The Cysteine-Desulfurase Iscs Promotes the Production of the Rhodanese Rhda in the Persulfurated Form

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FEBS Letters 579 (2005) 6786–6790 FEBS 30176

The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form

Fabio Forlania, Angelo Ceredaa, Andrea Freuerb, Manfred Nimtzc, Silke Leimku¨hlerd, Silvia Pagania,* a Dipartimento di Scienze Molecolari Agroalimentari, Facolta` di Agraria, Universita` di Milano, Via Celoria 2, Milano 20133, Italy b Institut fu¨r Pﬂanzenbiologie, TU Braunschweig, 38106 Braunschweig, Germany c Institut fu¨r Strukturbiologie, Gesellschaft fu¨r Biotechnologische Forschung (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany d Institut fu¨r Biochemie und Biologie, Universita¨t Potsdam, 14476 Potsdam, Germany

Received 20 September 2005; revised 3 November 2005; accepted 5 November 2005

Available online 21 November 2005

Edited by Miguel De la Rosa

Azotobacter vinelandii RhdA [17] appears to be a unique Abstract After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfu- member of the rhodanese homology superfamily [1,18,19]. rated form (RhdA-SSH). We identified L-cysteine as the most The RhdA active-site motif (HCQTHHR) is not currently effective sulfur source in producing RhdA-SSH. An E. coli solu- found in rhodanese-like proteins, and generates an unusually ble extract was required for in vitro persulfuration of RhdA, and strong positive electrostatic field [1] that favors stabilization the addition of pyridoxal-50-phosphate increased RhdA-SSH of a persulfide bond on the catalytic cysteine residue production, indicating a likely involvement of a cysteine desulfur- (Cys230), the only cysteine residue in this protein. After heter- ase. We were able to show the formation of a covalent complex ologous expression in Escherichia coli, A. vinelandii RhdA is between IscS and RhdA. By combining a time-course fluores- purified in a mixture of persulfurated RhdA (RhdA-SSH) cence assay and mass spectrometry analysis, we demonstrated and of the sulfane sulfur-deprived RhdA. The presence of the transfer of sulfur from E. coli IscS to RhdA. these forms can be assessed by 15N NMR spectroscopy [20], 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. as well as by intrinsic fluorescence measurements on the isolated protein [18], and by mass spectrometry [21]. The observa- Keywords: Rhodanese; Sulfane sulfur transfer; Cysteine tion that these forms were found in variable ratios after desulfurase overexpressions of RhdA in rich medium (LB), prompted us to investigate the nature of the sulfur source for RhdA-SSH production in E. coli. In the present work, RhdA was expressed in E. coli in minimal medium to determine the nature of the sulfur 1. Introduction sources used for producing RhdA-SSH. We found that L- cysteine was the most effective sulfur source. Addition of 0 Rhodaneses (thiosulfate:cyanide sulfurtransferase, E.C. pyridoxal-5 -phosphate (PLP) to the E. coli cell extracts in- 2.8.1.1) are widespread enzymes that in vitro catalyze the creased the production of RhdA-SSH in vitro, suggesting transfer of a sulfane sulfur atom from thiosulfate to cyanide the involvement of PLP-dependent cysteine desulfurases via a protein-bound persulfide group. They are either com- [16], such as IscS, in this process. Indeed, by combining posed of a single catalytic rhodanese domain, or are a fusion mass spectrometry analysis and a time-course fluorescence of two rhodanese domains, with the C-terminal domain con- assay, we demonstrated that E. coli IscS can efficiently trans- taining the putative catalytic cysteine [1–6]. The biological role fer sulfur to RhdA. The formation of a RhdA–IscS complex, of rhodaneses is still largely debated, and the observed abun- under defined interaction conditions, suggests molecular rec- dance of potentially functional rhodanese-like proteins also ognition between the sulfur donor IscS and the acceptor within the same genome (PFAM accession number: RhdA. PF00581; http://www.sanger.ac.uk/Software/Pfam/) suggests their involvement in distinct biological processes. Proposed functions for rhodaneses include cyanide detoxification, for- 2. Materials and methods mation of prosthetic groups in iron–sulfur cluster proteins, and sulfur transfer for thiamine, thiouridine or molybdopterin 2.1. Bacterial strains, cell culture, and treatments biosynthesis [7–11]. Cellular sulfur trafficking requires special- Overexpression of His-tagged RhdA was carried out in E. coli ized systems to avoid production of sulfur in a toxic form, and BL21[pRep4, pQER1] [18]. To define the sulfur source for RhdA per- formation of persulfide groups on a protein scaffold represents sulfuration, cells were grown in 100 ml of M9 minimal medium [22] supplemented with Basal Medium EagleÕs vitamin solution (10 ml/l, a biological strategy to sequester sulfur in an activated form BME, Sigma–Aldrich) and antibiotics according to the plasmid for biosynthetic purposes [12–16]. requirements. When the culture reached A600 0.6, the medium was replaced with fresh M9-BME medium supplemented with 1 mM IPTG, 2mM L-leucine, 2 mM L-isoleucine and 2 mM of the selected sulfur * Corresponding author. Fax: +390250316801. source (MgSO4, L-methionine, L-cysteine or thiosulfate). The possible E-mail address: [email protected] (S. Pagani). toxicity of cysteine was by-passed by addition of 2 mM L-leucine and

