Báo cáo khoa học: Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies potx

Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies Sonia Melino 1 , Daniel O. Cicero 1,2 , Maria Orsale 1 , Fabio Forlani 3 , Silvia Pagani 3 and Maurizio Paci 1,2 1 Dipartimento di Scienze e Tecnologie Chimiche and 2 INFM, Sez. B, University of Rome ‘Tor Vergata’, Italy; 3 Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [ 15 N]Cys, and changes in 1 Hand 15 N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1 H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo. Keywords: 15 N-NMR; Azotobacter vinelandii; rhodanese; selenodiglutathione; sulfurtransferase. The rhodanese from Azotobacter vinelandii (RhdA) is a sulfurtransferase which catalyzes in vitro the production of thiocyanate, transferring the sulfane sulfur atom from thiosulfate to cyanide, by a double displacement mechanism (thiosulfate–cyanide sulfurtransferase, EC 2.8.1.1) [1–3]. The best studied rhodanese is that from bovine liver (Rhobov). Studies on its catalytic mechanism in vitro have shown that, during the transfer of sulfane sulfur from thiosulfate to cyanide, this enzyme cycles between two stable intermediates, a sulfur-loaded (ES) and a sulfur-free form (E). Physical properties of these intermediates have been demonstrated to be different by a variety of solution methods [4–6], but crystallographic data do not appear to show appreciable flexibility in the rhodanese when ES crystals are soaked with cyanide [7,8]. Thermodynamic calculations [9] on the two forms of Rhobov reveal that the ES form is about 8 calÆmol )1 more stable than the E form. It has been suggested that the conformational changes in rho- danese may form the basis of its activity. The physiological role of this class of enzyme is still unclear, but its wide distribution among eukaryotes and prokaryotes suggests that it is involved in essential metabolic pathways. The proposed roles include cyanide detoxification [3], restoration of iron-sulfur centres in Fe-S proteins such as ferredoxin [10,11], and sulfur metabolism [3,12]. It has recently been found to be involved in selenium trafficking [13]; selenium uptake in the persulfide position of the bovine enzyme is achieved by reaction with selenodiglutathione (SDG), the primary metabolite of selenite. The selenium-loaded enzyme (ESe) has been proposed to be the carrier of selenium for selenophosphate synthase. It has been hypothesized that a rhodanese-like enzyme may behave as a transferase for the regulation of selenium concentration in vivo [13]. Recombinant RhdA is one of the most recently expressed prokaryotic enzymes [14], and its 3D structure has been elucidated [8]. In contrast with Rhobov, which has four cysteine residues, RhdA has only one (Cys230), which is the residue involved in the catalytic mechanism. This is a fundamental advantage in the study of rhodanese-like proteins. The interconversion between the ES and E form has been studied by NMR spectroscopy in parallel with fluorescence methods [4]. Selective [ 15 N]Cys labelling of RhdA was performed in order to investigate, by NMR spectroscopy changes in the status of the active site when the enzyme cycles between the two forms. Analysis of high-resolution 1 H-NMR spectra of the ES and E form has revealed some differences that are diagnostic of the two forms. In this work, we propose the use of an alternative method, 1D NMR spectroscopy, to investigate the interconversion between the ES and E form in solution and to monitor the state of the enzyme by addition of substrates or inhibitors. Correspondence to M. Paci, Dipartimento di Scienze e Tecnologie Chimiche, Universita ` di Roma ‘Tor Vergata’, Via della Ricerca Scientifica, 00133-Rome, Italy. Fax: + 39 0672594328, Tel.: + 39 0672594446, E-mail: paci@uniroma2.it Abbreviations: RhdA, rhodanese of Azotobacter vinelandii;E,sulfur- free form of rhodanese; ES, sulfur-loaded form of rhodanese; ESe, selenium-loaded form of rhodanese; HSQC, heteronuclear single quantum coherence; Rhobov, bovine rhodanese; SDG, selenodiglutathione. Enzyme: rhodanese; thiosulfate–cyanide sulfurtransferase (EC 2.8.1.1). (Received 9 July 2003, revised 25 August 2003, accepted 5 September 2003) Eur. J. Biochem. 