Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS͞IscU complex: Implications for the mechanism of iron-sulfur cluster assembly

Shin-ichiro Kato*, Hisaaki Mihara*, Tatsuo Kurihara*, Yasuhiro Takahashi†, Umechiyo Tokumoto†, Tohru Yoshimura*, and Nobuyoshi Esaki*‡

*Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan; and †Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan

Communicated by Takayoshi Higuchi, Kyoto University, Kyoto, Japan, March 4, 2002 (received for review February 14, 2002) IscS and IscU from Escherichia coli cooperate with each other in the formation of an IscS͞IscU covalent complex (14, 15). IscS and biosynthesis of iron-sulfur clusters. IscS catalyzes the desulfuriz- IscU form the covalent bond complex when mixed with each ation of L-cysteine to produce L-alanine and sulfur. Cys-328 of IscS other in the presence of L-cysteine, the substrate for IscS (14, 15). attacks the sulfur atom of L-cysteine, and the sulfane sulfur derived The sulfur atom derived from L-cysteine is transferred to IscU from L-cysteine binds to the S␥ atom of Cys-328. In the course of the and forms a covalent bond with IscU. These observations suggest cluster assembly, IscS and IscU form a covalent complex, and a that the sulfur transfer from IscS to IscU occurs in an early stage sulfur atom derived from L-cysteine is transferred from IscS to IscU. of the cluster assembly, and its mechanistic analysis is essential The covalent complex is thought to be essential for the cluster in dissecting the cluster assembly process. However, at present, biogenesis, but neither the nature of the bond connecting IscS and neither the nature of the covalent bond (whether the bond is a IscU nor the residues involved in the complex formation have been disulfide bond or a polysulfide bond) nor the residues involved determined, which have thus far precluded the mechanistic anal- have been clarified. The composition of the complex has not yses of the cluster assembly. We here report that a covalent bond even been determined. Accordingly, it has been impossible to is formed between Cys-328 of IscS and Cys-63 of IscU. The bond is describe the sulfur transfer process at a molecular level. In the a disulfide bond, not a polysulfide bond containing sulfane sulfur present study, we have shown that IscS and IscU are covalently between the two cysteine residues. We also found that Cys-63 of bound to each other by a disulfide bond and that the residues IscU is essential for the IscU-mediated activation of IscS: IscU forming this bond are Cys-328 of IscS and Cys-63 of IscU. We induced a six-fold increase in the activity of also found a novel phenomenon that IscU promotes the cysteine IscS, whereas the IscU mutant with a serine substitution for Cys-63 desulfurase activity of IscS, which requires Cys-63 of IscU. Based had no effect on the activity. Based on these findings, we propose on these findings, we propose a mechanism for the sulfur transfer a mechanism for an early stage of iron-sulfur cluster assembly: the reaction from IscS to IscU. sulfur transfer from IscS to IscU is initiated by the attack of Cys-63 of IscU on the S␥ atom of Cys-328 of IscS that is bound to sulfane Experimental Procedures sulfur derived from L-cysteine. Materials. His⅐bind resin was purchased from Novagen. Lysyl ron-sulfur are widely distributed in almost all organ- endopeptidase and 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) Iisms and play essential roles in energy , DNA were purchased from Wako (Osaka, Japan). CDP-Star was repair, transcriptional regulation, and biosynthesis of nucleotides purchased from Roche (Basel, Switzerland), Sep-Pak and Cen- tricon YM-10 were from Millipore, and Capcell Pak C18 SG300 and amino acids (1, 2). Although their prosthetic groups, iron- ϫ sulfur clusters, have been the focus of genetic, biochemical, and (4.6 mm 250 mm) was from Shiseido (Tokyo, Japan). The Silver stain kit was purchased from Bio-Rad. All other chemicals biophysical studies, little is known about the mechanism of their were of analytical grade from Nacalai Tesque (Kyoto). biosynthesis and repair. Recent studies demonstrated that the assembly of the clusters is mediated by proteins encoded by the Expression and Purification of Proteins. The iscU was amplified isc (iron-sulfur cluster) operon in prokaryotes (3–7), such as from the Kohara miniset clone No. 430 (16) by PCR and inserted Escherichia coli and Azotobacter vinelandii, and counterparts of into the NdeI and XhoI sites in pET21a(ϩ) to yield pUH12, these proteins in eukaryotes (8, 9). Among these proteins, IscS which was used for the expression of IscU-His . The recombinant and IscU are believed to function in an early stage of the cluster 6 contains six histidine residues at the C terminus of IscU. assembly. IscS, which is a homodimeric pyridoxal phosphate- For the coexpression of IscU-His and IscS, a DNA fragment dependent cysteine desulfurase, catalyzes the production of 6 containing the iscU-iscS coding region was obtained by PCR and sulfur and L-alanine from L-cysteine via an -bound inserted into the NdeI and XhoI sites in pET21a(ϩ) to yield persulfide intermediate on the active-site cysteine residue, Cys- pFH6. To produce three mutants of IscU (C37S, C63S, and 328 in the case of IscS from E. coli (10, 11). The sulfur atom C106S) and a C328A mutant of IscS, the QuikChange site- derived from L-cysteine is then transferred to IscU and eventu- ally incorporated into iron-sulfur clusters. E. coli IscU has three conserved cysteine residues, Cys-37, Cys-63, and Cys-106 and Abbreviations: ABD-F, 4-fluoro-7-sulfamoylbenzofurazan; TBP, tributylphosphine. provides a scaffold for the IscS-directed sequential assembly of ‡To whom reprint requests should be addressed. E-mail: [email protected]. 2ϩ 2ϩ labile [Fe2S2] and [Fe4S4] clusters (12, 13). The publication costs of this article were defrayed in part by page charge payment. This Recently, it was demonstrated that the sulfur atom derived article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. from L-cysteine is transferred directly from IscS to IscU via the §1734 solely to indicate this fact.

