Title: The SufE Protein and the SufBCD Complex Enhance SufS Cysteine Desulfurase Activity as Part of a Sulfur Transfer Pathway for Fe-S Cluster Assembly in Escherichia coli
Abstract: The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo. The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo. Numerous processes within the cell require the mobilization of elemental sulfur from l-cysteine. These processes include Fe-S cluster assembly as well as the synthesis of molybdopterin, thiamine, biotin, and thionucleosides in tRNA (1Mihara H. Esaki N. Appl. Microbiol. Biotechnol. 2002; 60: 12-23Crossref PubMed Scopus (225) Google Scholar). Often sulfur mobilization occurs via a cysteine desulfurase enzyme that converts l-cysteine to sulfane sulfur and l-alanine in a process that requires pyridoxal 5′-phosphate as a cofactor (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar). The sulfane sulfur, present as a persulfide intermediate on the active site cysteine of the desulfurase, is then transferred to various sulfur acceptors depending on the physiological pathway. One of the earliest identified cysteine desulfurases is the NifS protein of Azotobacter vinelandii, which is involved in Fe-S assembly in the nitrogenase enzyme (2Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar). A. vinelandii also contains another NifS homologue, IscS, which is involved in Fe-S cluster assembly in enzymes other than nitrogenase (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). The Gram-negative bacterium Escherichia coli contains three NifS homologues, IscS, CsdA (also known as CSD), and SufS (also known as CsdB). All three E. coli enzymes have been purified and shown to exhibit cysteine desulfurase activity (4Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 5Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 6Mihara H. Kurihara T. Yoshimura T. Esaki N. J. Biochem. (Tokyo). 2000; 127: 559-567Crossref PubMed Scopus (126) Google Scholar, 7Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The three NifS homologues are present at separate loci and are co-expressed with different accessory proteins. The question arises as to why three NifS homologues are present in E. coli. Possibly they carry out divergent functions within the cell or are regulated differentially to provide similar functions under different conditions. One way to examine the functions of the three NifS homologues is to characterize the activities of the accessory proteins co-expressed with each homologue. In E. coli, IscS is part of an operon that includes IscR, IscU, and IscA. The IscR transcriptional repressor regulates the isc operon by sensing changes in Fe-S cluster assembly status (8Schwartz C.J. Giel J.L. Patschkowski T. Luther C. Ruzicka F.J. Beinert H. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14895-14900Crossref PubMed Scopus (333) Google Scholar). IscU is a Fe-S assembly scaffold protein used to construct nascent Fe-S clusters (9Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar). A conserved cysteine residue on IscU accepts sulfur from IscS during Fe-S cluster building (10Smith A.D. Agar J.N. Johnson K.A. Frazzon J. Amster I.J. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2001; 123: 11103-11104Crossref PubMed Scopus (173) Google Scholar, 11Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar). IscA also can form Fe-S clusters and may function as an alternate scaffold (13Krebs C. Agar J.N. Smith A.D. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. Biochemistry. 2001; 40: 14069-14080Crossref PubMed Scopus (210) Google Scholar, 14Ollagnier-de-Choudens S. Mattioli T. Takahashi Y. Fontecave M. J. Biol. Chem. 2001; 276: 22604-22607Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Downstream of the isc operon is the hscB-hscA-fdx operon. Both hscA and hscB encode molecular chaperone proteins while fdx encodes a ferredoxin, and all three proteins play a part in isc-mediated Fe-S cluster assembly (15Hoff K.G. Ta D.T. Tapley T.L. Silberg J.J. Vickery L.E. J. Biol. Chem. 2002; 277: 27353-27359Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 17Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (228) Google Scholar). In some organisms, the hscB-hscA-fdx operon is co-expressed with isc but in E. coli the two operons are regulated separately (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 19Zheng M. Wang X. Templeton L.J. Smulski D.R. LaRossa R.A. Storz G. J. Bacteriol. 