270, 4208–4215 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03818.x Materials and methods Preparation of the protein The plasmid pQER1 [14], containing the gene coding for RhdA with an N-terminal His-tag, was used to transform the BL-21(DE3)[pREP4] Escherichia coli strain [14], and overexpression of the recombinant protein was induced by addition of isopropyl thio-b- D -galactoside to a mid-expo- nential culture. RhdA was purified by chromatography on a Ni/nitrilotriacetate/agarose column (Qiagen). The His- tagged protein was eluted by addition of 200 m M imidazole and precipitated in 75% saturated ammonium sulfate. The protein concentration was determined using A 0.1% 280 ¼ 1.3 [2], and the molecular mass of 31 kDa was estimated by SDS/PAGE. Rhodanese activity was measured by the discontinuous colorimetric assay described by So ¨ rbo [15]. The presence of the His-tag did not affect enzymatic activity. The sulfur-free form (E) was prepared by adding a 10-fold molar excess of cyanide to ES rhodanese in 50 m M Tris/HCl (pH 7.4)/0.3 M NaCl followed by a 10-min incubation at room temperature. Excess cyanide and thiocyanate were removed by loading the protein solution on to a Centricon-3 (3000 molecular mass cut-off; Amicon). As a control, ES (to which no cyanide was added) was analogously treated. The conversion of ES into E was monitored by measuring the increase in fluorescence quantum yield that accompanies the removal of the persulfide sulfur [4]. Production of [ 15 N]Cys-containing RhdA His-tagged RhdA protein labelled with [ 15 N]Cys was expressed by growing the transformed BL21[pREP4] E. coli strain in medium containing: 2 mgÆmL )1 succinic acid; 0.9 mgÆmL )1 magnesium acetate tetrahydrate; 10 mgÆmL )1 K 2 HPO 4 ;2mgÆmL )1 sodium acetate trihy- drate; 1 mgÆmL )1 ammonium chloride; 0.01 mgÆmL )1 CaCl 2 ; 0.004 mgÆmL )1 FeCl 2 ;0.05mgÆmL )1 nicotinic acid; 0.05 mgÆmL )1 thiamin; 0.1 lgÆmL )1 biotin; 0.125 mgÆmL )1 guanosine, cytosine and uracil; 0.08 mgÆmL )1 thymine; 0.4 mgÆmL )1 L -alanine, L -glutamic acid, L -glutamine, L -arginine and glycine; 0.25 mgÆmL )1 L -aspartic acid; 0.1 mgÆmL )1 L -asparagine, L -histidine, L -isoleucine, L -lysine, L -proline, L -threonine, L -tyrosine and L -valine; 0.25 mgÆmL )1 L -methionine; 1.6 mgÆmL )1 L -serine; 1mgÆmL )1 L -leucine; 0.05 mgÆmL )1 L -tryptophan, L -cys- tine, L -phenylalanine and L -cysteine; 1% glycerol; 0.1 mgÆmL )1 ampicillin; 0.025 mgÆmL )1 kanamycin. A 10-mL volume of BL21[pREP4]/pQER1 culture grown overnight in Luria–Bertani medium was added to 500 mL expression medium (filtered through 0.2-lm nylon filter), and incubated (for 6 h) at 37 °Cinanorbitalshakerto A 600 ¼ 1. The culture was then induced with 1 m M isopropyl thio-b- D -galactoside, followed by a 10-min incu- bation. The cell suspension was harvested by centrifugation, washed twice with 500 mL 0.9 mgÆmL )1 NaCl, and resus- pended in the presence of 1 m M isopropyl thio-b- D - galactoside, in 500 mL of the expression medium in which both cystine and cysteine were replaced by 15 N-labelled cystine (0.2 mgÆmL )1 ) (Isotec, Sigma-Aldrich, UK). After 2.5 h of induction at 37 °C, cells were harvested by centrifugation and stored at )80 °C. The procedure used for purification of the labelled protein was as described above. After purification, the enzyme was assayed as previously described [14]. The procedure used for the expression and purification of the uniformly 15 N-labelled protein will be reported elsewhere. The sample obtained gives well-resolved and intense 15 N-NMR spectra; the results will be reported elsewhere. Preparation of the selenium-substituted rhodanese RhdA in the E form was prepared by adding KCN to the enzyme solution, with a molar ratio of E to KCN of 1 : 10, in 50 m M Tris/HCl buffer, pH 7.4. After the reaction, the protein solution was dialyzed at 4 °C for 12 h. The selenium-loaded RhdA, E–Se, was prepared from the persulfide-free enzyme by reaction with a solution of selenite and glutathione (GSH), in a molar ratio of 1 : 4, respect- ively, in 50 m M Tris/HCl, pH 7.4, containing 1 m M EDTA, as described previously [13]. NMR spectroscopy NMR measurements were performed at an RhdA concen- tration of % 0.1–0.4 m M in 50 m M Tris/HCl (pH 7.25)/ 0.3 M NaCl and at 20 °C on a Bruker AVANCE instru- ment, operating at a proton frequency of 700 MHz equipped with a z-gradient triple