5948–5952 ͉ PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.082123599 Downloaded by guest on September 30, 2021 Fig. 1. Covalent complex formation between IscS and IscU and between two IscU subunits. (A) An IscU-containing fraction from a His⅐bind column was treated (lane 1) or not treated (lane 2) with 0.1% 2-mercaptoethanol and analyzed by SDS͞PAGE. Lane ‘‘M’’ denotes marker proteins with sizes given in the right margin. Proteins were stained with Coomassie brilliant blue. (B) Gel filtration analysis of a copurified fraction containing IscU and IscS. (Upper) The elution profile of the fraction by using a Superose 12 column. (Lower) Nonreducing SDS͞PAGE analysis of Superose 12 fractions. Proteins were visualized by silver staining.

directed mutagenesis kit was used with appropriate mutagenic Other Analytical Methods. Western-blot analysis was carried out by primers. E. coli BL21(DE3)pLysS cells harboring an expression using anti-rabbit IgG conjugated to alkaline phosphatase and a plasmid were cultured aerobically in 500 ml of LB broth sup- CDP-Star detection reagent. S0 was determined by cyanolysis plemented with 1 mM isopropyl-␤-D-thiogalactopyranoside, 200 according to the method of Wood (17). The activity of IscS was ␮g͞ml of ampicillin, and 40 ␮g͞ml of chloramphenicol at 37°C determined as described (18). Protein was measured with the for12horat26°C for 20 h in the case of coexpression. The cells protein assay CBB solution (Nacalai Tesque) by using BSA as a were harvested by centrifugation (10,000 ϫ g); suspended in a standard. N-terminal amino acid sequences were determined buffer containing 20 mM Tris⅐HCl (pH 7.9), 0.5 M NaCl, and 5 with a Shimadzu PPSQ-10 protein sequencer. The nucleotide mM imidazole; and disrupted by sonication. The cell debris was sequences were determined with an ABI Prism 310 DNA removed by centrifugation (10,000 ϫ g), and the supernatant sequencer. Protein bands in an SDS͞PAGE gel were quantified solution was applied to a nickel-chelating column (Ni-charged with the NIH IMAGE software. IDA agarose) (7 ml) and washed with 100 ml of a binding buffer [20 mM Tris⅐HCl (pH7.9)͞0.5 M NaCl͞5 mM imidazole] and 250 Results ⅐ ͞ ͞ ml of a washing buffer [20 mM Tris HCl (pH 7.9) 0.5 M NaCl 60 IscS and IscU Are Covalently Linked by a Disulfide Bond. IscU-His6 mM imidazole]. Proteins retained in the column were eluted was recombinantly expressed in E. coli BL21(DE3)pLysS har- with 25 ml of an elution buffer [20 mM Tris⅐HCl (pH 7.9)͞0.5 M boring pUH12, and the cell-free extract was subjected to nickel- ͞ NaCl 1 M imidazole]. chelating column chromatography. IscU-His6 was copurified with a 45-kDa protein, which was identified as IscS by N-terminal Identification of Residues Forming a Covalent Bond Between IscS and amino acid sequencing, indicating the specific interaction be- IscU. The copurified IscS͞IscU complex (14 mg of protein) was tween the two proteins (data not shown). As a negative control, BIOCHEMISTRY incubated in an alkylation mixture (3 ml) containing 0.15 M we applied the cell extract not containing IscU-His6 onto the Tris⅐HCl (pH 7.8), 6 M urea, 2% SDS, 5 mM EDTA, and 4 mM nickel column and found that IscS did not bind to the column, iodoacetic acid for2hat37°C in the dark. The reaction mixture indicating that IscS binds to the column only through an inter- was dialyzed, concentrated with Centricon YM-10, and digested action with IscU-His6 (data not shown). ␮ with lysyl endopeptidase (15 g) at 37°Cfor4hin1mlofa We next overproduced both IscS and IscU-His6 in E. coli reaction mixture containing 0.1 M Tris⅐HCl (pH 8.0), 4 M urea, BL21(DE3)pLysS harboring pFH6 to obtain a large amount of ͞ and 5 mM EDTA. The peptides were purified with a Sep-Pak C18 the IscS IscU complex for biochemical analyses. IscS was co- cartridge, concentrated by evaporation, and resuspended in 300 purified with IscU-His6 by nickel-column chromatography as ␮l of 30% acetonitrile containing 0.1% trifluoroacetic acid. The judged by SDS͞PAGE (Fig. 1A, lane 1). We carried out SDS͞ samples were applied onto the Capcell Pak C18 HPLC column PAGE analysis for the same sample under nonreducing condi- equilibrated with 20% acetonitrile containing 0.1% trifluoro- tions to examine whether the sample included proteins bound to acetic acid, and the column was washed with the same buffer. each other with a disulfide bond (or polysulfide bond) (Fig. 1A, The peptides were separated with a 50-min linear gradient from lane 2). The fraction was found to contain 64- and 38-kDa 20% to 100% acetonitrile at a flow rate of 1 ml͞min and proteins, in addition to 45- and 19-kDa proteins corresponding monitored by UV absorption at 215 nm. Each peak was recov- to monomeric IscS and IscU, respectively. N-terminal sequence ered, evaporated, and resuspended in 200 ␮l of a labeling analyses revealed that the 64-kDa protein is a covalently asso- mixture containing 0.1 M Tris⅐HCl (pH 8.0), 5 mM EDTA, and ciated IscS͞IscU heterodimer and the 38-kDa protein is an IscU 0.5 mM ABD-F. The labeling reaction was performed for 5 min homodimer with subunits covalently bound to each other. at 50°C in the presence or absence of 1 mM tributylphosphine The eluate from the nickel-chelating column was subjected to (TBP). The relative fluorescence of each fraction was measured gel filtration to determine the subunit composition of the native at 520 nm with excitation at 380 nm, and a fraction that gave high proteins. Proteins were eluted as three resolved peaks corre- fluorescence intensity was reanalyzed by HPLC. The peptides sponding to an ␣2␤2 complex of IscS and IscU (about 150 kDa), were recovered, and the primary structures were determined dimeric IscU (about 45 kDa), and monomeric IscU (about 20 with an automated protein sequencer. kDa) (Fig. 1B). We analyzed these proteins by SDS͞PAGE