2001; 183: 4562-4570Crossref PubMed Scopus (661) Google Scholar, 20Seaton B.L. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2066-2070Crossref PubMed Scopus (83) Google Scholar). In vivo the isc operon together with the hscB-hscA-fdx genes have been shown to be important for the assembly of a variety of Fe-S enzymes (18Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (228) Google Scholar, 21Schwartz C.J. Djaman O. Imlay J.A. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9009-9014Crossref PubMed Scopus (258) Google Scholar). The second E. coli NifS homologue, CsdA, is encoded adjacent to the ygdK gene, which encodes a homologue of SufE. Little is known about the in vivo role of CsdA. CsdA is the most efficient of the three E. coli NifS homologues at providing sulfur to MoaD for synthesis of molybdopterin in vitro (22Leimkuhler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and may be involved in molybdopterin synthesis in vivo. The SufS cysteine desulfurase is co-expressed with five additional gene products, SufA, SufB, SufC, SufD, and SufE (23Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar). Besides its cysteine desulfurase activity, SufS has also been shown to exhibit a strong selenocysteine lyase activity in vitro (5Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), although SufS does not appear to be important for selenium metabolism in vivo (24Mihara H. Kato S. Lacourciere G.M. Stadtman T.C. Kennedy R.A. Kurihara T. Tokumoto U. Takahashi Y. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6679-6683Crossref PubMed Scopus (52) Google Scholar). SufA is a homologue of IscA and can form Fe-S clusters in vitro (25Ollagnier-de Choudens S. Nachin L. Sanakis Y. Loiseau L. Barras F. Fontecave M. J. Biol. Chem. 2003; 278: 17993-18001Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). SufC has the sequence hallmarks of the ATPase subunit of ABC transporters and has been shown to exhibit ATPase activity (26Rangachari K. Davis C.T. Eccleston J.F. Hirst E.M. Saldanha J.W. Strath M. Wilson R.J. FEBS Lett. 2002; 514: 225-228Crossref PubMed Scopus (59) Google Scholar). The biochemical functions of SufB, SufD, and SufE are unknown, although fluorescence anisotropy and yeast two-hybrid assays indicate that SufB and SufD interact with SufC (26Rangachari K. Davis C.T. Eccleston J.F. Hirst E.M. Saldanha J.W. Strath M. Wilson R.J. FEBS Lett. 2002; 514: 225-228Crossref PubMed Scopus (59) Google Scholar, 27Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (223) Google Scholar). Most of the suf genes are well conserved in a variety of microorganisms, including cyanobacteria, as well as in higher plants (28Ellis K.E. Clough B. Saldanha J.W. Wilson R.J. Mol. Microbiol. 2001; 41: 973-981Crossref PubMed Scopus (87) Google Scholar, 29Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Previous experiments are consistent with a role for the Suf proteins in Fe-S assembly. In E. coli, mutations in sufS or sufD result in the loss of a Fe-S cluster in the FhuF iron reductase (30Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar), thereby preventing the use of ferrioxamine B as a sole iron source. Similarly, mutations in SufB, SufC, or SufD impair Erwinia chrysanthemi growth on ferric chrysobactin as a sole iron source, a phenotype that has been attributed to loss of an unidentified Fe-S enzyme (27Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (223) Google Scholar). In Arabidopsis thaliana, disruption of a SufB homologue leads to accumulation of protoporphyrin IX, indicating an undefined role for SufB in chlorophyll biosynthesis in that organism (31Moller S.G. Kunkel T. Chua N.H. Genes Dev. 2001; 15: 90-103Crossref PubMed Scopus (176) Google Scholar). Most suf mutants are synthetically lethal with isc mutants in E. coli, indicating overlap in the roles of the two operons (29Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). E. coli deletion mutants of suf also show decreased growth and loss of some Fe-S enzyme activity under oxidative stress conditions (23Nachin L. El Hassouni M. Loiseau L. Expert D. Barras F. Mol. Microbiol. 