufD SufS SufE oxygen-containing environments disrupted iron and sulfur excess of ferric iron and sulfide and purification onto an metabolism and was a main driving force in the acquisition anion exchange column . Recently, in vivo experiments of additional (more) SUF proteins by the SufB–SufC core show that SufBC D complex after an early step purification . Thus, there would have been an evolutionary trajectory displays a typical 2Fe–2S UV-visible spectrum, reinforcing in which suf grew in complexity from an operon encoding the idea that SufB might be a 2Fe–2S protein rather than a only sufB–sufC through the sequential recruitment of other 4Fe–4S protein . Interestingly, SufB 2Fe–2S cluster is genes such as sufD, sufS and sufE. more stable and resistant to H O, O and iron chelator than 2 2 2 The SUF system has been the subject of in depth bio- the 2Fe–2S of IscU in agreement with its function under chemical, genetic and regulatory studies, especially in E. oxidative conditions . Both 2Fe–2S and 4Fe–4S holo- coli and Erwinia chrysanthemi [22, 30–32]. From that we forms of SufB are competent for transfer for intact cluster know that SufB, SufC, and SufD can interact with each other to diverse proteins such as SufA, ferredoxin (Fdx) and aco- forming SufB C, SufC D and SufBC D complexes; SufS nitase [38–40]. The N terminus of SufB from E. coli and its 2 2 2 2 2 interacts with SufE forming a 1:1 complex and similarly close relatives contains a putative Fe–S cluster motif (CXX- SufS interacts with SufU. Finally, SufSE complex interacts CXXXC) that was proposed early to be the site of Fe–S to SufBC D complex. cluster assembly . However, the cysteine triple mutant can still assemble a Fe–S cluster in vitro after chemical reconstitution suggesting that these cysteine are not cluster Biochemistry of Suf proteins ligands (Layer et al. unpublished results). Recently, cysteines of this motif were unambiguously excluded as ligands . SufB Among the invariant cysteine residues in SufB, Cys405 (E. coli) is proposed to be one of the Fe–S ligand from structural SufB is the scaffold protein of the system, an essential player studies  and recent in vivo experiments  (see below). in the process. SufB is a difficult protein to manipulate Residues Glu434, His433 and/or Glu432 are proposed to in vitro as it tends to be insoluble and it exists under dif- be the other Fe–S ligands . As a scaffold, SufB is able ferent oligomerization states (Fig. 3). This likely explains to interact with the cysteine desulfurase SufSE complex that no structure of SufB is available. As a scaffold protein, through SufE protein. The interaction between SufB and SufB is able to assemble transiently a Fe–S cluster even SufE occurs only if SufC is present in agreement with the though its nature is not clearly known. Previous studies have existence of SufB C and SufBC D physiological complexes 2 2 2 established that E. coli SufB assembles a 4Fe–4S cluster (Fig. 3). When SufB (within SufBC D complex) is incubated after in vitro reconstitution [33, 34]. Both 4Fe–4S and linear with SufSE and l -cysteine and without reducing agent, up to 3Fe–4S clusters were observed on purified His–SufB after seven sulfur atoms can accumulate on SufB . Recently, in vivo co-expression with sufCDSE genes . However, two cysteine residues of SufB, which are strictly conserved we discovered that SufB can stabilize a 2Fe–2S cluster after cysteine residues, were identify as good sulfur acceptor sites anaerobic incubation of apo-SufB with a threefold molar from SufE: Cys254 and Cys405 . Cys254A mutation 1 3 Archaea Bacteria 584 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 Fig. 3 Possible interaction of Suf proteins. Interactions SufD SufD between Suf proteins that were SufC identified by biochemical and SufB SufB SufE biophysical studies SufU SufS SufB SufU SufB SufB SufD SufS SufS SufB SufA SufA SufB SufB SufE SufU SufC SufE SufA SufC SufC SufS SufS SufS SufE SufE SufS SufS SufD SufD SufE SufC SufC SufD SufS SufE SufB SufD SufB SufC SufC SufC SufC abolishes sulfur accumulation while and Cys405A mutation the ATPase activity of SufC was not shown to be important strongly diminishes sulfur binding. Interestingly, Cys254 for Fe–S assembly in vitro. However, deletion of sufC or residue is critical for the stimulation of the cysteine desul- mutation in the ATP binding site abolish in vivo function furase activity of SufSE by SufBC D complex . of the SUF pathway [12, 47, 48]. In particular, the as puri- fied His6–SufBC D–SufC(L40R) in which there is a point SufC mutation in the Walker A site of SufC (and thus no ATPase activity) displays a eightfold reduction of iron content rela- SufC is encoded along with SufB scaffold in all suf operons tive to the wild-type His6–SufBC D strongly suggesting that identified so far, in agreement with biochemical evidences the ATPase activity is necessary for iron acquisition in vivo showing that these two proteins interact to form a SufB C during Fe–S assembly . If the entire E. coli suf operon 2 2 complex (Fig. 3). While it is not clear if this interaction is expressed, SufC is able to associate with SufB and SufD is physiologically relevant in E. coli, it reflects the active forming the SufBC D complex (Fig. 3). SufCB complex in organisms that lack SufD and have a minimal sufBC operon such as Methanococcus vulcanius SufD and Blastocystis. SufC is a monomer in solution (Fig. 3) and is endowed with an ATPase activity [12, 42]. It contains SufD is a paralog of SufB (17% identity and 37% similar- all motifs that are characteristic of the ABC ATPases, like ity) and sequence homology suggests that its gene derives the Walker sites A and B as well as ABC signature [43–45]. from a duplication of an ancestral SufB sequence. This is in The basal ATPase activity of the SufC alone is quite low but agreement with phylogenetic analyses showing that SufB significantly enhanced when SufC is associated with either gene appears earliest in the evolutionary time among the SufB or SufD (180-fold with SufB and fivefold with SufD) suf genes. SufD from E. coli, has a sequence with no known . Some amino acids were identified as potentially impor - predicted motifs, and after purification from E. coli it con- tant for ATPase activity [Lys40, Lys152, Glu171, Asp173 tains any cofactor or prosthetic group. Even it is a paralog and H203 (E. coli SufC)] based on comparison with ABC of SufB, after incubation with an excess of iron and sulfide, ATPases, but there was no in vitro study associated. So far, SufD does not harbor any Fe–S cluster. SufD is stable as 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 585 purified and under an homogeneous dimeric form (Fig. 3). rate [52–55]. Recent investigations show that interaction of SufD is proposed to play a role in iron acquisition since dele- SufE–SufS elicits changes in structural dynamics of SufS tion of sufD diminishes the iron content of the SufB C sub- within its active site facilitating the desulfuration reaction 2 2 complex (like the SufC K40R mutation) . Early studies and also that a conformational change of SufE accompanies by F. Barras and Expert’ groups demonstrated a link between the interaction with SufS . Thus, coupled conforma- SufD and iron metabolism [12, 49]. However, so far, there is tional changes likely accompanies the SufS–SufE interac- no in vitro study showing that SufD binds iron either ferrous tion explaining the enhancement of the cysteine desulfurase or ferric, even transiently. SufD can interact with SufC and activity. This is described in more details in the structural SufB to form SufBC D complex and in the absence of SufB section below. Cysteine desulfurase activity of SufSE com- can form also a SufC D complex (Fig. 3) . plex is further enhanced by both SufB C and SufBC D 2 2 2 2 2 complexes [33, 53] and recently, residues Cys254, Gln285 SufS and Trp287 of SufB were