resonance probe. Data were processed and analysed on an IRIS O2 work station (Silicon Graphics) using NMRPIPE [16] and NMRVIEW [17]. Fluorescence measurements All fluorescence measurements were made using an LS50 Perkin–Elmer spectrofluorimeter equipped with a thermo- statically controlled stirrer cell holder. The temperature was maintained at 23 °C, and the protein concentration was kept constant at 6 l M . The excitation and emission bandwidths were 5 and 3 nm, respectively. The excitation wavelength was set at 286 nm, and the spectra were recorded from 300 to 400 nm. The changes in fluorescence intensity at 336 nm (F obs ) are given as DF %: DF% ¼ abs½ðF obs À F 0 Þ=F 0 Â100 where F 0 is the original fluorescence intensity of RhdA. ES and E were used as references. Fluorescence meas- urements were made in the presence of different fixed ion concentrations with an enzyme concentration of 6 l M in 50 m M Tris/HCl, pH 7.2. Results and Discussion 15 N labelling of Cys230 of RhdA and 15 N-NMR spectroscopy Incorporation of [ 15 N]Cys in the expression of RhdA protein was estimated to be % 10% from 1 H- 15 N hetero- nuclear single quantum coherence (HSQC) spectra, com- paring the 15 N-filtered spectrum of [ 15 N]Cys-RhdA with the unfiltered enzyme at a fixed delay and also with the 1 H- 15 N HSQC spectra of the uniformly 15 N-labelled RhdA Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4209 (unpublished). The low level of [ 15 N]Cys incorporation is probably due to transamination reactions and to the use of a nonauxotrophic strain of E. coli. In previous fluorescence experiments and crystallographic investigations, native RhdA was prepared as the persulfurated form at Cys230 [8,18]. We observed that RhdA is obtained as a mixture of ES and E. In fact, two different cross-peaks were observed in the 1 H- 15 N HSQC spectrum (Fig. 1A) of the over- expressed [ 15 N]Cys230–RhdA when protein purification was performed without addition of thiosulfate. We evalu- ated that, after purification, % 30% of the overexpressed RhdA was in sulfur-loaded form and 70% in the sulfur-free form (Fig. 1A). The fluorescence and MALDI-TOF data from the same NMR samples confirm the estimate of the ratio between the two forms of RhdA made from NMR (data not shown). Addition of a thiosulfate and cyanide excess, respectively, allowed identification of the 1 H- 15 N correlation peaks corresponding, respectively, to the ES and E form (Fig. 1B,C). In both cases, the samples were dialyzed before the NMR experiments to remove the excess reagents. The observed changes in chemical shift were % 0.26 and 1.0 p.p.m. for 1 Hand 15 N, respectively. Additional analysis of 1 H- 15 N HSQC spectra from a uniformly labelled sample of RhdA showed that about 20 peaks out of the 230 observed signals show different chemical shifts between the two forms of the protein (unpublished). These changes may reflect the conformational changes associated with sulfur loading, probably of residues located near the active site. 1 H-NMR spectroscopy 1 H-NMR spectra were obtained for the ES and E forms. They show a characteristic 1 H resonance and, in particular, differences can be seen in the region typical of the indolyl protons of tryptophan and imidazolyl protons of histidine. Water presaturation was performed before data acquisition, making it difficult to detect the fast exchanging NHs of histidines. Moreover, all resonances in this region showed 15 N resonances at 128–131 p.p.m. (data not shown), typical of the NH group of the side chain of tryptophans. These two observations led us to tentatively assign those reso- nances as belonging to tryptophans. Figure 2 shows the selected regions that are diagnostic of the different state of Fig. 1. 1 H- 15 NNMRHSQCspectraof[ 15 N]Cys230-labelled RhdA. (A) 1 H- 15 NHSQCspectrumof0.2m M [ 15 N]Cys230-labelled RhdA in 50 m M Tris/HCl (pH 7.2)/0.3 M NaCl, after purification at 20 °C. (B) Spectrum of [ 15 N]Cys230-labelled RhdA after treatment with 2 m M thiosulfate (ES form). (C) Spectrum of [ 15 N]Cys230-labelled RhdA after treatment with 2 m M KCN (E form). Fig. 2. Selected regions of 1 H-NMR spectrum of RhdA. The two characteristic 1 H spectra of the ES and E forms of the enzyme are shown in (A) and (B), respectively. The transition between the two forms can be observed as a shift in the peaks (the NH of the indole ring of a Trp, from 11.6 to 12.1 ppm) on conversion of the enzyme from the E to the ES form on addition of thiosulfate ions (E/thiosulfate, 1 : 10) (C). 