2001; 39: 960-972Crossref PubMed Scopus (156) Google Scholar, 27Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (223) Google Scholar). In addition, transcription of the E. coli suf operon is induced by oxidative stress through the OxyR hydrogen peroxide sensor (19Zheng M. Wang X. Templeton L.J. Smulski D.R. LaRossa R.A. Storz G. J. Bacteriol. 2001; 183: 4562-4570Crossref PubMed Scopus (661) Google Scholar) and by iron starvation through loss of repression by Fur (30Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar, 32Lee J.H. Yeo W.S. Roe J.H. J. Microbiol. (Korea). 2003; 41: 109-114Google Scholar). To learn more about the roles of the individual Suf proteins, we have purified the suf gene products and determined that SufE stimulates the activity of the SufS cysteine desulfurase. In addition, the SufB, SufC, and SufD proteins interact in a stable complex and can further increase the desulfurase activity of SufS in a SufE-dependent manner. These results indicate that interactions with accessory proteins can enhance the activity of NifS homologues above that observed with the cysteine desulfurase alone. We propose that regulated sulfur transfer conferred by SufE and SufBCD may be important under iron-limited and oxidative stress conditions. Strains and Media—(His)6-SufE and SufABCDSE were expressed in TOP10 (Invitrogen), and (His)6-SufA and (His)6-SufS were expressed in BL21(DE3)plysS (Invitrogen). All of the bacterial growth was in Lennox Broth (10 g of tryptone,5 g of yeast extract,5 g of NaCl/liter). Ampicillin was used at 100 mg/liter, and chloramphenicol was used at 30 mg/liter. l-Arabinose was added to 0.2% final concentration by weight, and isopropyl-1-thio-β-d-galactopyranoside was added to 1 mm final concentration. All of the chemicals used were obtained from Sigma unless indicated otherwise. Plasmid Construction—All of the PCR reactions were carried out using MG1655 chromosomal DNA as template. SufABCDSE was PCR-amplified using two primers (5′-GAGGTAAATCGATGGATCCGCATTCAGGAAC-3′ and 5′-GTTCACCTGAATTCAAAACACTCCTGTGC-3′). The EcoRI-digested PCR fragment was ligated into the NcoI (bluntended with Klenow fragment) and EcoRI sites of pBADmychisC (Invitrogen) to generate pGSO164. SufE was amplified using two primers (5′-GAGGCACCATGGCTTTATTGCCGGATAA-3′ and 5′-CCTTTTAGTTTAGCTGAATTCAGCGGCTTTG-3′), digested with NcoI and EcoRI, and cloned into the corresponding sites of pBADmychisC to generate pGSO165. SufS was amplified using two primers (5′-GGAGGTGCAAGATGAGATCTTCCGTCGACAAAGT-3′ and 5′-CCATAGTGAATTCCTGTTATCCCAGCAAACGG-3′), digested with BglII and EcoRI, and cloned into the corresponding sites of pRSETB (Invitrogen) to generate pGSO166. SufA was PCR-amplified using two primers (5′-GTTGCTTCAGAATTCCGAGACATAGTACCGCC-3′ and 5′-GAGGTAAATCGATGGATCCGCATTCAGGAAC-3′), digested with EcoRI and BamHI, and cloned into the corresponding sites of pRSETB to generate pGSO167. Plasmids were designed such that (His)6-SufA and (His)6-SufS were fused with a (His)6-tag at their N termini while (His)6-SufE was fused with a Myc and (His)6-tag at the C terminus. Mutation of SufE Cys51 to Ser was performed using the pGSO165 construct, two mutagenic primers (5′-CAAAATAGCATTCAGGGCAGCCAGAGTCAGGTGTGG-3′ and 5′-CCACACCTGACTCTGGCTGCCCTGAATGCTATTTTG-3′), and the QuikChange site-directed Mutagenesis kit (Stratagene) according to the manufacturer's protocol to generate pGSO168. The nucleotide sequences of all of the plasmid inserts were confirmed. Protein Expression and Purification—Strains carrying the (His)6 expression constructs were induced by isopropyl-1-thio-β-d-galactopyranoside ((His)6-SufA and (His)6-SufS) or l-arabinose ((His)6-SufE) when the cultures obtained an A 600 of 0.4–0.6. After 3 h of expression at 37 °C, cells were harvested by centrifugation. All of the cells were lysed in 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 5 mm imidazole, 1 mm phenylmethylsulfonyl fluoride, 1× EDTA-free protease inhibitor tablet (Roche Applied Science) via sonication ((His)6-SufA and (His)6-SufS)) or with a high pressure cell disrupter (Constant Systems LTD) at 10,000 p.s.i. ((His)6-SufE). Following centrifugation at 20,000 × g for 30 min, cleared lysate was loaded on a nickel-nitrilotriacetic acid Superflow (Qiagen) column on a fast