identified to be critical for the enhancement of the cysteine desulfurase activity of SufSE SufS is a PLP-dependent dimeric cysteine desulfurase by SufBC D . (Fig. 3) that mobilizes sulfur from l -cysteine substrate, resulting in an enzyme-bound persulfide intermediate at SufU Cys-364 (E. coli numeration) in the active site. It belongs the group II desulfurase enzyme family and have low basal SufU is present in many bacteria, in particular members of activity with regard to group I of cysteine desulfurase family the phylum Firmicutes (Bacillus subtilis, Enterococcus fae- such as IscS. Several structural features distinguish group II calis), and in some Mycobacteria (M. tuberculosis) (Fig. 2). enzymes from group I explaining their differences in activ - In B. subtilis, it is essential for survival [58, 59]. The SUF ity. In particular, a key structural difference between SufS pathway of the organisms that contain SufU has SufB, SufC, and IscS is that the extended lobe of SufS containing the SufD and SufS but lacks the mandatory sulfur acceptor active site loop has an 11-residue deletion. The shortening SufE. Strikingly, genomic analysis showed that SufU and of this region in SufS structurally restricts the flexibility SufE tend not to co-occur (i.e., nearly all species containing of the SufS Cys364-anchoring extended lobe. In contrast, sufU lack a copy of the sufE gene, and vice versa). B. subtilis the corresponding loop of IscS is longer and disordered in SufU diverges structurally from the SufE-like proteins in that most structures of IscS due to its flexibility . Therefore, it has two additional cysteine residues that are poised near group II cysteine desulfurases require a specific sulfur shut- the sulfur acceptor site (Cys41 in B. subtilis SufU). D43A tle protein for full activity. Furthermore, SufS binds tightly mutation of SufU results in purification of small amounts to SufE (K : 0.36 µM) and the resulting 1:1 complex dis- of Fe–S cluster, proposed to be bound by the three cysteines plays a much larger cysteine desulfurase activity [52, 53]. . The ability of SufU(D43A) to bind small amounts of Molecular investigations demonstrated that sulfur enters at Fe–S cluster led to propose SufU as an Fe–S scaffold protein SufS, in the form of persulfide on Cys364, and that thanks for the SUF system in Firmicutes [59, 60]. In agreement to a transpersulfuration reaction sulfur is transferred to the with this idea, recombinant purified wild-type SufU that is invariant SufE Cys51 residue . The sulfur transfer from devoid of Fe–S clusters, binds upon in vitro reconstitution a SufS to SufE proceeds via a ping-pong mechanism that may 4Fe–4S cluster under sub-stoichiometric amounts. The clus- be important for limiting sulfur transfer under oxidative con- ter can be transferred to the isopropylmalate isomerase Leu1, ditions [55, 56]. forming catalytically active Fe–S-containing Leu1 . SufU interacts with SufS (Fig. 3) and activate sulfur transfer SufE by enhancing SufS activity about 40-fold in vitro [59, 61]. Therefore, it was proposed that SufU functions as an Fe–S SufE protein exists under a monomeric form in solution cluster scaffold protein tightly cooperating with the SufS (Fig. 3). As mentioned above, SufE protein interacts with cysteine desulfurase. This assignment of SufU as a scaffold the SufS dimer in a 1:1 stoichiometry forming in solution a was consistent with the extensive homology between SufU SufS E complex (J. Pérard, unpublished results) (Fig. 3). and the IscU. However, several observations suggest these 4 4 When SufE interacts with SufS, the cysteine desulfurase two proteins have different roles. (1) Sequence alignments activity is increased by an order of magnitude [52, 54]. reveal small but important differences between IscU and The slowest step in the desulfurase activity corresponds to SufU. SufU proteins contain an insertion of 18–21 residues the nucleophilic attack of the Cys364 thiolate on the sub- between the second and third cysteine residue, and SufU has strate cysteine-PLP ketimine adduct. The invariant Cys51 also replaced a key histidine residue (H105 of IscU) used for of SufE acts as a co-substrate for SufS and accepts the sul- cluster binding. (2) IscU does not enhance the activity of fur from Cys364 of SufS, thereby enhancing the catalytic its cognate desulfurase IscS to the same level as SufU does 1 3 586 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 for SufS [52, 58, 61, 62]. (3) The three cysteine residues provides specific mechanistic advantages for cluster transfer of B. subtilis SufU (Cys41, Cys66, Cys128) together with to 4Fe–4S targets proteins under physiological conditions as 2+ the Asp43 constitute a binding site for Zn , that is suggested from genetic data . In conclusion, all studies 17 −1 tightly bound to SufU (K of 10 M ) . Substitution of on SufA are in agreement with the notion that SufA is a these amino acids disrupts zinc binding. The enhancement Fe–S carrier rather than a Fe–S scaffold protein dedicated 2+ of SufS activity by SufU requires Zn to be bound to SufU. to maturation of 4Fe–4S proteins. Individual Ala-substitutions of Cys41, Cys66, Cys128 and Asp43 eliminate sulfurtransferase activity . It is impos- sible to reconstitute an Fe–S cluster on a zinc-bound SufU Structural and biophysical analyses of Suf that was shown to stabilize the protein . Based on all proteins these results and considering that there is no need to get two distinct scaffolds (SufU and SufB) on a same SUF pathway, SufS the reasonable current model of SufU function is that it acts as a sulfur transfer partner for SufS but is not a bona fide There are five crystal structures of SufS protein (PDB scaffold protein . The precise role of zinc as a struc- numbers: 5J8Q; 4W91; 1T3I; 5DB5; 1I29) whose three tural and/or catalytic element during sulfur transfer reaction published (Fig. 4) [71–73]. The first crystal structure was remains to be uncovered. obtained in 2002 with SufS from E. coli (initially named CsdB) . The Cys364 residue, which is essential for the SufA activity of SufS toward l -cysteine is clearly visible on a loop of the extended lobe (Thr362–Arg375) in all enzyme SufA is a member of the A-type carrier (ATC) family of forms studied, in contrast to the corresponding disordered Fe–S cluster carrier proteins including IscA and ErpA . loop (Ser321–Arg332) of the T. maritima NifS-like pro- SufA is a dimer in solution (Fig. 3) and it shares with IscA tein, which is closely related to IscS. The extended lobe of the ability to bind 2Fe–2S and 4Fe–4S clusters after chemi- SufS has an 11-residue deletion compared with that of IscS cal reconstitution [65, 66]. When purified anaerobically leading to a restricted flexibility of the Cys364-anchoring after co-expression in vivo with its cognate partner proteins extended lobe in SufS. Structure of SufS from Synechocys- from the suf operon (SufBCDSE) it contains a 2Fe–2S clus- tis sp. is very similar to that of E. coli SufS . It shows ter . Like most of ATC proteins, SufA contains three that the loop on which the catalytic Cys372 resides is well- strictly conserved cysteine residues (C XC XC for ordered and also shorter by 11 residues in comparison to 50 114 116 E. coli SufA) which are proposed for a long time to act as IscS from T. maritima. Sequence comparisons establish that ligands of the Fe–S cluster based on mutagenesis studies all SufS proteins have loops of similar length. The catalyti- on eukaryotic homologues . However, structural data cally essential cysteine of SufS is located in a deep cleft, strongly suggest another coordination mode (see below) 5 Å away from PLP, in a region of the polypeptide chain . SufA can transfer its cluster to downstream apo-pro- with limited flexibility. This might explain why the activ - teins such as biotin synthase, aconitase (4Fe–4S enzymes) ity is so weak and why the