4210 S. Melino et al.(Eur. J. Biochem. 270) Ó FEBS 2003 the enzyme. The shift in resonance from 11.6 to 12.1 p.p.m. is a sensitive check of the transition from the ES to the E form (Fig. 2A,B). Moreover, during this transition, a high- field shift of the resonance of a methyl group was also observed, i.e. the peak at 0.6 p.p.m. disappeared and a new peak appeared at 1.25 p.p.m. Figure 2C shows the beha- viour of the same methyl resonance upon reconversion of E into ES by addition of thiosulfate. Identification of these resonances requires extensive assignment work, but some educated guesses may be made before the assignment is complete. It has been reported that addition of cyanide in soaking experiments on RhdA crystal results in the removal of the persulfide S atom bound to Cys230, and this reaction induces conformational changes in the Cys230 and Trp195 side chains, which disrupts the Arg235 side chain [8]. Close inspection of the crystal structure reveals that the methyl groups of Leu238 and Leu180 face Arg235 and Trp195, the residues affected by the conformational changes around the active site, thus these are likely candidates to be affected by the change in the persulfurated state of the protein. Previous studies have shown that a number of residues surrounding the catalytic Cys230 are able to generate a strong positive electrostatic field which reaches an estimated value of 18 kTÆe )1 under standard physiological conditions (pH 7.5, ionic strength 0.15 M ) [19]. Therefore we studied the interaction of the active site of RhdA with negative ions by 1 H-NMR spectroscopy, monitoring whether there is transition between the forms of the enzyme after addition of these ions. Figure 3C,D shows the spectrum of the ES form after addition of phosphate and hypophosphite ions. No changes in chemical shift wereobserved up to amolar ratio of RhdA (ES) to ion of 1 : 10 at pH 7.2. The results indicate that, in solution, the catalytic Cys230 residue was not affected by the presence of these ions, up to the concentrations used. Fluorescence experiments As also observed for Rhobov, RhdA shows an intrinsic fluorescence with a maximum at 336 nm, resulting from six tryptophan residues present in the polypeptide chain [4,5,20]. Fluorescence spectroscopy is particularly useful in the study of rhodanese as it can report on modifications of the active site cysteine. In fact, formation of a persulfide group in the active site quenches the intrinsic fluorescence of the protein without affecting its shape (Fig. 4A). This has been attributed to local perturbation or long-range energy transfer [20]. Therefore we carried out a fluores- cence quenching study to monitor the change in confor- mation of RhdA. The results before and after addition of hypophosphite ions are shown in Fig. 4B,C. There was a small effect of quenching on the E form after addition of a very high concentration of hypophosphite ions (Fig. 4C). It is probable that these anions are electrostatically attracted by the positively charged side chains of the residues around the active site and bind in their proximity, influencing the intensity of the fluorescence of the trypto- phan residues and resulting in a fluorescence quenching effect. Fig. 3. Selected regions of the 1 H-NMR spectrum of RhdA of the two forms of the enzyme, ES (A) and E (B), with 1 H-NMR spectra of RhdA (ES) after the addition of phosphate (C) and hypophosphite ions (D). A molar ratio of RhdA (ES) to ion of 1 : 10 in 50 m M Tris/HCl, pH 7.2 was used for (C,D). Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4211 Previous studies also showed that fluorescence changes in RhdA seem to be modulated by phosphate anions, when the protein was purified in phosphate buffer at pH 6.0 [19]. In phosphate buffer, recovery of the intrinsic fluorescence after the addition of KCN, to produce sulfur-free RhdA, was significantly lower than in the presence of Tris/HCl (18% vs. 46%) [19]. The strong positive electrostatic field of the active site may be decreased in intensity by a large excess of phosphate ions, resulting in a decrease in the stability of the persulfide bond. Crystallographic studies of the ES form of RhdA after the addition of 5 m M phosphate or hypophos- phite anions reported that these compounds completely remove the persulfide sulfur atom from Cys230 and in particular the hypophosphite anion was observed in the catalytic pocket. In contrast, no phosphate anions were observed near the active site [19]. An explanation for these different results may be found in the different behaviour of the protein in solution. We used these different experimental conditions, i.e. the molar ratio of ions, pH and incubation times, because our goal was to determine the behaviour of different anions compared with cyanide to evaluate the different affinities for the protein. A large excess of, and long exposure to, phosphate, as used in the previous study [19], may have a different effect on the stability of the S-S bond. Previously characterized rhodaneses, including the bovine liver enzyme [9] and the enzyme from E. coli [21], are typically inhibited reversibly and competitively with respect to thiosulfate by most anions (acetate, sulfate and phos- phate anions) at very high concentration. Our results clearly indicate that the removal of the persulfide group from Cys230 is due to a selective reaction with a sulfane sulfur acceptor, such as cyanide, in conditions close in pH and ionic strength to physiological, and not to the simple presence of anions. However, the limited survey of anions performed in the present study does not allow us to rule out the possibility that low molecular mass mimics of active site groups of normal protein acceptors may also be able to replace cyanide. Reaction of RhdA with SDG Selenium uptake in the persulfide position of RhdA was monitored by fluorescence and NMR spectroscopy after reaction with SDG. This compound was prepared by the reaction of GSH with selenite as previously reported [13] and based on earlier studies [22,23], suggesting that SDG and its subsequent reduction to glutathionyl selenide anion [24,25] are key intermediates in the selenium metabolic pathway. It has been observed that in vitro the labile SDG may react with Rhobov at neutral pH to generate an ESe form [13]. The intrinsic fluorescence of RhdA before and after addition of the GSH/selenite solution leads to the selenium-loaded form of RhdA (ESe) (Fig. 5). In fact, quenching of the intrinsic fluorescence corresponding to DF of 22% at 336 nm was observed after incubation of RhdA (E) with SDG solution [RhdA (E)/SeO3-2/GSH, 1 : 5 : 20] at 37 °C for 10 min, whereas a DF of 11% was observed on addition of a 10-fold molar excess of thiosulfate with the same E form (Fig. 5). No further changes in intrinsic fluorescence of the ESe form were observed after addition of an excess of thiosulfate, confirming the presence of the loaded form of the enzyme (data not shown). On the other hand, fluorescence experiments with the ES form of RhdA showed no changes after addition of SDG. Intrinsic fluorescence was measured by adding selenite or GSH to RhdA [at a molar ratio of RhdA (E) to SeO 3 2– of 1 : 5 and Fig. 4. Fluorescence spectra of RhdA in the presence of anions. (A) The sulfur-loaded state of 6 l M RhdA in 50 m M Tris/HCl, pH 7.2, (solid line), and the sulfur-free form of RhdA (dashed line) with thiosulfate (E/thiosulfate, 1 : 100) (dotted line); (B) ES form (solid line) in the presence of hypophosphite ions with a molar ratio of RhdA to ion of 1:1; 1:10; 1:20;1:50;1:100; (C) E form inthe presence of hypophosphite ions at the molar ratio used in (B). 4212 S. Melino et al.(Eur. J. Biochem. 270) Ó FEBS 2003 RhdA (E) to GSH of 1 : 20], and the quenching (DF) observed was 4% for both (data not shown). This indicates that the labile SDG compound, produced by reaction between selenite and GSH, reacts with RhdA at neutral pH to generate an ESe rhodanese. The quenching of intrinsic fluorescence for selenium loading of RhdA is higher than that observed after treatment with thiosulfate because of the higher quenching properties of selenium than of the persulfide bond. These results are in agreement with those of Cannella et al. [20], who prepared a selenium derivative of Rhobov by using the synthetic substrate selenosulfate and examined its spectroscopic properties. Selenium binding to the protein was also