protein liquid chromatography system (Amersham Biosciences) and eluted with a step gradient of 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 250 mm imidazole. (His)6-SufA and (His)6-SufS were dialyzed against 25 mm Tris-Cl, 100 mm NaCl, pH 7.4, concentrated, and stored at -70 °C. Purified (His)6-SufE was dialyzed against 25 mm Tris-Cl, 100 mm NaCl, pH 7.4, and then was digested with trypsin (1:400 ratio by weight of trypsin to (His)6-SufE). The digested SufE lacked the Myc and (His)6 tag but contained the full sequence of the native protein, as confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and N-terminal amino acid sequencing. The digested SufE was treated with 10 mm DTT, 1The abbreviations used are: DTT, dithiothreitol; BSA, bovine serum albumin; CD, circular dichroism; DLS, dynamic light scattering. further purified on a Mono Q column (Amersham Biosciences), concentrated, and stored at -70 °C. SufEC51S was purified as described for SufE. Similar yields were obtained for the SufEC51S mutant protein as compared with native SufE, and it was as resistant to proteolysis as the native SufE protein, indicating that it was correctly folded. The entire suf operon was expressed simultaneously to purify Suf-BCD. Cells expressing the entire suf operon induced by the addition of l-arabinose at A 600 of 0.4–0.6 turned grayish-black after the 3-h expression period, possibly because of the accumulation of iron sulfides within the cell. Cell pellets from the SufABCDSE expression were lysed in 25 mm Tris-Cl, pH 8.0, 50 mm NaCl, 10 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 1× EDTA-free protease inhibitor tablet using a high pressure cell disrupter at 10,000 p.s.i. Following centrifugation at 20,000 × g for 30 min, cleared lysate was loaded onto a Mono Q column and eluted with a linear gradient of 1 m NaCl. Fractions containing SufBCD were pooled, dialyzed overnight in 25 mm Tris-Cl, pH 7.4, 150 mm NaCl, and concentrated. The concentrated fractions then were separated on a Superdex 200 gel filtration column (Amersham Biosciences). Purified SufBCD was concentrated and stored at -70 °C. Purified IscS and NifS from A. vinelandii were kindly provided by D. R. Dean. Size Determination—A Superdex 200 gel-filtration column was used to determine size by gel-filtration chromatography. Concentrated protein samples in 50 mm NaH2PO4, pH 7.0, 150 mm NaCl were loaded onto the column in 1-ml volumes to minimize dilution effects. Molecular weights were calculated by plotting log molecular weight versus the ratio of the elution volume/void volume for the standard proteins. Sizes were also determined by dynamic light scattering (DLS) using a DynaPro instrument (Protein Solutions) and Dynamics software. Cysteine Desulfurase Activity Assays—Total sulfide was measured by a previously published protocol (11Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Reactions were carried out anaerobically in 25 mm Tris-Cl, pH 7.4, 100 mm NaCl using 500 nm cysteine desulfurase ((His)6-SufS, IscSA. vinelandii, or NifSA. vinelandii) and varying ratios of (His)6-SufA, SufBCD, SufE, and/or BSA. A molecular mass of 165 kDa (as measured by DLS) was used to calculate molar concentrations of the SufBCD complex. Pyridoxal 5′-phosphate was added to 10 μm, and reactions were initiated by dilution of a 10 mm l-cysteine/10 mm DTT stock to a 100 μm final concentration of each in a total reaction volume of 800 μl. Reactions were allowed to proceed for 20 min at 27 °C and then were quenched by the addition of 100 μl of 20 mm N,N-dimethyl-p-phenylenediamine in 7.2 m HCl. The addition of 100 μlof30 mm FeCl3 in 1.2 m HCl and incubation for 20 min led to the formation of methylene blue. Precipitated protein was removed by 30-s centrifugation at 20,000 × g, and methylene blue was measured at 670 nm. Na2S was used as a standard for calibration. Sulfur Transfer Assays—Reactions consisted of 1 μm (His)6-SufS mixed with 20 μm (His)6-SufA, SufE, SufBCD, and/or BSA in 25 mm Tris-Cl, pH 7.4, 100 mm NaCl, 100 μm DTT. Reactions were initiated by the addition of l-[35S]cysteine (150 mCi/mmol, Amersham Biosciences) to a final concentration of 30 μm. The final reaction volume was 35 μl. After a 30-s incubation, samples were loaded onto preequilibrated G50 ProbeQuant