limiting step of the reaction is and Fdx (2Fe–2S protein) [39, 67]. Cluster transfer from pre- the formation of the persulfide at the catalytic cysteine. assembled 2Fe–2S SufA to Fdx is more efficient than cluster Very recently, high-resolution crystal structure of the B. transfer from 4Fe–4S SufB C and SufBC D to Fdx. The dif- subtilis (Bs) homodimer in its product-bound state (i.e., 2 2 2 ference in transfer efficiency between SufA and complexes in complex with pyridoxal-5-phosphate, alanine, Cys361- may be due to the fact that 2Fe–2S cluster of SufA can be persulfide) was obtained . Like for other SufS proteins, directly transferred to Fdx while 4Fe–4S of complexes has BsSufS monomer forms a tightly intertwined homodimer to undergo first a conversion step (4Fe–4S to 2Fe–2S) prior with another monomer across the crystallographic symmetry to transfer to Fdx . It is also possible that the structure of axis. In addition, the interface and architecture of the BsSufS SufA may promote more rapid release of the 2Fe–2S cluster homodimer closely resemble those of E. coli SufS. as compared to complexes. SufA cannot transfer its cluster to SufBC D but on the other hand can receive cluster from SufE/SufU SufBC D . Even though SufBC D can transfer Fe–S 2 2 cluster to Aconitase (4Fe–4S) without requirement of SufA There are three crystal structures of SufE protein (PDB , recent studies demonstrated that the cluster transfer numbers: 1NI7; 1MZG; 1WL0) [74, 75] under monomeric to aconitase from SufBC D or SufB C proceed through form (Fig. 4). Escherichia coli SufE displays 35% iden- 2 2 2 a Fe–S SufA intermediate if apo-SufA is present during tity with E. coli CsdE (YgdK) (PDB id 1NI7). CsdE is a the Fe–S transfer . This suggests that SufA is impor- sulfur acceptor protein from CsdA cysteine desulfurase tant for maturation of 4Fe–4S proteins and that SufA likely and together they form a complex like SufSE . The 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 587 Fig. 4 Overview of Suf protein Name Organism Pdb code Resolution Structure Reference structures 2.5 Å Kitaoka, S et al. 2006 SufC E. coli 2D3W 1.75 Å Badger, J et al. 2005 SufD E. coli 1VH4 Mihara, H et al. 2002 SufS E. coli 1I29 1.8 Å Goldsmith-Fischman, S et al. 2004 E. coli 1MZG 2.0 Å SufE Kornhaber, GJ et al. 2006 SufU B. sublis 2AZH NMR 2D2A 2.7 Å Wada, K et al. 2005 SufA E. coli Wada, K et al 2009 2.2 Å SufC D E. coli 2ZU0 2 2 2.96 Å Hirabayashi, K et al. 2015 SufBC D E. coli 5AWF structures of E. coli SufE (RX) and CsdE (NMR) are will see below that indeed, interaction of SufS with SufE strikingly similar, but in spite of their strong structural leads to a similar phenomenon . conservation, there are differences in the protein dynam- Concerning SufU, there is only one structure from B. ics in the vicinity of the sulfur-acceptor site in these two subtilis (PDB code: 2AZH) (Fig. 4). The structure shows proteins, that may be responsible for a differential binding the presence of the zinc atom bound to SufU that displays specificity for the desulfurase or for downstream sulfur- a tetra-coordination by the four conserved residues, Cys41, acceptor proteins. E. coli SufE structure shows that the Cys66, Cys128, and Asp43 . active cysteine Cys51, forming persulfide, occurs at the tip of a loop, where its side-chain is buried from solvent SufSE complex exposure in a hydrophobic cavity . This orientation of SufE active site cysteine loop might be an advantage since There is no SufS–SufE three-dimensional structure making it may protect the protein from oxidation. However, SufE it difficult to understand the SufS–SufE sulfur transfer reac- Cys51 must come into close proximity to active Cys364 of tion at the molecular level and the origin of the stimulating SufS for transpersulfuration reaction, and therefore, SufE effects of SufE on the SufS cysteine desulfurase activity. protein must undergo a conformational change allowing However, recently some HDX-MS and deuterium trapping a flexibility of its loop require for sulfur transfer mecha- experiments have been carried out on E. coli SufE and SufS nism with SufS. Examination of the structure of the rest- proteins as a reporter of protein–protein interaction zones ing SufE shows a variety of interactions that hold the and conformational changes, providing mechanistic insights active site loop folded down into the interior of SufE and into the sulfur transfer and enhancement of the cysteine reveals that the Asp74 residue would play a key role for desulfurase activity . These studies indicate that SufE maintaining such a structure. Amide hydrogen/deuterium interacts with SufS via two peptides: peptide 38–56 (a sur- exchange mass spectrometry (HDX-MS) analysis of the face loop containing Cys51) and peptide 66–83 (that forms SufE D74R mutant revealed an increase in solvent acces- one side of a structural groove into which Cys51 thiolate is sibility and dynamics in the loop containing the active oriented) (Fig. 5a). Interaction of SufE–SufS induces some site Cys51 used to accept persulfide from SufS . In conformational changes on SufE, in particular at the level addition, SufE D74R mutant is a better sulfur acceptor for of the Cys51 loop whose solvent accessibility is increased SufS than wt SufE. Therefore, D74R substitution induces upon SufS binding . SufE carrying D74R mutation (see a conformational change in SufE, making the Cys51 active above), localized in peptide 66–83, prevents hydrogen bond site loop more dynamic for sulfur transfer mechanism. with peptide 38–56, destabilizing interaction between the Since Asp74 is located in the peptide 66–83 of SufE that active site loop and the interior groove. This induces a SufE interacts with SufS , it is proposed that D74R muta- conformational change by making the Cys51 active site loop tion mimics SufE–SufS interaction leading conformational more dynamic. In addition, it was shown that this mutation changes that are propagated to the Cys51 loop allowing promotes higher interaction of SufE with SufS . There- transpersulfuration reaction between SufS and SufE. We fore, this mutation enhances the ability of SufE to accept 1 3 588 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 Fig. 5 Interactions studies of SufS–SufE by HDX-MS. a Effect of The model represents SufS protein. At the bottom of b is represented SufS on SufE protein by HDX trapping assays. The model represents linear SufS sequence with important peptides whose accessibility to SufE protein before (left panels, two orientations) and after (right deuterium is modified by SufE interaction. The persulfide C364–SSH panels, two orientations) interaction with SufS. At the bottom of a is indicated (in yellow) closed to the PLP cofactor labeled in red. is represented linear SufE sequence with important peptides whose Interaction between SufS and SufE implicated deuterium protec- Apo accessibility to deuterium is modified by SufS interaction. The inter - tion of peptide 225–236 (green) and 356–366 (magenta). Interaction action between SufE and SufS implicated deuterium protection of between SufS and SufE implicated deuterium protection of pep- Apo alk peptide 38–56 (cyan) containing C51 and peptide 66–83 (magenta) tides 225–236 (green), 262–274 (orange) and 356–366 (magenta). of SufE. The C51 flipping process in the presence of SufS (repre- Interaction between SufS and SufE implicated deuterium pro- per alk sented by the black hatched arrow) leads to C51 solvent accessibility tection of peptides 225–236 (green), 262–274 (orange) and 356–366 and the formation of a groove (black arrow) (manual representation (magenta) and increase accessibility of peptide 243–255 (cyan) by pymol). b Effect of SufE on SufS protein by HDX trapping assays. sulfur from SufS. All this suggests that SufE active Cys51 cysteine was alkylated mimicking a sulfur-accepting con- becomes accessible for sulfur transfer after