detected by NMR spectroscopy. Experiments on 15 N-Cys-labelled RhdA were performed under the same conditions as for the fluorescence experi- ments. Figure 6 shows the HSQC spectra of the [ 15 N]Cys- labelled RhdA (E) in the presence of GSH/selenite. The spectrum indicates the formation of a new form of the Cys residue. The 1 H- 15 N cross-peak of the catalytic cysteine was shifted ( 15 N, 118.5 p.p.m.; 1 H, 8.56 p.p.m.) upon reaction with the SDG. Moreover, no changes in the 1 Hand 15 N resonances of the [ 15 N]Cys230 were observed after the addition of the selenite/GSH mixture to the sulfur-loaded RhdA (data not shown). Figure 7 shows the 1 H-NMR spectral regions highly indicative of the persulfurated and sulfur-free states of the enzyme after addition of GSH alone, selenite alone, or selenite/GSH equivalent to adding SDG. The 1 H-NMR spectra of RhdA show that no loading occurred on addition of either GSH or selenite alone to the E form (Fig. 7B,C). In contrast, the addition of selenite/GSH to the E form induces changes in the 1 H-NMR resonance pattern similar to those observed by addition of thiosulfate. No specific interactions with the catalytic site of the enzyme were found in presence of the GSH or selenite ions alone, indicating that the loading of selenium to the ESe form occurs by a specific reaction with SDG. These results confirm the hypothesis that rhodanese-like proteins may function as components of the delivery system for reactive selenium in vivo. Prospective studies over the last few years have suggested that Se intake may protect against cancer [26,27]. Several mechanisms have been proposed to explain the anticarci- nogenic effects of Se compounds [26]. One hypothesis is that Se compounds induce apoptosis in initiated premalignant cells, i.e. SDG induces p53 [28]. Furthermore, Ghose et al. [29] recently reported that SDG induces apoptosis in oral cell cultures. Induction of apoptosis has been attributed to SDG because of the observation that it alters the redox status of the cell by manipulating the level of a cellular reducing agent, such as thioredoxin, which has been implicated in growth control in other contexts and is overexpressed in many tumours [30]. In fact, SDG has been shown to be a specific oxidant of reduced thioredoxin and inhibitor of thioredoxin reductase in a cell-free system [31,32]. It has been shown that Rhobov has an affinity that is 1000-fold higher for the reduced form of thioredoxin than Fig. 6. 1 H- 15 N-NMR HSQC spectrum of the selectively [ 15 N]Cys230- labelled RhdA spectrum after addition of SDG. The [ 15 N]Cys ESe was obtained by addition of the SDG solution at 0.1 m M [ 15 N]Cys230- RhdA (E) [RhdA (E)/SeO 3 2– /GSH, 1 : 10 : 40) in 20 m M Tris/HCl/ 0.3 M NaCl, pH 7.4. Fig. 5. Fluorescence changes induced by SDG on RhdA. Sulfur-free form (E) of 5 l M RhdA in 50 m M Tris/HCl (pH 7.4)/1 m M EDTA (solid line); E form after addition of thiosulfate 50 l M (dotted line) or SDG solution (to a final concentration of 25 l M SeO 3 2– and 100 l M GSH) (dashed line). Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4213 for cyanide [33], so it seems reasonable to suppose that SDG may have an indirect effect in vivo on the thioredoxin system through the rhodanese system. Moreover, the rhodanese- like proteins may participate in detoxification of molecules such as thiosulfate, selenite and SDG, raising interest about the biological role of these proteins. Our results show that a simple 1 H-NMR spectrum can be used as a sensitive and fast monitor of sulfur or selenium loading of RhdA. 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Cell Biol. 30, 973–977. Ó FEBS 2003 Rhodanese interaction with ions and selenium loading (Eur. J. Biochem. 270) 4215 . Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies Sonia Melino 1 , Daniel O. Cicero 1,2 , Maria Orsale 1 , Fabio Forlani 3 , Silvia Pagani 3 and Maurizio. addition of thiosulfate 50 l M (dotted line) or SDG solution (to a final concentration of 25 l M SeO 3 2– and 100 l M GSH) (dashed line). Ó FEBS 2003 Rhodanese interaction with ions and selenium loading. Azotobacter vinelandii; rhodanese; selenodiglutathione; sulfurtransferase. The rhodanese from Azotobacter vinelandii (RhdA) is a sulfurtransferase which catalyzes in vitro the production of thiocyanate,