Kato et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 5949 Downloaded by guest on September 30, 2021 Fig. 2. Determination of cysteine residues participating in the disulfide bond formation. (A) Peptide eluted at 37.1 min was treated with ABD-F in the absence (a and b) or presence (c and d) of TBP and analyzed by C18 column chromatography. Peptides were monitored by UV absorption at 215 nm (a and c)orby fluorescence at 520 nm (b and d). Effect of mutation(s) in IscS (B) or IscU (C) on the interaction and the covalent bond formation between IscS and IscU. Interactions were examined by copurification experiments with a nickel-chelating column as described in Experimental Procedures. Proteins were separated by SDS͞PAGE without the use of 2-mercaptoethanol and detected by Western blot analysis with anti-IscS or anti-IscU antiserum.

under nonreducing conditions. The fractions of the ␣2␤2 com- with iodoacetic acid to alkylate free sulfhydryl groups and next plex almost exclusively contained a covalently associated IscS͞ digested with lysyl endopeptidase. The peptides were separated IscU heterodimer, and the amounts of monomeric IscS and IscU and purified by RP-HPLC, and a peptide containing the disul- were negligibly small, indicating that the tetramer comprises two fide bond was identified by labeling the relevant cysteine residues covalent IscS͞IscU heterodimers. IscU appeared as a covalently with ABD-F after reducing the disulfide bond with TBP to linked dimer in the IscU dimer fractions. produce free sulfhydryl groups. As a result, a peptide eluted at To determine whether the covalent linkages between IscS and 37.1 min was exclusively labeled. The treatment with TBP caused IscU and between the subunits of the IscU dimer were disulfide the cleavage of the peptide into two peptides, which were eluted bonds or polysulfide bonds, S0 in the IscS͞IscU complex and the at 22.5 min (Fig. 2A, c and d, Peak 1) and 33.8 min (Fig. 2A, c IscU dimer was measured by cyanolysis. The amount of S0 in 1.5 and d, Peak 2). Both of them showed fluorescence derived from mg of the copurified sample was determined to be 1.2 nmol. The ABD-F (Fig. 2A, d). These peptides were not obtained without copurified sample consisted of 42% IscU dimer, 38% IscS͞IscU treatment with TBP (Fig. 2A, a and b). The N-terminal amino complex, 16% IscU monomer, and 4% IscS monomer based on acid sequences of Peaks 1 and 2 were determined to be TYGXG- quantification of the SDS͞PAGE bands. Thus, the amount of S0 SAIAS and DLAVSSGSAX, respectively. The former sequence in the IscS͞IscU complex is estimated to be at most 0.13 mol per corresponds to Thr-60-Ser-69 in IscU, and the latter corresponds mol of the IscS͞IscU heterodimer assuming that all S0 was to Asp-319-Cys-328 in IscS. The ‘‘X’’ in the amino acid sequences derived from the IscS͞IscU complex [1.2 ϫ 10Ϫ9͞{1.5 ϫ 10Ϫ3 ϫ denotes the cysteine residue that was modified with ABD-F and 0.38͞(45,095 ϩ 14,784)} ϭ 0.13; the molecular weights of the therefore not identified with an automated protein sequencer. subunits of IscS and IscU-His6 are 45,095 and 14,784, respec- These results indicate that a disulfide bond is formed between tively]. The amount of S0 in the IscU dimer is estimated to be Cys-63 of IscU and Cys-328 of IscS, which is the active site 0.056 mol per mol of the dimer at most. Accordingly, the vast residue producing a persulfide intermediate in the reaction with majority of the IscS͞IscU complexes and the IscU dimers have L-cysteine. disulfide bonds, not polysulfide bonds, with S0 between two We next examined whether the disulfide bond between Cys-63 cysteine residues. of IscU and Cys-328 of IscS is an absolute requirement for the association of IscU with IscS or whether other amino acid Formation of the Disulfide Bond Between Cys-63 of IscU and the residues mediate the specific interaction between IscS and IscU. Active-Site Cys-328 of IscS. We next identified the residues con- With this aim, we replaced Cys-328 of IscS with alanine by stituting the disulfide bond between IscS and IscU. We also site-directed mutagenesis. We examined whether IscS-C328A examined whether the disulfide bond is an absolute requirement interacts with IscU-His6 by copurification experiments with a for the interaction between IscS and IscU or whether other nickel-chelating column. As shown in Fig. 2B, the C328A mutant amino acid residues also mediate the specific interaction. of IscS was copurified with IscU-His6: IscS-C328A was identified To identify the cysteine residues forming the disulfide linkage in the eluate by Western-blot analysis with an anti-IscS Ab. Thus, between IscS and IscU, the IscS͞IscU complex was first treated Cys-328 of IscS is not an absolute requirement for the specific