activation due to formation) implicates deuterium protection of peptides a conformational change induced by SufS through peptide 225–236, 262–274 and 356–366. Finally, interaction 66–83 of SufE.between SufS (SufS containing persulfide) and SufE per alk SufE interaction to SufS induces localized dynamic implicates deuterium protection of peptides 225–236, perturbations on SufS (Fig. 5b) involving PLP binding 262–274 and 356–366 and an increased accessibility of site and active site cysteine 364 loop, however, without peptide 243–255. All these results suggest that the pres- inducing global conformational changes on SufS. Indeed, ence of SufE (1) promotes external aldimine formation interaction between SufS and SufE implicates deuterium between PLP and l -cysteine, and therefore, persulfide for- protection of peptides 225–236 and 356–366. Interac- mation Cys364 of SufS and (2) diminishes the persulfide tion between SufS and SufE (where the SufE catalytic stabilization facilitating the nucleophilic attack by SufE alk 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 589 Cys-51 on the SufS Cys-364 persulfide for direct sulfur SufC transfer. Finally, these studies demonstrate that once SufS and SufE interact some subtle dynamics exist which are There are two crystal structures of monomeric SufC pro- the molecular basis explaining the sulfur transfer between tein from Thermus thermophilus HD8 and E. coli (PDB SufS and SufE reaction and the enhanced cysteine des- numbers: 2D2F; 2D3W) (Fig. 4) [79, 80]. The SufC subu- ulfurase activity. nit has two domains, as observed in the members of the Recently, crystallographic structure of the E. coli ABC ATPase family: a catalytic α/β domain that contains CsdA–CsdE complex was solved . Since all active the nucleotide-binding Walker A and Walker B motifs, and cysteine-containing regions are well ordered it was pos- a helical domain specific to ABC ATPases containing an sible to compare the structure of the complex to that of ABC signature motif. The two domains are connected by a structures of free CsdA and CsdE proteins [75, 78]. In Q-loop that contains a strictly conserved glutamine residue comparison with SufS–SufE complex some similarities (Fig. 6). The overall architecture of the SufC structure is can be drawn. Like for the SufE within the SufS–SufE similar to other ABC ATPases structures, but there are sev- complex, in the CsdA–CsdE structure, the CsdE Cys-61 eral specific motifs in SufC. Indeed, the structure of SufC loop region moves and becomes exposed. It undergoes an reveals an atypical nucleotide binding conformation at the 11 Å shift upon interaction with CsdA becoming oriented end of the Walker B motif. Three residues following the toward Cys-358 of CsdA for sulfur transfer. The distance end of the Walker B motif form a novel 3 helix (type of between the two active cysteines of CsdA and CsdE is secondary structure) which is not observed in other ABC estimated to be 6 Å. Given that the transition state of the ATPases. Due to this novel 3 helix, the conserved glu- transpersulfuration reaction contains three sulfur atoms tamate residue (Glu169 in T. thermophiles, Glu171 in E. and that the disulfide bond length is 2.1–2.3 Å, these two coli) involved in ATP hydrolysis is flipped out. Although captured cysteines of CsdA and CsdE in the structure this unusual conformation is unfavorable for ATP hydrolysis, are likely of the intermediate stage. Despite the change it is stabilized by several interactions around the novel 3 in CsdE conformation, there are no noticeable structural helix. Glu and Asp residues (Glu169 and Asp171 in T. ther- changes to the CsdA cysteine desulfurase backbone in mophiles, Glu171 and Asp173 in E. coli) form salt-bridges the CsdA–CsdE complex like for SufS in the presence with a Lysine (Lys150 in T. thermophiles, Lys152 in E. coli); of SufE. and there are several water molecules that form a strong HDX-MS experiments were also initiated on B. subtilis hydrogen bond network. This makes the novel 3 helix of SufS and SufU. Binding of BsSufU to BsSufS induces SufC a rigid conserved motif . conformational changes in both proteins . These In addition, compared to other ABC ATPase structures, experiments demonstrate that SufU induces an opening a significant displacement occurs at a linker region between of the active site pocket of SufS allowing the Cys361 loop the ABC α/β domain and the α-helical domain. The linker of SufS to move freely . conformation is stabilized by a hydrophobic interaction Fig. 6 Structure detail of SufC protein (pdb code 2D3W). Walker-A Critical domains and important C-ter regions are illustrated into the Catalyc α/β domain C-ter structure. The ATP binding site are indicated by black arrow Walker-A and critical amino acid residues K40 H-mof E171 were indicated in blue. Picture N-ter ATP binding site H-mof H203 is obtained by Chimera (1.10.2) H203 K40 Walker-B D173 D-Loop D173 K152 D-Loop N-ter E171 K152 Pro-Loop ABC Walker-B signature ABC Q-Loop Pro-Loop Q-Loop signature α-helical domain 1 3 590 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 between conserved residues around the Q loop. Finally, the The structure of the SufD homodimer in the SufC D 2 2 surface of SufC has a cleft different from those observed in complex is almost identical to that reported for SufD other ABC ATPase structures. These results suggest that homodimer crystallized alone. Its C-terminal part interacts SufC interacts with its partners, SufB and SufD, in a manner with SufC via extensive hydrophobic interactions as well different from that of ABC transporters. as hydrogen bonds and one salt-bridge. Interestingly, the SufD residues involved in the hydrophobic interactions are SufD conserved not only in SufD orthologues but also in SufB sequences. The helices in the C-terminal helical domain of There is one crystal structure of SufD protein (PDB number: SufD interact with the β6 strand, the α2 and α3 helices and 1VH4) (Fig. 4) . SufD displays a novel structure and the Q-loop of SufC, which are located between the α/β and forms a crystallographic dimer. It shares 20% identity with helical domains of SufC. SufC and SufD interact through SufB, and therefore, likely share a similar fold. This novel extensive hydrophobic interactions as well as by eight structure of SufD is a flattened right-handed beta-helix of hydrogen bonds and one salt-bridge. Although the overall nine turns with two strands per turn; the N- and C-termini structure of SufC in the SufC D complex is similar to that 2 2 form helical subdomains. Homodimerization of SufD dou- of the monomeric form previously reported, several signifi- bles the length of the beta-helix (to 80 Å) and two highly cant structural changes occur in the ATP-binding segments conserved residues, Pro347 and His360, interact at the dimer upon complex formation. Importantly, the unique salt bridge interface. There are several highly conserved residues in the observed in the monomeric E. coli SufC between Glu171 C-terminal subdomain (Tyr374, Arg378, Gly379, Ala385, in the Walker B motif and Lys152 is cleaved, allowing the Phe393), whose role is unknown. All these residues men- rotation of the Glu171 side-chain toward the ATP-binding tioned are conserved in SufB, supporting the hypothesis that pocket. His203, another key residue for the activity of ABC it is able to homodimerize in a similar manner to SufD and ATPases, is shifted ± 5 Å toward Glu171. These structural that in vivo SufB and SufD may form a functional heterodi- changes remodel the catalytic pocket of SufC to be suit- mer analogous to the SufD homodimer. This heterodimer able for ATP binding and hydrolysis and result in a SufC SufD–SufB exists within the SufBC D complex  with local structure that more closely resembles that of active a structure of SufD almost identical to the reported SufD ABC-ATPases. Thus, as a monomer SufC is in a latent heterodimer. SufD is also able to interact with SufC to form form associated with a weak ATPase activity, whereas in a SufC D sub-complex . complex with SufD it represents a competent active form. 2 2 These observations are consistent with the kinetic experi- SufC D complex ments reporting that ATPase activity of SufC is enhanced 2 2 by SufD [46, 82]. Finally, in the SufC D structure the two 2 2 As mentioned before SufC and SufD interact forming a SufC subunits are spatially separated. Cross-linking experi- SufC D complex [46, 50] whose stoichiometry was deter- ments performed in solution indicate that the two SufC 2 2 mined by mass spectrometry and light scattering experi- subunits can associate with each other in the presence of 2+ ments. Electron microscopy and X-ray crystallography Mg and ATP . Therefore, a transient dimer forma- structures of the SufC D complex from E. coli were deter- tion of SufC can occur during ATP binding and hydrolysis 2 2 mined (Fig. 4) . Knowing that the minimalist suf operon and likely elicits a significant conformational change of the contains only sufB and sufC genes, this structure has prob-entire SufC D complex. 