columns (Amersham Biosciences) and spun for 2 min at 1700 × g. Samples were separated on a non-reducing 10–20% Trisglycine gel, dried onto filter paper, and exposed overnight to a phosphorimaging screen or film. Control transfer reactions without DTT showed no increase in labeling as compared with samples with DTT. Purification of the Suf Proteins—The SufS cysteine desulfurase and its accessory proteins, SufA, SufB, SufC, SufD, and SufE, were purified to characterize their biochemical functions (Fig. 1A). (His)6-SufA, (His)6-SufS, and SufE were expressed and purified separately. (His)6-SufS was purified as a dimer with an absorption maximum at 420 nm as has been reported for native SufS (5Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). SufE was expressed as a Myc and (His)6 fusion, but the tag was later removed by limited proteolysis. The molecular weight of SufE was analyzed using two different techniques. SufE eluted from a gel-filtration column as a single peak at a molecular mass of 19.8 kDa (Fig. 1B). This molecular mass is between the predicted molecular masses for a SufE monomer (15.8 kDa) and dimer (31.6 kDa). However, a monodispersed species with a molecular mass of 32 kDa was detected using DLS, indicating that SufE can exist in a homodimeric form. These differences in SufE molecular mass and oligomerization probably reflect differences between the two techniques. The SufE protein eluted from the gel-filtration column in a very dilute peak; therefore, its molecular weight was measured at a concentration of ∼0.1 mg/ml. In contrast, the molecular mass was measured by DLS using a SufE concentration of 4 mg/ml. These results suggest that SufE can form monomers at low protein concentrations and homodimers at higher protein concentrations. The SufB, SufC, and SufD proteins were expressed simultaneously with all of the suf genes from a single expression construct. During isolation of Suf proteins from cells expressing the SufABCDSE construct, we found that SufB, SufC, and SufD always co-purified on both anion-exchange and gel-filtration resins (Fig. 1A). In addition, the SufBCD proteins co-purified during ammonium sulfate precipitation and on a variety of hydrophobic interaction resins. In fact, the individual SufB, SufC, and SufD proteins could only be completely separated from the complex using reverse phase high performance liquid chromatography, indicating tight association. This result is consistent with previous yeast two-hybrid (27Nachin L. Loiseau L. Expert D. Barras F. EMBO J. 2003; 22: 427-437Crossref PubMed Scopus (223) Google Scholar) and fluorescence anisotropy (26Rangachari K. Davis C.T. Eccleston J.F. Hirst E.M. Saldanha J.W. Strath M. Wilson R.J. FEBS Lett. 2002; 514: 225-228Crossref PubMed Scopus (59) Google Scholar) studies that suggest interactions between SufC and the SufB and SufD proteins. The SufBCD complex was in a folded conformation based on its CD spectra and also possessed ATPase activity (data not shown). Analysis of the SufBCD complex using gel filtration resulted in a major species of 172 kDa (Fig. 1C). A similar monodispersed molecular mass of 165 kDa was obtained for the Suf-BCD complex by DLS. A small amount of SufBCD was also seen in a higher molecular mass peak of 324 kDa during gel filtration, which corresponds to a multimer of the complex. Several protein stoichiometries could match the 172-kDa molecular mass measured for the SufBCD complex, but on a denaturing gel the proteins appear to be present in equimolar amounts (Fig. 1A). Further experiments are necessary to elucidate the exact stoichiometry of the SufBCD complex. In this study, we simply refer to the 172-kDa complex of the SufB, SufC, and SufD proteins as SufBCD. SufE Enhances SufS Cysteine Desulfurase Activity—Our purified (His)6-SufS has a specific activity of 0.008 units/mg toward l-cysteine at 37 °C. This activity is slightly less (2–3-fold) than the 0.019 units/mg activity obtained for native SufS using a different assay that contained 500-fold higher levels of reductant and 120-fold higher levels of l-cysteine (5Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In general, the in vitro cysteine desulfurase activity of isolated SufS is low compared with the activities of the isolated IscS or CsdA enzymes (6Mihara H. Kurihara T. Yoshimura T. Esaki N. J. Biochem. (Tokyo). 