0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.11.013 F. Forlani et al. / FEBS Letters 579 (2005) 6786–6790 6787

2mM L-isoleucine [23]. After 4-h growth, the cells were harvested by centrifugation (5000 · g, 15 min) and washed in 100 ml water. Cell-free extracts were always obtained as previously described [24].

2.2. Protein preparations Overespressed His-tagged RhdA was purified by Ni-NTA affinity chromatography [24], and gelfiltered using a G25 column equilibrated in 50 mM Tris–HCl, 100 mM NaCl (pH 8). Sulfane sulfur-deprived RhdA was prepared by incubating purified RhdA with a 10-fold molar excess of KCN for 15 min at room temperature, and by removing excess KCN by gelfiltration. Cys230-blocked RhdA (RhdA-mBBr) was obtained by incubation of 156 lM sulfane sulfur-deprived RhdA in 50 mM Tris–HCl, 100 mM NaCl buffer (pH 8) with 5 mM monobromobimane (mBBr, thiolyte, Calbiochem, Darmstadt, Germany). After 2 h in the dark at room temperature with continuous stirring, the reaction mixture was gelfiltered on a G-25 Sephadex column. The binding of mBBr to RhdA was assessed by fluorescence [25]. Recombinant IscS was purified from BL21(DE3) E. coli strain harboring pSL209 [11]. The persulfide form of IscS (IscS-SSH) was prepared by incubation of IscS with an excess of L-cysteine for 5 min at 4 C. For the purifica- tion of IscS-SSH, the incubation mixture was gelfiltered using a PD10 column (GE healthcare) equilibrated with 100 mM Tris–HCl, pH 7.2. Protein concentration was determined by the method of Bradford [26].

2.3. Electrospray ionization mass spectrometry (ESI-MS) Sulfane sulfur-deprived RhdA (20 lM) was incubated overnight at room temperature with 2 lM IscS and 2 mM L-cysteine in 50 mM Tris–HCl 100 mM NaCl (pH 8). After the incubation, the buﬀer was exchanged with 5 mM ammonium acetate, and 1–3 ll aliquots were di- luted 1:1 with methanol, made 10 lM in formic acid, and applied to nanospray gold-coated glass capillaries placed orthogonally in front of the entrance hole of a QTOF2 mass spectrometer (Micromass, Man- chester, UK). Approximately 1000 V were applied to the capillary and the resulting multiple charge ions were separated by the time-of-ﬂight analyzer. The raw spectra were deconvoluted using the Max.Ent1 software package (Micromass).

2.4. MALDI/TOF peptide mass fingerprinting Protein bands were cut out from a 1D SDS-gel, reduced and carbox- amidomethylated, and then subjected to in-gel tryptic digestion. The resulting peptides were extracted, desalted using ZipTip devices (Milli- pore, Bedford, USA) and analyzed by MALDI/TOF-MS using a Bru- Fig. 1. Analysis of RhdA persulfuration. The figures of extent of ker Ultraflex time-of-flight mass spectrometer (Bruker Daltonics, RhdA persulfuration were calculated for all experiments by the Bremen, Germany). sequential fluorescence measurements depicted in the inset:to6lM RhdA (full line) was added either KCN (0.25 mM; dashed line) or 2.5. Spectroscopic characterization Na2S2O3 (1.5 mM; dotted line). (A) Data from four sets of separated Fluorescence measurements were carried out in a Perkin–Elmer LS- growth experiments with the addition of the indicated sulfur sources 50 instrument, and data were analyzed as previously described [18]. (bars indicate standard deviation). RhdA from cultures grown in the The following formula was used to calculate the extent of RhdA per- presence of the selected sulfur source was purified from cell-free sulfuration: extracts using a batch procedure of nickel-affinity chromatography. The affinity-bound RhdA recovered by elution with 300 mM imidazole F F RhdA-SSH ð%Þ¼ CN 0 100; in 50 mM Tris–HCl, 300 mM NaCl buffer (pH 8) was used for the F CN F THIO fluorescence measurements. (B) 100 lM RhdA in 300 lL 50 mM Tris– HCl, 300 mM NaCl (pH 8) was incubated 2 h at 30 C in the presence where F0 is the intrinsic fluorescence (kexc = 280 nm; kem = 340 nm) of of 2 mM L-cysteine (2), with addition of 50 lgofE. coli (MG1655 the isolated RhdA, FCN and FTHIO the intrinsic fluorescence of the protein after cyanide and thiosulfate addition, respectively. strain) protein extract in the absence (3), and in the presence of 10 lM PLP (4). Untreated RhdA is shown as control (1). Before fluorescence measurements the mixtures were gel-filtered. Bars indicate standard deviation from three independent experiments. 3. Results