2 2 ably no physiological significance but it likely mimics the As a conclusion from the SufC D structure, mainly 2 2 quaternary structure of a SufB C or a Su fBC D complex information got from SufC are of significant importance 2 2 2 considering the sequence similarity between SufB and SufD for Fe–S biogenesis process. The SufC sequence possesses proteins. Therefore, the SufC D complex structure consti- several motifs: those that contribute to ATP binding and 2 2 tutes an informative structure for the understanding of Fe–S hydrolysis (Walker A, Walker B, and ABC signature), one biogenesis. for dimerization (D-loop), and one for interaction with part- In the structure of S ufC D , though each SufC subunit ner proteins (Q-loop). These properties are encountered also 2 2 is bound to each subunit of SufD homodimer, one SufC in the SufBC D structure. subunit was mostly disordered. Since the SufC D com- 2 2 plex exhibits an apparent twofold symmetry, the invisible SufBC D complex segments of the SufC subunit were modeled. The model structure of the SufC D complex is in agreement with the As mentioned before SufB, SufC, and SufD interact with 2 2 3D-reconstitution image of the complex derived from neg- each other generating a SufBC D complex whose stoichi- ative-stain electron microscopy confirming the quaternary ometry was determined by mass spectrometry . Forma- structure of the SufC D complex. tion of the SufBC D complex results from the controlled 2 2 2 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 591 share a common domain organization: an N-terminal heli- expression from the intact sufABCDSE operon (and not from cal domain, a core domain which consists of a right-handed incubation between SufB, SufC and SufD purified proteins). parallel β-helix, and a C-terminal helical domain to which Under these conditions, no SufC D complex is detected 2 2 SufC interacts. Important structural change of the SufBC D and small amount of SufB C complex is observed but still 2 2 complex occur, initiated by SufC dimerization in the pres- contaminated with SufD (stoichiometry 0.5) . This likely 2+ ence of Mg and ATP. Thanks to a fluorescent experiment, indicates that the physiological and active complex for Fe–S Cys405 of SufB, a strictly conserved amino acid buried biogenesis in E. coli is the ternary SufBC D complex. This at the heterodimer interface between the SufB and SufD is in agreement with in vivo and in vitro studies which show heterodimer, was shown to become exposed during ATP that SufBC D complex plays a central role in Fe–S assembly hydrolysis. His360 of SufD, localized close to Cys405 of and is the platform for Fe–S cluster assembly [12, 38, 39, SufB, likely undergoes similar exposure upon conformation 53]. For a long time, getting structural information of the 2+ change. Finally, two Hg ions are present in the structure SufBC D complex was impossible, and therefore, consid- at the interface of the SufB–SufD heterodimer. One bound ered as a real challenge. Recently, the structure of the E. coli to Cys405 in SufB, and the other bound to Cys358 in SufD, SufBC D complex was solved at 2.95 Å resolution (Fig. 4) which is located adjacent to His360 of SufD. These ions can . It consists of one SufB subunit, two SufC subunits, and bind the authentic Fe–S binding site, and therefore, these one SufD subunit with a stoichiometry of 1:2:1, consistent three residues Cys405 in SufB, H360 and Cys358 in SufD with previous biochemical experiments . This structure were proposed as good candidate for Fe–S cluster ligation does not reveal any cofactors such as Fe–S cluster and/or . We will see that in vivo experiments excluded Cys358 FADH that bind the SufBC D complex after anaerobic puri- 2 2 of SufD (see below). fication . Negative-stain electron microscopy and small As a conclusion, the main insight brought by the SufBC D angle X-ray scattering (SAXS) data from the as-isolated structure in comparison to SufC D structure is that SufC 2 2 SufBC D complex in solution are in agreement with the forms a transient head-to-tail dimer within the complex dur- crystal structure . As expected from the SufC D struc- 2 2 ing the catalytic step of ATP binding and hydrolysis and ture, the SufBC D complex shares a common architecture that SufC dimerization drives huge structural changes of the with SufC D where one SufD subunit is replaced by the 2 2 SufB–SufD heterodimer, leading to the exposure of Cys405 SufB subunit and SufB interacts with both SufD and SufC. of SufB inside the heterodimer interface (and likely H360 Thus, each of the SufC subunits is bound to a subunit of the of SufD). At this stage, the Fe–S assembly story would be SufB–SufD heterodimer (termed SufC and SufC ). SufB SufD the following. In the resting state, the SufC in the complex SufC and SufC have almost identical structures. On SufB SufD is ready for ATP binding, and the nascent cluster-assembly the whole, structure of SufBC D is very similar to that of site at the SufB and SufD interface is buried inside the com- SufC D  as follow: (1) the two SufC subunits are bound 2 2 plex. Upon ATP binding, SufC forms the head-to-tail dimer (one with SufB and one with SufD) but spatially separated and its dynamic motion is transmitted to the SufB–SufD (more than 40 Å) with their ATP-binding motifs facing heterodimer where the invariant residue Cys405 in SufB and one another. Each SufC subunit can transiently associate likely the His360 in SufD, become exposed to the surface to 2+ with each other in the complex in the presence of Mg and construct the nascent Fe–S cluster. ATP as shown by disulfide cross-linking experiment; (2) the overall structure of SufC subunits in the SufBC D com- SufA plex is similar to that of monomeric SufC (51) with signifi- cant structural changes around the ATP-binding pocket: (a) There is one crystal structure of E. coli SufA protein (PDB the salt bridge observed in the monomeric SufC between number: 2D2A) (Fig. 4) . The structure corresponds to Glu171 and Lys152 is cleaved in the complex, leading to the an apo-form of the protein, without Fe–S cluster. SufA shares rotation of the Glu171 side chain toward the ATP-binding 48% sequence identity with IscA but SufA exists in crystals pocket; (b) His203 is shifted about 4 Å toward Glu171 in the as a homodimer, in contrast to the tetrameric organization complex. These structural changes rearrange the catalytic of apo-IscA . Furthermore, the C-terminal segment pocket of SufC to be suitable for ATP binding and hydroly- containing two essential cysteine residues (Cys–Gly–Cys), sis. These findings are consistent with kinetic experiments which is disordered in the IscA structure, is clearly visible showing that the SufC ATPase