2000; 127: 559-567Crossref PubMed Scopus (126) Google Scholar). However, all of the previous experiments were carried out with purified SufS in the absence of the other suf gene products. Because the addition of IscU to IscS enhances the desulfurase activity of IscS (12Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar), it seemed possible that accessory proteins could alter the enzyme activity of SufS. To determine whether the SufA, SufBCD, or SufE proteins affect the cysteine desulfurase activity of SufS, we measured (His)6-SufS activity in the presence of the accessory proteins. The addition of SufE increased the desulfurase activity of (His)6-SufS by nearly an order of magnitude (8-fold) while the addition of (His)6-SufA, SufBCD, or a nonspecific BSA control had little effect (Fig. 2A). Maximum enhancement was observed at a 6-fold molar excess of SufE (Fig. 2A), possibly reflecting a need for SufE in excess to fully promote (His)6-SufS activity or indicating that some fraction of the SufE protein is not fully active. (His)6-SufA, SufBCD, and SufE did not exhibit any measurable desulfurase activity in the absence of (His)6-SufS (data not shown). The SufBCD Complex Magnifies the SufE Enhancement of SufS Activity—To determine whether the other Suf proteins affect the SufE-dependent enhancement of (His)6-SufS activity, we also measured desulfurase activity in the presence of various Suf protein combinations (Fig. 2B). Although the addition of (His)6-SufA or BSA to a mixture of SufE and (His)6-SufS had no further effect on SufS activity, the addition of the SufBCD complex to SufE and (His)6-SufS increased desulfurase activity from 8- to ∼32-fold (Fig. 2B). The enhancement of desulfurase activity by SufE and SufBCD is specific for (His)6-SufS as the addition of SufE and SufBCD to the NifS and IscS cysteine desulfurases from A. vinelandii had little effect on their activities (Fig. 3). Cys51 Is Essential for SufE Function—It has been shown that IscS transfers sulfur to the Fe-S scaffold protein IscU via Cys63 on IscU and that this residue is required for IscU-dependent enhancement of IscS activity (12Kato S. Mihara H. Kurihara T. Takahashi Y. Tokumoto U. Yoshimura T. Esaki N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5948-5952Crossref PubMed Scopus (115) Google Scholar). An examination of the SufE amino acid sequence for conserved residues that might be involved in its function revealed the presence of a highly conserved cysteine at position 51 (Fig. 4A). This residue represents a candidate to accept sulfur mobilized from l-cysteine by (His)6-SufS. We substituted this conserved cysteine with a serine, isolated the mutant protein, and assayed its effect on (His)6-SufS desulfurase activity. As shown in Fig. 4B, the SufEC51S mutant lacks the ability to enhance (His)6-SufS desulfurase activity. SufBCD enhancement of (His)6-SufS activity also was abolished in the presence of SufEC51S, further highlighting the importance of the Cys51 residue for SufE function (Fig. 4B). SufE Functions as a Sulfur Acceptor Protein—Because SufE and SufBCD enhancement of (His)6-SufS activity could involve specific transfer of sulfane sulfur to one of these proteins, we tested the ability of SufS to transfer sulfur to the Suf accessory proteins. We observed clear mobilization of 35S from l-[35S]cysteine to SufE via (His)6-SufS (Fig. 5A). In contrast, 35S labeling of (His)6-SufA by (His)6-SufS was essentially the same as that observed with the control protein BSA, indicating little to no specific transfer to this protein (Fig. 5A). Although there was more labeling of the SufBCD complex than (His)6-SufA, the labeling was substantially less intense than the labeling of SufE, suggesting that there is no specific sulfur transfer between (His)6-SufS and SufBCD. When SufBCD is added to (His)6-SufS in the presence of SufE, we observed a diminished level of SufE monomer labeling and more high molecular weight labeling (Supplemental Fig. 1A). The high molecular weight species result from increased disulfide bonding among (His)6-SufS, SufE, and SufB that occurs when the proteins are mixed in the presence of l-cysteine (Supplemental Fig. 1B). The SufEC51S mutant is not labele