5950 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.082123599 Kato et al. Downloaded by guest on September 30, 2021 of IscU, the residue forming the disulfide bond with the active- site residue of IscS, is essential for the activation of IscS. Discussion In the present study, we have shown that IscS and IscU specif- ically interact with each other to produce a 1:1 complex and that Cys-328 of IscS and Cys-63 of IscU form a disulfide bond in the complex. Formation of the disulfide bond is supposed to be facilitated by noncovalent interactions between amino acid residues other than these cysteine residues, considering that the specific interaction between IscS and IscU is achieved even in the absence of these cysteine residues (Fig. 2 B and C). Specific interaction between IscS and IscU was also shown by the following observation. Although E. coli has two more with cysteine desulfurase activity, CSD (18) and CsdB (19), only IscS was shown to interact with IscU by surface plasmon resonance analysis (data not shown). The covalent complex of IscS and IscU is produced only in the presence of L-cysteine, the substrate for IscS, implying that the target of IscU is IscS with sulfane sulfur derived from L-cysteine on its active-site Cys-328 (12, 14, 15). Because a disulfide bond was found between Cys-328 of IscS and Cys-63 of IscU, it is most likely that the S␥ atom of Cys-63 of IscU attacks the S␥ atom of Fig. 3. Activation of cysteine desulfurase activity of IscS by IscU. Cysteine Cys-328 carrying the sulfane sulfur derived from L-cysteine. It is desulfurase activity was measured in the presence of various amounts of also conceivable that the attack is crucial for the IscU-mediated IscU-His (E), IscU-C37S (ᮀ), IscU-C63S (Œ), and IscU-C106S (■). The reaction 6 activation of IscS, taking account of the indispensability of mixture contained 20 mM L-cysteine, 50 mM DTT, 20 ␮M pyridoxal 5Ј- phosphate, 125 mM Tricine-NaOH (pH 8.0), 0.2 ␮M IscS, and wild-type or Cys-63 for the activation (Fig. 3); the attack by Cys-63 probably mutant IscU. facilitates the release of the sulfane sulfur from Cys-328 of IscS. To increase the turnover rate of the IscS-catalyzed cysteine desulfurase reaction, IscU must be dissociated from IscS imme- interaction between IscS and IscU. As expected, the 64-kDa diately after its attack on Cys-328 to make this residue competent band corresponding to the covalent IscS͞IscU complex was not again for the next cycle of catalysis. Dissociation of IscU from observed by SDS͞PAGE for IscS-C328A because of the lack of Cys-328 of IscS is probably facilitated by DTT in our assay the disulfide bond between IscS-C328A and IscU-His6. system. We also constructed His6-tagged versions of IscU-C37S, IscU- It will be interesting to examine what molecules are respon- C63S, and IscU-C106S and analyzed whether these mutant sible for the dissociation of IscU from IscS in vivo. Glutathione proteins were able to associate with IscS. As shown in Fig. 2C, may be involved in reduction of the disulfide bond. An hsp70- IscS was copurified with every IscU mutant, as judged by type molecular chaperone Hsc66 and a cochaperone Hsc20 Western blot analysis of the eluates with the anti-IscS Ab. Thus, encoded by the isc operon have been shown to interact with IscU none of the three cysteine residues of IscU is absolutely required (14, 20, 21). Thus, in addition to molecules required for reduc- for the specific interaction between IscS and IscU. However, as tion of the disulfide bond between IscS and IscU, these molec- expected, Cys-63 is essential for the formation of the covalent ular chaperones may be involved in the dissociation process. That a significant amount of the covalent IscS͞IscU complex was complex of IscS and IscU: the 64-kDa band corresponding to the BIOCHEMISTRY covalent complex was not observed when IscU-C63S was used obtained in our copurification experiments may reflect the in for the copurification experiment. Cys-37 and Cys-106 of IscU vivo situation that the molecules required for the dissociation of were not required for the formation of the covalent complex. It IscU from IscS were not sufficient compared with overproduced IscS and IscU. is noteworthy that Cys-63 is also essential for the formation of the Based on the findings in the present study, we would like to covalent IscU dimer: Western blot analysis of the eluate for propose a mechanism for the sulfur transfer from IscS to IscU. IscU-C63S with the anti-IscU Ab showed the absence of the Previous reports have suggested that the sulfur atom derived 38-kDa band, which corresponds to the IscU dimer. from L-cysteine is transferred from IscS to IscU and covalently Taken together, these results demonstrated that the disulfide bound to IscU (14, 15). We show here that IscU exists in two bond was specifically formed between Cys-328 of IscS and Cys-63 forms, a monomeric form and a covalent dimeric form (Fig. 1B), of IscU and that the specific interaction between IscS and IscU and either of these two forms of IscU is supposed to receive the was achieved by amino acid residues other than these cysteine sulfur atom. If the monomeric form of IscU reacts with IscS, the residues through noncovalent interactions. sulfane sulfur is probably transferred to a cysteine residue located in the vicinity of Cys-63; the S␥ atom of Cys-328 of IscS Activation of Cysteine Desulfurase Activity of IscS by IscU. We found is attacked by Cys-63 of IscU, and the sulfane sulfur released that IscU enhances the cysteine desulfurase activity of IscS and from Cys-328 is most likely accepted by a nearby residue. If the this activation requires the action of Cys-63 of IscU (Fig. 3). This dimeric form of IscU is the molecule that attacks IscS, Cys-63 of was a novel observation with respect to IscS activation. We IscU is supposed to be the primary site for sulfur transfer from observed a maximum of a 6-fold increase in the activity with IscS to IscU (Fig. 4). In this scheme, the disulfide-bridged IscU stoichiometric amounts of IscU-His6 (0.2 mM). The addition of dimer reacts with persulfide-containing IscS, resulting in the excess IscU-His6 (3 times the IscS concentration) did not result formation of a disulfide linkage between Cys-328 of IscS and in any further stimulation. The IscU-C37S and IscU-C106S Cys-63 of one of the two subunits of the IscU dimer, releasing mutants promoted 4.5- and 6-fold increases in the IscS activity, the other subunit of IscU with persulfide on Cys-63. Because IscS when added in stoichiometric amounts. In contrast, IscU-C63S is a dimeric protein, there are two binding sites for IscU in each did not stimulate the activity of IscS at all, indicating that Cys-63 IscS dimer. On the basis of the x-ray crystallographic data for two

Kato et al. PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 5951 Downloaded by guest on September 30, 2021 Fig. 4. Proposed scheme for the formation of the covalent IscS͞IscU complex involving the sulfur transfer from Cys-328 of IscS to Cys-63 of IscU. The bracketed ␣2␤4 hexamer of the IscS͞IscU complex is supposed to be produced transiently in the reaction. Details of the processes indicated by dotted arrows are not known. The type of iron-sulfur clusters on IscU serving as iron-sulfur cluster precursor(s) has not been determined.

IscS homologs, CsdB from E. coli (22) and an NifS-like protein IscU monomers are released concomitantly with the formation from Thermotoga maritima (23), two subunits of IscS are prob- of one heterodimeric complex of IscS and IscU. ably associated with each other so that the two Cys-328 residues are located on opposite sides of the dimer (Fig. 4). If this is the This work was supported in part by a Grant-in-Aid for Scientific case, two IscU dimers must react with one IscS dimer to produce Research on Priority Areas (B) 13125203 (to N.E.) from the Ministry of ␣ ␤ ͞ Education, Culture, Sports, Science, and Technology, by a Grant-in-Aid the 2 2 heterotetramer of the IscS IscU complex because two for Scientific Research 13480192 (to N.E.), and by Grants-in-Aid for Cys-63 residues in one IscU dimer are not able to attack Encouragement of Young Scientists 12780470 (to T.K.) and 13760069 (to two Cys-328 residues of the IscS dimer simultaneously. Thus, two H.M.) from the Japan Society for the Promotion of Science.

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