activity is enhanced by the in one molecule (the α1 subunit) of the SufA homodimer. presence of SufB and SufD [46, 82]; (3) structure of the Although this segment is disordered in the other molecule SufD subunit is almost identical to that of one subunit of the (the α2 subunit), computer modeling suggests that the four SufD homodimer . cysteine residues of the Cys–Gly–Cys motif (Cys114 and Concerning specific features encountered in the SufBC D Cys116 in each subunit) are positioned in close proximity complex. The structures of SufB and SufD are similar and (3.1–6.7 Å) at the dimer interface allowing in SufA dimer 1 3 592 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 coordination of an Fe–S cluster. More recently, the crystal proteins. The in vivo complementation assays reveal criti- structure of a 2Fe–2S cluster-bound form of Thermosyn- cal amino acids on SufC: Lys40, Glu171 and His203  in echococcus elongatus IscA showed a different coordination agreement with previous experiments showing that Lys40 is mode. Indeed, the structure is formed by an asymmetric, essential for SufC ATPase activity and Fe–S formation on domain-swapped tetramer formed by two α and two β subu- SufBC D complex . The second strategy confirms these nits, in which the 2Fe–2S cluster is coordinated by two con- results since mutants in these amino acids have white cells formationally distinct α and β subunits, with asymmetric . Altogether, these results on SufC show that residues cluster coordination by Cys37, Cys101, Cys103 from α and Lys40, Glu171 and His203 are essential for the assembly of Cys103 from β . Later, the domain swapping has been Fe–S cluster on SufBC D (Table 1). attributed to a crystallization artifact . Very recently, a For SufD, the in vivo complementation assay and cells nice work performed by NMR demonstrates that the 2Fe–2S color reveals that His360 is critical for Fe–S metabolism and cluster on the human ISCA2 homodimer is coordinated tran- Fe–S assembly on Suf BC D [37, 41] in agreement with pre- siently by Cys144 and Cys146 of each monomer and that vious data that indicated His360 residue essential for SufD this form evolves to a more thermodynamically species in function [37, 50] (Table 1). By this technics, no other residue which the 2Fe–2S cluster is ligated by Cys79 and Cys144 of SufD were identified as important. Even Cys358, that 2+ . It is possible that a similar coordination exist on SufA was shown to be involved in the binding site for one Hg containing a 2Fe–2S cluster. ions in the SufBC D structure  and that is localized at the SufD–SufB interface, does not prove to be necessary in the complementation assay . It is also not involved In vivo studies on SufBC D in Fe–S assembly on SufBC D since cells overproducing 2 2 SufBC D(C358A) proteins are blackish-green . Since the beginning of Fe–S assembly study, many in vivo For SufB, the in vivo complementation assay revealed experiments were carried out on the suf operon. They mainly that Cys254, Cys405, Arg226, Asn228, Gln285, Trp287, consisted in studying the in vivo impact (Fe–S enzymes Lys303 and Glu434 are critical for growth (Table 1) . activity, bacterial growth, sensitivity to oxidants and iron Gln285 and Lys303 take part of a putative tunnel ranging chelator…) after inactivation of a single suf gene and through the α-helix core domain of SufB connecting Cys254 allowed to demonstrate that suf operon is involved under and Cys405 in SufB. Interestingly, deletion of the entire oxidative stress and iron limitation [12, 24, 42, 49, 87]. In CxxCxxxC canonical motif has no effect on the complemen- the next lines, we will focus on the impact of point muta- tation showing that the three cysteines of this motif are dis- tions in sufB, sufC or sufD genes within the suf operon on pensable in vivo and thus not the Fe–S ligands. Interestingly the Fe–S assembly process in E. coli. To detect the effect of also, is the partial complementation of UT109 strain with the mutation in vivo, two strategies were used. One strategy was double SufB Glu432A/His433A protein that contains muta- to perform complementation assays using an E. coli mutant tions at the SufB–SufD interface . The second strategy, strain that can survive without Fe–S clusters . In this E. coli strain (UT109) the chromosomal suf and isc oper- Table 1 Critical amino acid of the SufBC D complex for Fe–S ons are deleted (ΔsufABCDSE ΔiscUA-hscBA). Deletion assembly and binding and their proposed function of both operons in E. coli is lethal in general; but, UT109 SufB harbors the plasmid pUMV22 that carries three genes for Cys254 Sulfur entry the mevalonate (MVA) pathway, which allows UT109 to Cys405 Final sulfur acceptor and Fe–S ligand grow with an absolute dependence on MVA supplementation Asp432 Potential Fe–S ligand . Upon introduction of functional sufAB and sufCDSE His433 Potential Fe–S ligand genes (via plasmids) the cells become able to grow nor- Asp434 Fe–S ligand mally even in the absence of MVA. Therefore, this strategy Q285 Sulfur production on SufSE and highlights crucial amino acid of the SUF system for Fe–S sulfur channeling metabolism in E. coli. The second strategy consists to assess W287 Sulfur production on SufSE Fe–S assembly in vivo on SufBC D using the color of host K303 Sulfur channeling cells overproducing SufBC D complex that contain mutation SufD on SufB, SufC or SufD proteins. Cells are blackish-green His360 Iron acquisition, Fe–S ligand when active SufBC D complex is overproduced and white SufC for an inactive complex, unable to form a Fe–S cluster . Lys40 ATP hydrolysis Therefore, this strategy highlights crucial amino acid for Glu171 ATP hydrolysis Fe–S assembly on SufBC D complex. For both strategies, a His203 ATP hydrolysis series of mutations was generated on SufB, SufC and SufD 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 593 as expected, reveals that SufB Cys405 is important for SufB (Fig. 7), hypothesis that have to be experimentally tested in function since complex containing Cys405A mutation has a near future. white cells, indicating that this residue is indispensable for There are still some remaining questions. How and when cluster assembly. No experiments were performed with iron is delivered to the Fe–S assembly site? is there a specific mutations on residues Cys254, Arg226, Asn228, Gln285, iron donor protein for the SUF system? Genetic experiments Trp287, Lys303 and Glu434. strongly suggest a link between SufD and iron metabolism [12, 35, 49]. However, so far such an hypothesis was not validated in vitro. A flavin is co-purifying under anaerobic Proposed model for Fe–S biogenesis by SUF conditions with SufBC D complex with a stoichiometry of 1 flavin per complex . Only the reduced form of the fla- Based on biochemical, biophysical, structural and in vivo vin (FADH2) binds to the complex, FAD is unable to. We experiments the current model for Fe–S assembly on demonstrated in vitro a ferric reductase activity of the flavin 3+ 2+ SufBC D complex is the following (E. coli numeration). (Fe –Fe ) on small chelates (ferric citrate) and proposed It is likely that the Fe–S assembly is initiated by ATPase that it can be involved during Fe–S assembly in the reduction activity of SufC. Indeed, in the resting state, the SufC is of ferric iron . Recently, it was proposed that the flavin 0 2− ready for ATP binding, and the nascent cluster-assembly can provide electrons for persulfide cleavage (S to S ) even site at the SufB and SufD interface is buried inside the though this was not demonstrated experimentally . Thus, complex. Upon ATP binding, SufC transiently forms a the actual hypothesis is that FADH serves to reduce iron. dimer that elicits a significant conformational change of the Considering that SUF system is involved under oxidative entire SufBC D complex. In particular, the invariant resi- stress and iron limitation another