3.1. Production of RhdA in the persulfurated form (the sulfane sulfur donor to RhdA in the in vitro catalyzed Heterologous expression of A. vinelandii RhdA in E. coli reaction). in a minimal medium containing either sulfate, L-methionine, We tested whether L-cysteine can act as direct sulfane sulfur L-cysteine and thiosulfate as sulfur source resulted in differ- donor to purified RhdA [24] in the presence and in the absence ent levels of RhdA-SSH (Fig. 1A). The level of RhdA per- of other cellular components. As shown in Fig. 1B, the presence sulfuration was determined by sequential fluorometric of the E. coli soluble extract is required for RhdA persulfura- measurements, as depicted in the inset to Fig. 1A. Among tion in vitro (Fig. 1B; cfr 2 vs. 3). Addition of pyridoxal-50- the sulfur sources tested, L-cysteine was most effective in phosphate (PLP) to the reaction mixture further improved increasing RhdA-SSH production, followed by thiosulfate the yield of RhdA-SSH (Fig. 1B; 4), indicating that the reaction 6788 F. Forlani et al. / FEBS Letters 579 (2005) 6786–6790 was catalyzed by one of the PLP-dependent cysteine desulfurases present in E. coli [16].

3.2. Sulfur transfer from IscS to RhdA At least three different cysteine desulfurases have been identified so far in E. coli, namely IscS, CsdA and SufS [16]. Con- sidering that in the A. vinelandii genome an IscS-like protein (in addition to the NifS protein coded by the nif operon) has been identified and characterized, and that a mutation in iscS is lethal in this organism [27], we focused our attention on the molecular interaction between RhdA and E. coli IscS. E. coli and A. vinelandii IscS show a sequence identity of 74%, and share the same consensus sequence (SSGSACTS) around the catalytic cysteine [16]. To study the sulfur transfer from IscS to RhdA in vitro we developed a time-course fluorescence assay to monitor changes of RhdA intrinsic fluorescence in various reaction mixtures in the presence of 12 mM L-cysteine, a substrate concentration in the range of that used in standard activity assay for cysteine desulfurases [28]. Clear changes of RhdA fluorescence were de- tected in the presence of IscS and L-cysteine (Fig. 2, full line), but not when L-cysteine was omitted (Fig. 2, dashed-dotted line). In this latter case, the RhdA fluorescence decreased only after the addition of thiosulfate. When RhdA was treated with Fig. 3. ESI-MS spectra of RhdA. Molecular mass range of the L-cysteine and bovine serum albumin (BSA), no change in deconvoluted ESI spectra of: RhdA and IscS in the presence of L- fluorescence was observed, and again a decrease in fluores- cysteine (A), RhdA after incubation with 0.1 mM Na2S2O3 (B), sulfane cence was only observed after the addition of thiosulfate sulfur-deprived RhdA (C). The transfer of a sulfur atom to the single (Fig. 2, dotted line). cysteine residue of RhdA can be readily observed by a mass increase of 32 Da characteristic of the persulfide loaded protein. Monobromobimane (mBBr) was used to block the reactive Cys230 in RhdA, thus producing RhdA-mBBr with less than 2% residual thiosulfate:cyanide sulfurtransferase activity. No fluorescence changes were measured on RhdA-mBBr also after incubation with equimolar amounts of persulfurated IscS- the addition of thiosulfate (Fig. 2, dashed line). SSH, without the addition of extra L-cysteine (data not After incubation of RhdA with IscS and L-cysteine for 350 s, shown), ruling out the involvement of non-protein thiols dur- the extent of RhdA persulfuration was determined to be 72%. ing the sulfur transfer process to RhdA. Considering that in our experimental system the acceptor The transfer of sulfane sulfur from IscS to RhdA was con- RhdA was present in a 5-fold molar excess over IscS, it seems firmed by electrospray mass spectrometry (ESI-MS) (Fig. 3). that IscS was able to perform multiple cycles of sulfur transfer. The mass spectra of RhdA upon incubation with IscS in the Eighty percent persulfuration of RhdA was obtained after presence of L-cysteine (Fig. 3A) show a main species at 31083 Da, revealing an increase of 32 Da over the molecular mass of cyanide-treated RhdA (31051 Da, Fig. 3C). The 31083 Da species was also seen after incubation of RhdA with thiosulfate (Fig. 3B). The additional peak approximately 19 Da up mass (Fig. 3A) can be explained by the presence of þ + NH4 and/or Na adducts in the less concentrated sample.

3.3. Inter-protein complex formation upon interaction between IscS and RhdA When equimolar amounts of RhdA and IscS were incubated overnight, and then separated by SDS–PAGE under not- reducing conditions, a protein band of Mr 80000 was observed (Fig. 4, lane 1). Western blot analysis revealed that this protein band was recognized by anti-RhdA antibody (not shown). MS-peptide mapping of the tryptic peptides obtained after Fig. 2. Time-course fluorescence measurements. The intrinsic fluores- in-gel digestion from the gel slice showed that the protein band cence changes (kexc = 280 nm, kem = 340 nm) following the addition of 0.4 lM purified IscS were monitored in 1 ml of 50 mM Tris–HCl, of Mr 80000 was indeed a complex between RhdA and IscS 100 mM NaCl, pH 8, containing: 2 lM sulfane sulfur-deprived RhdA, (data not shown). All major signals obtained after MALDI/ either in the absence (dashed-dotted line) or in the presence of 12 mM TOF fingerprinting could be assigned to peptides originating L-cysteine (full line); 2 lM RhdA-mBBr in the presence of 12 mM L- either from RhdA or IscS, unequivocally demonstrating the cysteine (dashed line). As a control: 0.4 lM BSA (instead of IscS) was presence of a complex of both proteins in the gel band. Forma- added to sulfane sulfur-deprived RhdA in the presence of 12 mM L- tion of the RhdA–IscS complex did not occur when RhdA- cysteine (dotted line). Addition of IscS or BSA is denoted by (*), whereas addition of 0.5 mM Na2S2O3 by (#). mBBr replaced RhdA (Fig. 4, lane 4). The inter-protein F. Forlani et al. / FEBS Letters 579 (2005) 6786–6790 6789

Under these conditions sulfane sulfur loading of RhdA via cysteine desulfurase reaction could be limited, but formation of the hetero-complex indicated an intimate contact between the interacting proteins, representing the first step in the sulfur exchange process. L-cysteine concentrations higher than 0.8 mM likely favor sulfur transfer to RhdA, and the release of RhdA-SSH. The in vivo substrate(s) of RhdA-SSH are thus far not identified, but in vitro investigations demonstrated that RhdA-SSH can function as sulfur carrier to deliver sulfur in an ‘‘activated’’ form for 2Fe–2S cluster synthesis [21]. The present investiga- Fig. 4. SDS–PAGE analysis of RhdA/IscS complex formation. The tion indicates a role of the rhodanese RhdA in assisting elemen- samples, incubated overnight at 25 C and resolved by SDS–PAGE tal sulfur mobilization (mediated by cysteine desulfurase), thus without the use of 2-mercaptoethanol, contained: 15 lM RhdA and 15 lM IscS previously incubated in the absence (lane 1) and in the minimizing the loss of sulfur to cellular reductants [15], and presence of L-cysteine (0.75 mM: lane 6; 1.5 mM: lane 7; 3 mM: lane 8; maintaining, in conditions of high sulfur mobilization, the sul- 6 mM: lane 9; 12 mM: lane 10); RhdA-mBBr and IscS (lane 4). Runs fur in a protected/activated (and non-toxic) form. Although of the individual protein in the absence of L-cysteine are also shown our results were obtained in an ‘‘heterologous system’’, they (lane 2: RhdA; lane 3: IscS; lane 5: RhdA-mBBr). The gel was stained provide novel insights for defining the cellular function of with Coomassie Blue. Interprotein complexes are identified as: ++, rhodaneses, that cannot be limited to cyanide scavenging in (IscS)2; *+, IscS/RhdA complex; **, (RhdA)2; +, IscS; *, RhdA. Mr, denotes the position of the molecular weight standards. view of their broad distribution and of recurrence within the same genome. complex formation was dependent on the L-cysteine concen- Acknowledgements: This work was supported by Consorzio Interuni- tration present during the interaction of RhdA with IscS. As versitario Biotecnologie (to S.P.). The mobility of researchers among shown in Fig. 4 (lanes 6–10), the RhdA–IscS complex was de- laboratories involved in the work was funded by Ministero dellÕUniver- tected only at L-cysteine concentration lower than 0.8 mM. sita` and DAAD in the frame of Programma Vigoni (to S.P. and S.L.). We thank Dr. Elisa Bonacina for the experimental assistance and Prof. Franco Bonomi for helpful comments. 4. Discussion

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