possibility would be that due Cys405 in SufB and likely the His360 in SufD, become the reduced flavin serves as a sensor of oxidative conditions exposed to the surface to construct the nascent Fe–S cluster. (hypothesis never considered so far). The binding site of the The building of the Fe–S is possible by arrivals of sulfur FADH is still unknown despite several experiments using and iron ions. SufS catalyzed desulfurization of l -cysteine mutants in SufB ; and therefore, the assignment of the with the formation of persulfide on its Cys364. Nucleo- FADH binding site requires further studies. Another next philic attack of SufE Cys51 thiolate allows transpersulfu- challenge in the future in the Fe–S assembly field involving ration reaction to occur and formation of a persulfide on SUF system is to get structural information of an integrated SufE Cys51. A second transpersulfuration between SufE and system containing SufSE–SufBC D proteins. The SufSE SufB generates a persulfide on SufB Cys254 residue that complex interacts with SufBC D complex to provide sul- serves as the first sulfur acceptor site on SufB. Then, sulfur fur atoms for Fe–S cluster assembly. Sulfur atoms enter migrates from SufB Cys254 to SufB Cys405. SufB Cys405 SufBC D complex via SufB protein. Some ITC experiments is > 25 Å away from SufB Cys254. An internal hydrophilic demonstrated a flip–flop mechanism of allosteric regulation tunnel ranging through the β-helix core domain of SufB where binding of one SufE to one active site of SufS dimer just between SufB Cys254 and SufB Cys405 may help dur- diminishes further SufE binding to the second active site ing sulfur transfer between these two cysteines. Residues . One can wonder under which oligomerization state Lys303 and Gln285 might be directly involved (with SufB SufS–SufE complex interacts with SufBC D complex for Glu236, SufB Glu252, SufB His265, SufB Thr283, SufB sulfur transfer: S ufSE, SufS E , SufSE? It is reasonable 2 2 2 Thr326 and SufB Lys328). If this putative sulfur tunnel is to hypothesize that a stoichiometric SufSE complex is rel- involved in sulfur transfer from Cys254 to Cys405 (that is evant for interaction with SufBC D since only one sulfur an interesting hypothesis) that would be the first time that entry to SufB is require (Fig. 7). Another important question sulfur transfer reaction occurs without transpersulfuration is related to the event that drives the interaction between mechanism, which usually is used for sulfur as a strategy to SufSE and SufBC D complexes? As a consequence, it is travel long distances under a non-toxic form. SufB Cys405 urgent to stabilize and get structural information on the huge is the final sulfur acceptor and a good candidate for one SufSEBC D complex. This would allow to trap Fe–S inter- of the Fe–S cluster ligands. SufD His360 is likely another mediate, and therefore, identify Fe–S coordination sites and one. SufB Glu434 and SufB His433 or SufB Glu432 may fully understand the Fe–S assembly mechanism. be also involved during Fe–S assembly or as Fe–S ligands 1 3 594 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 L-Cysteine -SSH ATP ADP + Pi C364 SufB -SSH SufD H433 C254 H360 E434 E432 SufC SufC SufSE 2Fe-2S Apo target Iron, 4Fe-4S target e (FADH ) FAD Fig. 7 Current proposed mechanism for Fe–S assembly by the SUF trons (FADH2) allows building of Fe–S cluster on the complex at system. SufBC D complex is in a relaxation mode. (1) The mecha- the SufB–SufD interface. SufD H360, SufB C405, E434 and H433 nism is initiated by ATPase activity of SufC. Upon ATP binding, or E432 can be involved in Fe–S coordination or Fe–S formation. (7) SufC transiently forms a dimer that elicits a significant conforma- SufSE release allows transfer of the SufBC D cluster to SufA that can tional change of the entire SufBC D complex. The SufB C405 and maturate 4Fe–4S target proteins. The original apo-SufBC D complex 2 2 likely SufD H360 become exposed to the surface. This confirma- is regenerated and ready for a new cycle. Critical amino acid are rep- tion of the complex is favorable to recruit SufE–SufS complex. (2) resented with bowls. Amino acids localized at the SufB–SufD inter- Cysteine desulfurase activity (arrival of l -cysteine) generates a per- face (red square) are zoomed in the inset in the middle of the figure. sulfide on SufS (C364) that is transferred to C51 of SufE (3) and SufU, not represented, is suggested to play a SufE-like role then to C204 (4) and C405 (5) of SufB. (6) Arrival of iron and elec- Acknowledgements This article/publication is based upon work from 3. Lill R (2009) Nature 460:831–838. https ://doi.org/10.1038/natur COST Action CA15133, supported by COST (European Cooperation e0830 1 in Science and Technology). We acknowledge networking support from 4. Py B, Moreau PL, Barras F (2011) Curr Opin Microbiol 14:218– the COST Action FeSBioNet (Contract CA15133). 223. https ://doi.org/10.1016/j.mib.2011.01.004 5. Jacobson MR, Cash VL, Weiss MC, Laird NF, Newton WE, Dean DR (1989) Mol Gen Genet 219:49–57 Open Access This article is distributed under the terms of the Creative 6. Frazzon J, Dean DR (2003) Curr Opin Chem Biol 7:166–173 Commons Attribution 4.0 International License (http://creativ ecommons 7. Zheng L, Cash VL, Flint DH, Dean DR (1998) J Biol Chem .org/licenses/b y/4.0/), which permits use, duplication, adaptation, distri- 273:13264–13272 bution and reproduction in any medium or format, as long as you give 8. Lill R, Dutkiewicz R, Elsasser HP, Hausmann A, Netz DJ, Pierik appropriate credit to the original author(s) and the source, provide a link AJ, Stehling O, Urzica E, Muhlenhoff U (2006) Biochim Biophys to the Creative Commons license and indicate if changes were made. Acta 1763:652–667 9. Takahashi Y, Tokumoto U (2002) J Biol Chem 277:28380–28383 10. Balk J, Pilon M (2011) Trends Plant Sci 16:218–226. https ://doi. Referencesorg/10.1016/j.tplan ts.2010.12.006 11. Lill R, Hoffmann B, Molik S, Pierik AJ, Rietzschel N, Stehling O, Uzarska MA, Webert H, Wilbrecht C, Muhlenhoff U (2012) 1. Beinert H (2000) J Biol Inorg Chem. 5:2–15 2. Fontecave M (2006) Nat Chem Biol 2:171–174 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 595 Biochim Biophys Acta 1823:1491–1508. https ://doi.or g/10.1016/j. 38. Wollers S, Layer G, Garcia-Serres R, Signor L, Clemancey M, bbamc r.2012.05.009 Latour JM, Fontecave M, de Choudens SO (2010) J Biol Chem 12. Nachin L, Loiseau L, Expert D, Barras F (2003) Embo J 285:23331–23341. https ://doi.org/10.1074/jbc.M110.12744 9 22:427–437 39. Chahal HK, Dai Y, Saini A, Ayala-Castro C, Outten FW (2009) 13. Olson JW, Agar JN, Johnson MK, Maier RJ (2000) Biochemistry Biochemistry 48:10644–10653. https ://doi.org/10.1021/bi901 39:16213–16219 518y 14. Stehling O, Wilbrecht C, Lill R (2014) Biochimie 100:61–77. 40. Chahal HK, Outten FW (2012) J Inorg Biochem 116:126–134. https ://doi.org/10.1016/j.bioch i.2014.01.010https ://doi.org/10.1016/j.jinor gbio.2012.06.008 15. Sheftel A, Stehling O, Lill R (2010) Trends Endocrinol Metab 41. Yuda E, Tanaka N, Fujishiro T, Yokoyama N, Hirabayashi K, 21:302–314. https ://doi.org/10.1016/j.tem.20 Fukuyama K, Wada K, Takahashi Y (2017) Sci Rep 7:9387. 16. Selbach BP, Chung AH, Scott AD, George SJ, Cramer SP, https ://doi.org/10.1038/s4159 8-017-09846 -2 Dos Santos PC (2014) Biochemistry 53:152–160. https ://doi. 42. Nachin L, El Hassouni M, Loiseau L, Expert D, Barras F (2001) org/10.1021/bi401 1978 Mol Microbiol 39:960–972 17. Mashruwala AA, Pang YY, Rosario-Cruz Z, Chahal HK, Ben- 43. Wilken S, Schmees G, Schneider E (1996) Mol Microbiol son MA, Mike LA, Skaar EP, Torres VJ, Nauseef WM, Boyd 22:655–666 JM (2015) Mol Microbiol 95:383–409. https://doi.or g/10.1111/ 44. Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L (2005) mmi.12860 EMBO J 24:1901–1910. https ://doi.org/10.1038/sj.emboj .76006 18. Huet G, Daffe M, Saves I (2005) J Bacteriol 187:6137–6146 57 19. Charan M, Singh N, Kumar B, Srivastava K, Siddiqi MI, Habib 45. Schmitt L, Tampe R (2002) Curr Opin Struct Biol 12:754–760. S (2014) Antimicrob Agents Chemother 58:3389–3398. https https ://doi.org/10.1016/s0959 -440x(02)00399 -8 ://doi.org/10.1128/aac.02711 -13 46. Petrovic A, Davis CT, Rangachari K, Clough B, Wilson RJ, 20. Di Perri G, Bonora S (2004) J Antimicrob Chemother 54:593– Eccleston JF (2008) Protein Sci 17:1264–1274. https ://doi. 602. https ://doi.org/10.1093/jac/dkh37 7org/10.1110/ps.03465 2.108 21. Choby JE, Mike LA, Mashruwala AA, Dutter BF, Dunman PM, 47. Xu XM, Moller SG (2004) Proc Natl Acad Sci USA Sulikowski GA, Boyd JM, Skaar EP (2016) Cell Chem Biol 101:9143–9148 23:1351–1361. https: //doi.org/10.1016/j.chembi ol.2016.09.012 48. Gisselberg JE, Dellibovi-Ragheb TA, Matthews KA, Bosch 22. Roche B, Aussel L, Ezraty B, Mandin P, Py B, Barras F (2013) G, Prigge ST (2013) PLoS Pathog 9:e1003655. https ://doi. Biochim Biophys Acta 1827:455–469. https://doi.or g/10.1016/j.org/10.1371/journ al.ppat.10036 55 bbabi o.2012.12.010 49. Expert D, Boughammoura A, Franza T (2008) J Biol Chem 23. Johnson DC, Dean DR, Smith AD, Johnson MK (2005) Annu 283:36564–36572. https ://doi.org/10.1074/jbc.m8077 49200 Rev Biochem 74:247–281 50. Wada K, Sumi N, Nagai R, Iwasaki K, Sato T, Suzuki K, 24. Outten FW, Djaman O, Storz G (2004) Mol Microbiol Hasegawa Y, Kitaoka S, Minami Y, Outten FW et al (2009) J 52:861–872 Mol Biol 387:245–258. https://doi.or g/10.1016/j.jmb.2009.01.054 25. Lee KC, Yeo WS, Roe JH (2008) J Bacteriol 190:8244–8247. 51. Cupp-Vickery JR, Urbina H, Vickery LE (2003) J Mol Biol https ://doi.org/10.1128/jb.01161 -08 330:1049–1059 26. Nesbit AD, Giel JL, Rose JC, Kiley PJ (2009) J Mol Biol 52. Loiseau L, Ollagnier-de-Choudens S, Nachin L, Fontecave M, 387:28–41. https ://doi.org/10.1016/j.jmb.2009.01.055 Barras F (2003) J Biol Chem 278:38352–38359. https :/ /doi. 27. Desnoyers G, Morissette A, Prevost K, Masse E (2009) EMBO org/10.1074/jbc.m3059 53200 J 28:1551–1561. https ://doi.org/10.1038/emboj .2009.116 53. Outten FW, Wood MJ, Munoz FM, Storz G (2003) J Biol Chem 28. Lee JH, Yeo WS, Roe JH (2004) Mol Microbiol 51:1745–1755 278:45713–45719. https ://doi.org/10.1074/jbc.m3080 04200 29. Boyd ES, Thomas KM, Dai Y, Boyd JM, Outten FW (2014) 54. Ollagnier-de-Choudens S, Lascoux D, Loiseau L, Barras F, Forest Biochemistry 53:5834–5847. https:// doi.org/10.1021/bi5004 88r E, Fontecave M (2003) FEBS Lett 555:263–267 30. Outten FW (2015) Biochim Biophys Acta 1853:1464–1469. 55. Selbach BP, Pradhan PK, Dos Santos PC (2013) Biochemistry https ://doi.org/10.1016/j.bbamc r.2014.11.001 52:4089–4096. https ://doi.org/10.1021/bi400 1479 31. Mettert EL, Kiley PJ (2015) Biochim Biophys Acta 1853:1284– 56. Dai Y, Outten FW (2012) FEBS Lett 586:4016–4022. https://doi. 1293. https ://doi.org/10.1016/j.bbamc r.2014.11.018org/10.1016/j.febsl et.2012.10.001 32. Fontecave M, Choudens SO, Py B, Barras F (2005) J Biol Inorg 57. Singh H, Dai Y, Outten FW, Busenlehner LS (2013) J Biol Chem Chem 10(7):713–721 288:36189–36200. https ://doi.org/10.1074/jbc.m113.52570 9 33. Layer G, Gaddam SA, Ayala-Castro CN, de Choudens SO, 58. Selbach B, Earles E, Dos Santos PC (2010) Biochemistry Lascoux D, Fontecave M, Outten FW (2007) J Biol Chem 49:8794–8802. https ://doi.org/10.1021/bi101 358k 282:13342–13350 59. Albrecht AG, Netz DJ, Miethke M, Pierik AJ, Burghaus O, Peuck- 34. Tsaousis AD, de Choudens SO, Gentekaki E, Long S, Gaston ert F, Lill R, Marahiel MA (2010) J Bacteriol 192:1643–1651. D, Stechmann A, Vinella D, Py B, Fontecave M, Barras F et al https ://doi.org/10.1128/jb.01536 -09 (2012) Proc Natl Acad Sci USA 109:10426–10431. https://doi. 60. Riboldi GP, Verli H, Frazzon J (2009) BMC Biochem 10:3. https org/10.1073/pnas.11160 67109 ://doi.org/10.1186/1471-2091-10-3 35. Saini A, Mapolelo DT, Chahal HK, Johnson MK, Outten FW 61. Albrecht AG, Peuckert F, Landmann H, Miethke M, Seubert (2010) Biochemistry 49:9402–9412. https ://doi.org/10.1021/ A, Marahiel MA (2011) FEBS Lett 585:465–470. https ://doi. bi101 1546org/10.1016/j.febsl et.2011.01.005 36. Blanc B, Clemancey M, Latour JM, Fontecave M, de Choudens 62. Kato S, Mihara H, Kurihara T, Takahashi Y, Tokumoto SO (2014) Biochemistry 53:7867–7869. https://doi.or g/10.1021/ U, Yoshimura T, Esaki N (2002) Proc Natl Acad Sci USA bi501 2496 99:5948–5952 37. Hirabayashi K, Yuda E, Tanaka N, Katayama S, Iwasaki K, 63. Kornhaber GJ, Snyder D, Moseley HN, Montelione GT (2006) Matsumoto T, Kurisu G, Outten FW, Fukuyama K, Takahashi J Biomol NMR 34:259–269. https ://doi.or g/10.1007/s1085 Y et al (2015) J Biol Chem 290:29717–29731. https ://doi. 8-006-0027-5 org/10.1074/jbc.m115.68093 4 1 3 596 JBIC Journal of Biological Inorganic Chemistry (2018) 23:581–596 64. Vinella D, Brochier-Armanet C, Loiseau L, Talla E, Barras F 76. Loiseau L, de Choudens SO, Lascoux D, Forest E, Fontecave M, (2009) PLoS Genet 5:e1000497. https ://doi.org/10.1371/jour n Barras F (2005) J Biol Chem 280:26760–26769 al.pgen.10004 97 77. Dai Y, Kim D, Dong G, Busenlehner LS, Frantom PA, Outten FW 65. Ollagnier-de Choudens S, Nachin L, Sanakis Y, Loiseau L, Barras (2015) Biochemistry 54:4824–4833. https ://doi.org/10.1021/acs. F, Fontecave M (2003) J Biol Chem 278:17993–18001. https ://bioch em.5b006 63 doi.org/10.1074/jbc.m3002 85200 78. Kim S, Park S (2013) J Biol Chem 288:27172–27180. https://doi. 66. Ollagnier-de-Choudens S, Sanakis Y, Fontecave M (2004) J Biol org/10.1074/jbc.m113.48027 7 Inorg Chem 9:828–838 79. Kitaoka S, Wada K, Hasegawa Y, Minami Y, Fukuyama K, Taka- 67. Gupta V, Sendra M, Naik SG, Chahal HK, Huynh BH, Outten hashi Y (2006) FEBS Lett 580:137–143. https://doi.or g/10.1016/j. FW, Fontecave M, de Choudens SO (2009) J Am Chem Soc febsl et.2005.11.058 131:6149–6153 80. Watanabe S, Kita A, Miki K (2005) J Mol Biol 353:1043–1054 68. Jensen LT, Culotta VC (2000) Mol Cell Biol 20:3918–3927 81. Badger J, Sauder JM, Adams JM, Antonysamy S, Bain K, Berg- 69. Wada K, Hasegawa Y, Gong Z, Minami Y, Fukuyama K, Taka- seid MG, Buchanan SG, Buchanan MD, Batiyenko Y, Christopher hashi Y (2005) FEBS Lett 579:6543–6548 JA et al (2005) Proteins 60:787–796 70. Tan G, Lu J, Bitoun JP, Huang H, Ding H (2009) Biochem J 82. Tian T, He H, Liu XQ (2014) Biochem Biophys Res Commun 420:463–472. https ://doi.org/10.1042/bj200 90206 443:376–381. https ://doi.org/10.1016/j.bbrc.2013.11.131 71. Tirupati B, Vey JL, Drennan CL, Bollinger JM (2004) Biochem- 83. Cupp-Vickery JR, Silberg JJ, Ta DT, Vickery LE (2004) J Mol istry 43:12210–12219 Biol 338:127–137. https ://doi.org/10.1016/j.jmb.2004.02.027 72. Blauenburg B, Mielcarek A, Altegoer F, Fage CD, Linne U, 84. Morimoto K, Yamashita E, Kondou Y, Lee SJ, Arisaka F, Tsuki- Bange G, Marahiel MA (2016) PLoS One 11:e0158749. https :// hara T, Nakai M (2006) J Mol Biol 360:117–132 doi.org/10.1371/journ al.pone.01587 49 85. Mapolelo DT, Zhang B, Naik SG, Huynh BH, Johnson MK (2012) 73. Mihara H, Fujii T, Kato S, Kurihara T, Hata Y, Esaki N (2002) J Biochemistry 51:8071–8084. https ://doi.org/10.1021/bi300 6658 Biochem (Tokyo) 131:679–685 86. Brancaccio D, Gallo A, Piccioli M, Novellino E, Ciofi-Baffoni 74. Goldsmith-Fischman S, Kuzin A, Edstrom WC, Benach J, Shastry S, Banci L (2017) J Am Chem Soc 139:719–730. h t t p s : / / d oi . R, Xiao R, Acton TB, Honig B, Montelione GT, Hunt JF (2004) org/10.1021/jacs.6b095 67 J Mol Biol 344:549–565 87. Patzer SI, Hantke K (1999) J Bacteriol 181:3307–3309 75. Liu G, Li Z, Chiang Y, Acton T, Montelione GT, Murray D, 88. Tanaka N, Kanazawa M, Tonosaki K, Yokoyama N, Kuzuyama Szyperski T (2005) Protein Sci 14:1597–1608. https ://doi. T, Takahashi Y (2016) Mol Microbiol 99:835–848. https ://doi. org/10.1110/ps.04132 2705 org/10.1111/mmi.13271 1 3
JBIC Journal of Biological Inorganic Chemistry – Springer Journals
Published: Dec 26, 2017
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera