Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus Zhanna Bugaytsova* and E. Bo¨ rje Lindstro¨m Department of Molecular Biology, Umea ˚ University, Sweden The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bac- terium oxidizes elemental sulfur and other reduced inor- ganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N-terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40 °C and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions. Keywords: Acidithiobacillus caldus; tetrathionate hydrolase; protein purification; intracellular protein localization; reduced inorganic sulfur compounds. Acidithiobacillus caldus (formerly Thiobacillus caldus)isa moderately thermophilic bacterium with an optimum growth temperature of 45 °C and optimum pH sensitivity from 2 to 2.5 [1]. A. caldus was enriched originally from acidic water from coal spoil [2]. Since then, A. caldus variants have been detected in diverse locations such as acidic hot springs in Yellowstone National Park (WY, USA), acid mine drainage in Iron Mountain (CA, USA) [3] and exposed pyritic ores in South Africa, Uganda and Greece [4–6]. These bacteria have been found in substantial numbers in leaching bioreactors [4,7–10]. A. caldus differs from other acidithiobacilli by its inability to oxidize ferrous iron and Fe-sulfides. However, these bacteria oxidize elemental sulfur and other reduced inorganic sulfur com- pounds. A. caldus alone does not enhance the oxidative dissolution of sulfide minerals, but it increases the bioleach- ing rate in mixed cultures with iron-oxidizing bacteria [11,12]. A. caldus may minimize the formation of a sulfur layer on mineral surfaces, which otherwise has a passivating effect on the bioleaching by iron-oxidizing bacteria [12]. Physiological traits of A. caldus have been only described briefly [1,13,14] and little is known about the pathway and characteristics of the enzymes involved in the sulfur metabolism. Tetrathionate, S 4 O 6 2– , can be used as the sole energy source for A. caldus and also occurs as a metabolic intermediate in the oxidation of some reduced sulfur compounds [15]. Despite many trials to investigate the tetrathionate metabolism in acidithiobacilli, it is still unknown how the enzymatic decomposition of tetrathio- nate occurs. Hydrolytic enzymatic decomposition of tetra- thionate was proposed by Steudel et al.[16],Hazeuet al. [17] and Meulenberg et al. [18] in experiments with intact cells of A. ferrooxidans and A. acidophilum.Propertiesof purified tetrathionate-decomposing enzymes from A. thio- oxidans [19], A. ferrooxidans [20,21] and A. acidophilum [22] were also consistent with the hydrolysis of tetrathionate. Okuzumi [23] proposed dismutation of tetrathionate to trithionate and pentathionate, while Steudel et al. [16] suggested that the hydrolysis of tetrathionate in A. ferro- oxidans started with the formation of sulfane-monosulfo- nate according to the equation: S 4 O 2À 6 þ H 2 O ! HS 2 SO À 3 þ HSO À 4 ð1Þ The unstable sulfane-monosulfonate was proposed as an intermediate in chain elongation leading to the production of other polythionates (tri-, penta- and hexathionate). HS 2 SO 3 – is further dissociated to S 2 SO 3 2– and both forms chemically react with tetrathionate, leading to the formation of thiosulfate and pentathionate [24]. Meulenberg et al.[18] suggested that tetrathionate hydrolysis formed thiosulfate, sulfur and sulfate in equimolar amounts according to the equation: S 4 O 2À 6 þ H 2 O ! S 2 O 2À 3 þ S  þ SO 2À 4 þ 2H þ ð2Þ De Jong et al. [21,22], working with purified tetrathionate hydrolase from A. acidophilum and A. ferrooxidans, supported this reaction pathway. The inhibitor data of Hallberg et al. [13] were also consistent with this model. Correspondence to E. Bo ¨ rje Lindstro ¨ m, Department of Molecular Biology, Umea ˚ University, S-90187 Umea ˚ ,Sweden. Fax: + 46 90 772630, Tel.: + 46 90 7856750, E-mail: Borje.Lindstrom@molbiol.umu.se Abbreviation: pI, isoelectric point; ddH 2 O, distilled deionized water. *Present address: Department of Odontology, Umea ˚ University, S-90187 Umea ˚ , Sweden. (Received 24 September 2003, revised 23 October 2003, accepted 17 November 2003) Eur. J. Biochem. 271, 272–280 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03926.x In A. thiooxidans [19] and A. ferrooxidans strain Funis 2–1 [20], tetrathionate hydrolase decomposed tetrathionate to thiosulfate and sulfate by disproportionation: 4S 4 O 2À 6 þ 5H 2 O ! 7S 2 O 2À 3 þ 2SO 2À 4 þ 10H þ ð3Þ Previously, the periplasmic localization of tetrathionate decomposing enzymes was demonstrated for A. acidophi- lum, A. ferrooxidans and A. thiooxidans [18,19,21,22]. Hall- berg et al. [13] proposed a model of sulfur metabolism in A. caldus, whereby thiosulfate was oxidized to tetrathionate in the periplasmic space and tetrathionate hydrolysis took place in the cytoplasm or in association with the inside of the cell membrane. The purpose of this work was to purify and characterize tetrathionate hydrolase from A. caldus. The products of the enzymatic reaction were also determined. Materials and methods Bacteria and growth conditions A. caldus strain KU (DSM 8584; ATCC 51756) was grown at 45 °C and pH 2.5 in mineral salts solution that contained (in gÆL )1 ): (NH 4 ) 2 SO 4 ,3.0;Na 2 SO 4 Æ10H 2 O, 3.2; KCl, 0.1; K 2 HPO 4 ,0.05;MgSO 4 Æ7H 2 O, 0.5; Ca(NO 3 ) 2 ,0.01andthe following trace elements (in mgÆL )1 ): FeCl 3 Æ6H 2 O, 11.0; CuSO 4 Æ5H 2 O, 0.5; HBO 3 ,2.0;MnSO 4 ÆH 2 O, 0.8; CoCl 2 Æ 6H 2 O, 0.6 and ZnSO 4 Æ7H 2 O, 0.9. Potassium tetrathionate (5 m M K 2 S 4 O 6 ; Sigma, Switzerland) or 0.5% (w/v) elemen- tal sulfur (flowers of sulfur; Riedel-de Haen, Seelze, Germany) was used as an energy source. Cells were grown aerobically with CO 2 (2%, v/v) enriched air and harvested in the late exponential growth phase (D 440 between 0.240 and 0.250) by centrifugation at 10 000 g at 4 °C. Cells were washed twice with 50 m M formate buffer, pH 3.0. Tetrathionate hydrolase assay The reaction mixture used for the determination of tetra- thionate hydrolase activity contained cell extract and 1 m M S 4 O 6 2– in 50 m M formate buffer, pH 3.0. Two methods were used in this work to estimate the enzyme activity. Continuous assay (qualitative method). This assay was described by de Jong et al. [21] and based on the formation of undetermined intermediates with long sulfur chains that increased absorbance at 290 nm. In this study, the assay was used to monitor tetrathionate hydrolase activity in cell extracts during purification. Enzyme activity was measured at 40 °C in a Shimadzu UV-160 A spectrophotometer (Shimadzu Europa GmbH). Activity of tetrathionate hydrolase was defined as DA 290 min )1 Æmg protein )1 .Empi- rically, the amount of tetrathionate hydrolase activity ensuring the appearance of UV-absorbing intermediates in the buffer without ammonium sulfate was more than 10 times higher than what was used for the cyanolysis or HPLC assays. A discontinuous assay. This assay measures the activity of purified enzyme. The reaction was performed in a thermo- stat-controlled chamber at 40 °C. The reaction was started by addition of protein into 2 mL of preincubated reaction mixture and 500 lL aliquots were taken at 3–10 min intervals. Samples were placed immediately in liquid nitrogen followed by boiling for 3 min to stop the reaction. As the reaction may potentially form elemental sulfur, samples were centrifuged at 14 000 g for 30 min followed by filtration [0.2 lm poly(vinylidene difluoride) membrane, Pall Gelman Laboratory, Ann Arbor, MI, USA]. The amount of tetrathionate was determined either by cyano- lysis [25] or by ion-pair chromatography [26]. The HPLC system included Model LC-5 A pump with dual pistons (Shimadzu, Kyoto, Japan), a Model 7125 injection-valve equipped with 20-lL sample loop (Rheodyne, Cotati, CA, USA), a silica ODS separating column (SPHFR ODS 2, 250 mm · 4.6 mm inner diameter, Jones Chroma- tography, Lakewood, CO, USA) and Kipp & Zonen (Holland) recorder. The HPLC system included a model 9012 Solvent Delivery system and 9100 autosampler (Varian, Walnut Creek, CA, USA), a Spectro Monitor 3100 photometric detector (Milton Roy, Riviera Beech, FL, USA) and VARIAN STAR CHROMATOGRAPHY WORKSHOP , Version 5.3. The flow rate was 0.6 mLÆmin )1 and the UV- detector was set at 230 nm. The mobile phase was acetonitrile/water (20 : 80, v/v) at pH 5.0 and contained 6m M tetrapropylammonium hydroxide. Acetic acid was used to adjust the pH. HPLC-grade acetonitrile and methanol were purchased by Burdick & Jackson (Mushe- gon, MI, USA) and tetrapropylammonium hydroxide from Aldrich Chemie GmbH (Steinheim, Germany). One unit (U) of tetrathionate hydrolase was defined as the amount of protein required for the hydrolysis of 1 lmol of S 4 O 6 2– in 1min. Other enzyme assays Acid phosphatase activity was measured in citrate buffer, pH 4.8 by Acid Phosphatase Kit (Sigma Diagnostic, no. 104, St. Louis, MO, USA) according to the manufacturer’s instruction. The production of p-nitrophenol was measured spectrophotometrically at 405 nm. One unit of the acid phosphatase activity was defined as the amount of protein which catalysed the formation of 1 lmol of p-nitrophenol in 1 min. Glc6P dehydrogenase activity was determined in a reaction mixture containing 100 m M Tris/HCl buffer, pH 7.5, 2 m M Glc6P,5 m M MgCl 2 ,1 m M NADP + (Sigma) and enzyme. The production of NADPH was determined spectrophotometrically at 340 nm. One unit of Glc6P dehydrogenase was defined as the amount of protein that reduced 1 lmol of NADP + in 1 min. Succinate dehydro- genase activity was measured in the reaction mixture containing 0.2 M Na-phosphate buffer (pH 7.5), 0.4 M Na-succinate, 2.5 m M 2,6-dichloroindophenol (Sigma) and enzyme. The reduction of 2,6-dichloroindophenol was followed spectrophotometrically at 600 nm. One unit of succinate dehydrogenase activity was defined as the amount of protein that reduced 1 lmol of 2,6-dichloroindophenol in 1 min. Cellular fractionation Washed cells were sonicated using Vibra Cell (Sonics & Materials Inc., Danbury, CT, USA) and cell debris was Ó FEBS 2003 Tetrathionate hydrolase of A. caldus (Eur. J. Biochem. 271) 273 removed by centrifugation at 10 000 g for 10 min and the activity of the crude cell-free extract in 50 m M formate buffer, pH 3.0 was determined. The cell free extract was then centrifuged at 100 000 g for 60 min to separate the soluble from the membrane bound proteins. The membrane fraction was washed three times with distilled deionized water (ddH 2 O), resuspended in a protein elution buffer and incubated on ice for 30 min. Several protein elution buffers were used: 0.5% (v/v) N-lauroylsarcosine, 1% or 2% (v/v) SDS, 1% (v/v) Triton X-100 in 50 m M formate buffer, pH 3.0; 0.5% (v/v) Triton X-100 in 100 m M Tris, 5 m M EDTA, pH 7.0; and 0.2% (v/v) Tween-20 in 50 m M potassium phosphate buffer, pH 7.0. The suspension was centrifuged at 100 000 g for 60 min and the solubilized proteins were carefully removed and dialysed against 50 m M formate buffer, pH 3.0 or 50 m M phosphate buffer, pH 7.0. The tetrathionate hydrolase activity was determined using HPLC and expressed as percentage activity of cell-free extract. Differential cell fractionation was used to determine the localization of tetrathionate hydrolase. Cells harvested from an exponential phase culture were washed twice with 33 m M Tris/HCl buffer (pH 8.0) and resuspended in 33 m M Tris/ HCl buffer (pH 8.0) containing 0.25 M sucrose, 10 m M EDTA and 1 m M protease inhibitor phenylmethylsulfonyl fluoride (Sigma). Cells were treated with 12.5 lglysozyme per miligram of cells (dry weight) at 37 °C for 2 h followed by centrifugation at 10 000 g for 10 min to remove the spheroplasts. Membrane proteins were separated by cen- trifugation at 100 000 g for 40 min and the supernatant was labelled as the periplasmic fraction. The spheroplasts were dried, resuspended in the original volume of ddH 2 Oand disrupted by sonication. Spheroplast debris was removed by centrifugation at 10 000 g for 5 min. The supernatant was then centrifuged at 100 000 g for 40 min to separate the cytoplasmic (supernatant) from the membrane fraction. Proteins were released from membranes using various protein elution buffers as described above. The fractions were assayed for tetrathionate hydrolase, acid phosphatase (periplasmic marker), Glc6P dehydrogenase (cytoplasmic marker) and succinate dehydrogenase (cytoplasmic mem- brane marker). Purification of tetrathionate hydrolase All purification procedures were performed at 4 °C. Washed cells (6 g of wet weight) were resuspended in 150 mL formate buffer with 1 m M protease inhibitor, phenylmethanesulfonyl fluoride. Cells were disrupted by three passages through a French pressure cell (Aminco, Silver Spring, MD, USA) at 200 MPa. Cell debris and undisrupted cells were removed by centrifugation at 10 000 g for 10 min. After centrifugation at 100 000 g for 40 min, the supernatant was collected as cell free enzyme extract. The cell free extract (140 mL) was dialyzed against 6 L of 50 m M formate buffer, 1.0 M NaCl, pH 3.8 for 16 h. Precipitates were removed by centrifugation at 10 000 g for 10 min. The supernatant (130 mL) containing crude enzyme extract was applied on a SP Sepharose Fast Flow column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (bed volume 60 mL) equilibrated with a 50 m M formate, 1.0 M NaCl buffer, pH 3.8. A linear gradient of NaCl from 1.0–1.5 M in 50 m M formate buffer, pH 3.8, was used for elution of proteins at 2.0 mLÆmin )1 . Fractions (76 mL) containing tetrathionate hydrolyzing activity, measured by continu- ous assay, were pooled and concentrated with Centriprep Centrifugial Filter Devices YM-50 (Millipore, Bedford, MA, USA). The concentrated protein solution was stored at )20 °C until used. Protein analyses Protein concentration was determined by the method of Lowry et al. [27] using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The molecular mass and subunit structure of the tetra- thionate hydrolase were determined using MALDI-TOF MS and a Voyager DE Biospectrometyl Workstation (Applied Biosystems, CA, USA). For N-terminal sequence analysis, the concentrated protein was run on 10% SDS/PAGE. The 55-kDa protein band was transferred to a poly(vinylidene difluoride) membrane (BIO-RAD, Hercules, CA, USA) using a BIO- RAD Trans-Blot system. The protein band was visualized by staining in Ponceau S (Serva, Heidelberg, Germany), followed by excision and N-terminal sequence analysis by Edman degradation. The N-terminal sequence was deter- mined in the Protein Analysis Center, Karolinska Institutet (Stockholm, Sweden). PAGE SDS/PAGE was performed by the Laemmli [28] method using 10% (w/v) resolving and 5% (w/v) stacking gel. Before loading into SDS/PAGE, samples were mixed with SDS-buffer and preincubated at 60 °C for 10 min. Low Molecular Weight Electrophoresis Calibration Kit (Amer- sham Pharmacia Biotech) was used for the determination of molecular mass of tetrathionate hydrolase in SDS/PAGE. Proteins were stained by GelCode Blue Stain Reagent (Pierce, Rockford, IL, USA) and the SilverXpress Silver Staining Kit (Invitrogen) according to the manufacturer’s instructions. Nondenaturing PAGE was performed according to Ausubel et al. [29] using a 10% (w/v) gel and 200 m M acetic acid gel buffer, pH 3.7. Standard SDS/PAGE elec- trode polarity was reversed at the power supply. The gel was stained by GelCode Blue Stain Reagent. Isoelectric focusing was performed according to the method of O’Farrel [30] with an IPGphor (Amersham Pharmacia Biotech) and Hoeffer minigel device (Hoeffer, San Francisco, CA, USA). The first-dimension separation was performed with Immobiline DryStrip and ampholytes (Amersham Pharmacia Biotech) in a pH range of 6–11. The second-dimension separation was performed with 10% (w/v) gels (NuPAGE BiS-Tris System, Invitrogen) according to the manufacturer’s instructions. All materials for 2D electrophoresis were from Amersham Pharmacia Biotech. Isoelectric point (pI) was determined with pI calibration markers (Isoelectric Focusing Calibration Kit, Pharmacia, Little Chalfont, England) according to manu- facturer’s instructions. Proteins were visualized by staining with SilverXpress Silver Staining Kit. 274 Z. Bugaytsova and E. B. Lindstro ¨ m(Eur. J. Biochem. 271) Ó FEBS 2003 Results Differential fractionation and localization of tetrathionate hydrolase Based on studies with whole cells and metabolic inhibitors, tetrathionate hydrolase in A. caldus has been suggested to be localized in the cytoplasm or associated with the inner membrane [13]. However, in the work presented here 92% of tetrathionate hydrolase activity was located to the soluble fraction. Further fractionation to the periplasmic, cytoplas- mic, and membrane fractions revealed that tetrathionate hydrolase (96%) and acid phosphatase (70%) were associ- ated with the periplasmic fraction, while most of the Glc6P dehydrogenase (66%) was in the cytoplasm (Table 1). Succinate dehydrogenase activity was detected in the inner membrane fraction. These data indicated that the fraction- ation procedure was satisfactory and that the tetrathionate hydrolase is a periplasmic enzyme. Purification of the tetrathionate hydrolase from tetrathionate-grown A. caldus After cell lysis and dialysis, the crude enzyme extract was fractionated on a SP Sepharose Fast Flow column using a linear gradient from 1.0–1.5 M NaCl in 50 m M formate buffer, pH 3.8. Eluted fractions formed three major absorption peaks at 280 nm (data not shown). Tetrathio- nate hydrolase activity was found in fractions eluted at about 1.25 M NaCl. The active fractions were pooled, concentrated by Centriprep Centrifugal Filter YM-3, and resolved by SDS/PAGE. One major band with a molecular mass of 54 kDa and a few faint bands with molecular mass < 40 kDa were also detected. To remove the low molecular mass impurities, the concentration step used a Centriprep Centrifugal Filter YM-50. The remaining protein solution demonstrated a 17-fold purification and about 8% recovery of the enzyme activity (Table 2). This active enzyme preparation showed only one band when stained by GelCode Blue Stain Reagent and SilverXpress Silver Staining Kit in SDS gel electrophoresis (Fig. 1). Isoelectric focusing in the 2D gel detected a single spot and the pI of the tetrathionate hydrolase was 9.8. The molecular mass of the purified tetrathionate hydrolase was determined by SDS electrophoresis and specified by MALDI-TOF MS. The enzyme was a homodimer with the molecular mass of 103 kDa, consisting of two mono- mers of 52 kDa. The N-terminal sequence of tetrathionate hydrolase was determined as Gly-Ile-Thr-Pro-Val-Leu-Glu-Pro-Gly- Asn-Pro-Phe-Asp-Pro-Asp. When aligned with the partial genome available for A. ferrooxidans ATCC 23270 (http://www.tigr.org) no homology was found. Search of tetrathionate hydrolase against the protein databases of National Center for Biotechnology Information (NCBI), using the BLAST search program [31], demon- strated that this enzyme is not yet annotated and no significant similarities were revealed for any proteins in the database. Tetrathionate hydrolase activity in sulfur-grown A. caldus A. caldus was also grown for 3 days in liquid medium with elemental sulfur as the energy source to investigate if tertrathionate hydrolase was constitutive. The cells were harvested, filtered free from sulfur particles and disrupted using a French press. Tetrathionate hydrolase activity was not detected in the crude enzyme extract or in fractions eluted from the SP Sepharose Fast Flow column. In this case, the protein concentration in fractions eluted at 1.25 M NaCl was much lower when compared to the tetrathionate- grown cells (data not shown). Tetrathionate hydrolase was not detected in any of the fractions concentrated by Centriprep Centrifugal Filter YM-3. Table 1. Localization of tetrathionate hydrolase in A. caldus cell fractions. Distribution and activity of assayed enzymes are given as a percentage of the total protein concentration and total activity in the cell free extract. Tetrathionate hydrolase activity in fractions was measured by HPLC. Cell fraction Protein (%) Activity (%) Acid phosphatase Glc6P dehydrogenase Succinate dehydrogenase Tetrathionate hydrolase Periplasm 29.5 ± 2.1 69.6 ± 6.4 a 33.5 ± 3.5 0 96.4 ± 5.1 Cytoplasm 63 ± 5.6 30.5 ± 6.4 66.5 ± 3.5 0 4 ± 1.5 Membrane 8.5 ± 3.5 0 0 100 0 a Specific activity (mean ± SD) from two independent experiments. Table 2. Purification of tetrathionate hydrolase from A. caldus. One unit (U) is defined as the amount of tetrathionate hydrolase required for the conversion of 1 lmol tetrathionate in 1 min. Enzyme fraction Total protein (mg) Specific activity (UÆmg protein )1 ) Total activity (U) Fold Yield (%) Cell free extract 176.4 0.14 24.7 1 100 Crude extract 84 0.24 20.2 1.7 82 SP Sepharose a 0.84 2.34 2.0 16.7 8 a Protein solution obtained after SP Sepharose was concentrated by Centriprep Centrifugal Filter Devices YM-50. Ó FEBS 2003 Tetrathionate hydrolase of A. caldus (Eur. J. Biochem. 271) 275 Chemical stability of potassium tetrathionate the substrate of tetrathionate hydrolase To quantitate the substrate using the HPLC method, the effect of pH, temperature and ammonium sulfate in the reaction mixture was investigated. Four buffers between pH 2.0 and 8.5 at 20 °C were used to test the effect of pH. As shown in the HPLC chromatogram (Fig. 2) at pH 3.0, a single peak of tetrathionate was eluted at 9 min. At pH 7.5, several additional peaks were detected with shorter retention times. Parallel assays based on cyanolysis revealed no decrease in the concentration of tetrathionate in these samples, demonstrating that the level of detection in the cyanolytic assay was far inferior when compared to HPLC- based detection and did not reveal the loss of tetrathionate under neutral pH. The influence of temperature on the stability of 1 m M tetrathionate was also examined in 50 m M formate buffer, pH 3.0 and in ddH 2 O for 30 min. Tetrathionate was stable under these conditions at temperatures of up to 80 °C(data not shown). The interference of ammonium sulfate with the HPLC- assay of tetrathionate was also tested. The height of the tetrathionate peak decreased and a shoulder appeared in the HPLC-chromatograms as the ammonium sulfate concen- tration increased from 0–2 M (Fig. 3). Ammonium sulfate was excluded from the purification protocol of the enzyme for final characterization. Biochemical properties of tetrathionate hydrolase The enzymatic activity was tested with regard to pH (Fig. 4), temperature (Fig. 5), and the influence of Cu 2+ ions (Table 3). Maximum activity was found at pH 3 and between 40 and 45 °C. Copper at 0.01 m M greatly stimu- lated the activity. The presence of Fe 2+ ,Mn 2+ and Zn 2+ also stimulated tetrathionate hydrolase activity whereas Ca 2+ and Mg 2+ (data not shown) were slightly inhibitory. Products of tetrathionate hydrolysis by tetrathionate hydrolase The HPLC-chromatograms of the enzymatic reaction mixture (without ammonium sulfate) demonstrated the presence of three peaks. In addition to the tetrathionate peak eluted at 9 min, thiosulfate and pentathionate were detected as reaction products (Fig. 6). The pentathionate peak position was identical to that described by Miura & Kawaoi [26] in their analytical study using authentic standards. Elemental sulfur was not detected in the reaction mixture using the analytical method of Hazeu et al. [17]. Sulfate was detected in the reaction mixture qualitatively with BaCl 2 [32]. Effect of ammonium sulfate on tetrathionate hydrolase activity Tetrathionate hydrolase activity was determined in the presence of 2 M ammonium sulfate. While the tetrathionate peak (retention time 9 min) decreased, there was no Fig. 1. SDS/PAGE of tetrathionate hydrolase from A. caldus. Elec- trophoresis was carried out on a 10% polyacrylamide gel. Lane 1: molecular mass markers; lane 2: cell-free extract; lane 3: crude enzyme extract; lane 4: purified tetrathionate hydrolase after SP Sepharose and concentration by Centriprep Centrifugal Filter Device YM-50. The bands were stained by GelCode Blue Stain Reagent. Lane 5: purified tetrathionate hydrolase stained with SilverXpress Silver Staining Kit. Fig. 2. HPLC analysis of potassium tetrathionate stability at different pH. Potassium tetrathionate (1 m M )wasaddedto50m M formate buffer, pH 3.0 (solid line) and 50 m M phosphate buffer, pH 7.5 (dashed line). The retention time of tetrathionate was 9 min. 276 Z. Bugaytsova and E. B. Lindstro ¨ m(Eur. J. Biochem. 271) Ó FEBS 2003 evidence for the formation of thiosulfate and pentathionate (data not shown). Maximum activity of the enzyme determined by cyanolysis was observed at 1.5 M of ammo- nium sulfate concentration (Fig. 7). Discussion The principal aim of this investigation was to localize tetrathionate hydrolase in the cell, purify it and characterize its properties. Tetrathionate-grown A. caldus has been suggested to use a membrane-associated cytoplasmic hydrolase in its energy metabolism [13]. The result of the present study showed that tetrathionate hydrolase was soluble and the activity was associated with the periplasmic fraction. Tetrathionate hydrolase activity was 10-fold higher in several buffers with acidic pH ranging from 2.0–3.5 than in the buffers with neutral pH. No activity was observed above pH 6 (data not shown). The pH optimum of 3.0 is in a good agreement with the low pH of the periplasmic space [14]. The pI of the enzyme was 9.8, indicating that tetrathionate hydrolase is a basic protein as also reported by Tano et al.[19]forA. thiooxidans. The excess of positive charges of tetrathionate hydrolase may result from the neutralization of the overall negative charge in the periplasm of the acidophilic A. caldus cells. As in A. caldus,tetra- thionate hydrolase is periplasmic also in A. thiooxidans [19], A. ferrooxidans [21] and A. acidophilum [22]. Periplasmic localization contradicts the model for the metabolism of reduced inorganic sulfur compounds previously proposed for A. caldus by Hallberg et al.[13]. Due to the instability of tetrathionate in the presence of ammonium sulfate, as demonstrated by the HPLC chro- matograms, ammonium sulfate was not used in the enzyme Fig. 3. HPLC analysis of potassium tetrathionate stability in the pres- ence of ammonium sulfate. Potassium tetrathionate (1 m M )wasadded to 50 m M formate buffer, pH 3.0 that contained 0 M (solid line) and 2 M ammonium sulfate (dashed line). Fig. 4. pH profile of tetrathionate hydrolase from A. caldus. Purified tetrathionate hydrolase was preincubated for 15 min at 40 °Cintest buffers before addition of 1 m M tetrathionate. The enzyme activity was estimated by cyanolysis. Relative activities are presented as percentage of the maximum activity. The vertical bars indicate standard devia- tions. j,phospatebuffer;d, formate buffer; m, succinate buffer. Fig. 5. Temperature profile of tetrathionate hydrolase from A. caldus. Theassaymixture(50 m M formate buffer, pH 3.0 and 1 m M potassium tetrathionate) was preincubated at indicated temperatures for 10 min before the addition of the enzyme. The initial enzyme activity was estimated by cyanolysis. Relative activities are presented as percentage of the maximum activity. The vertical bars indicate standard deviations. Ó FEBS 2003 Tetrathionate hydrolase of A. caldus (Eur. J. Biochem. 271) 277 purification procedure. The present study is the first to demonstrate the purification of tetrathionate hydrolase without ammonium sulfate in the buffers. Dialysis of the crude enzyme extract precipitated about 20% of the total protein, probably because hydrophobic proteins aggregated in the presence of the high NaCl concentration (1 M ). Most of the remaining proteins in the extract did not bind to the cation exchange resin in the presence of NaCl and tetrathionate hydrolase was then eluted at 1.25 M NaCl. The high positive charge of the enzyme (pI 9.8) caused a strong binding to the cation resin and a rather high salt concentration was needed to displace the protein again. The final purification of the tetrathionate hydrolase from A. caldus to homogeneity was performed using one chromatographic step. The relatively low purification yield of the enzyme may be due to several reasons. Copper and several other divalent ions stimulated the activity of the tetrathionate hydrolase and they and other cofactors may be present in the crude enzyme extract. They may help stabilize the protein but were removed during the purification steps, therefore, causing the low purification yield. Another reason for such a low recovery may be the loss of the enzymatic activity during dialysis, chromatography and concentration of protein which were carried out at 4 °C. Such effects have been demonstrated for other proteins and have been suggested to be due to unfolding of native protein structures at low temperatures [33,34]. Change in the tertiary structure at 4 °C could be significant for a protein with temperature optimum at around 40 °Corhigher. Table 3. Effect of copper on the activity of the tetrathionate hydrolase from A. caldus. Tetrathionate hydrolase was preincubated with copper for 30 min at 40 °C before addition of 1 m M tetrathionate. Specific activity (mean ± SD) from two independent experiments was meas- ured by HPLC using standard conditions described in the text. Compound Concentration (m M ) Specific activity (UÆmg protein )1 ) Activation (%) None – 0.7 ± 0.1 – CuCl 2 0.01 1.1 ± 0.1 157 0.1 2.2 ± 0.2 314 1.0 8.3 ± 1.7 1186 CuSO 4 0.01 0.9 ± 0.2 128 0.1 6.6 ± 0.3 942 1.0 9.3 ± 1.3 1328 Fig. 6. HPLC analysis of tetrathionate hydrolase products from A. caldus. Theassaymixture(50 m M formate buffer, pH 3.0 and 1 m M tetrathionate) was preincubated at 40°C for 10 min before addition of the enzyme. Products were analyzed immediately following the enzyme addition (solid line) and after 100 min incubation (dashed line). Determined peaks: 1, thiosulfate; 2, tetrathionate; 3, pentathionate. Fig. 7. Influence of ammonium sulfate on tetrathionate hydrolase activity. Tetrathionate hydrolase was added to 50 m M formate buffer, pH 3.0, containing ammonium sulfate. The mixture was preincubated for 10 min at 40 °C before starting the reaction with 1 m M tetra- thionate. Activities were determined by cyanolysis. Relative activities are presented as percentage of the maximum activity. Table 4. Properties of tetrathionate hydrolases from some bioleaching microorganisms. ND, not detected. Species Molecular mass (kDa) (number and mass of subunits) Optimal conditions for activity Localization pI ReferencepH Temperature (°C) A. thiooxidans, ON107 104 (2 · 58) 3.0–3.5 40 periplasm 9.68 [19] A. ferrooxidans, Funis-2 strain 49.6 (1 · 49.6) 3.5 50 plasma membrane ND [20] A. ferrooxidans, ATCC 19859 105 (2 · 52) 4.0 56 periplasm ND [21] A. acidophilum, DSM 700 100 (2 · 48) 2.5 65 periplasm ND [22] A. caldus, KU (ATCC 51756) 103 (2 · 52) 3.0 40 periplasm 9.8 This study 278 Z. Bugaytsova and E. B. Lindstro ¨ m(Eur. J. Biochem. 271) Ó FEBS 2003 The biochemical properties of tetrathionate hydrolase of A. caldus were similar to those of other acidithiobacilli (Table 4). The specific activity of tetrathionate hydrolase from A. caldus (2.3 UÆmg protein )1 ) is comparable with the activities for A. ferrooxidans (1.4 UÆmg protein )1 [22]), A. ferrooxidans, Funis-2 strain (1.6 UÆmg protein )1 [20]), and A. thiooxidans (4.8 UÆmg protein )1 [19]). The pH optimum of tetrathionate hydrolase from A. caldus is in general agreement with those previously determined for A. thiooxidans, A. ferrooxidans and A. acidophilum [18–22]. The temperature optimum of tetrathionate hydrolase is consistent with its moderately thermophilic character. However, several other enzymes from mesophiles have been characterized as thermotolerant with temperature optima in the range 50–65 °C (Table 4). The purified enzyme was a homodimer with a molecular mass of 103 kDa as deter- mined with MALDI-TOF MS. Except for tetrathionate hydrolase from A. ferrooxidans strain Funis-2 [20] all other tetrahionate hydrolases have been described as homodimers with a similar molecular mass. The N-terminal sequence of the tetrathionate hydrolase enzyme was determined for the first time in the present work. The stimulation of the tetrathionate hydrolase activity by sulfate ions has been demonstrated previously for A. thio- oxidans [19], A. ferrooxidans Funis 2–1 [20], A. ferrooxidans [21] and A. acidophilum [22]. In the case of A. ferrooxidans [21] and A. acidophilum [22], the activity of the tetrathionate hydrolase was so low that buffers containing 2 M ammo- nium sulfate were used during the whole purification procedures. It was demonstrated that sulfate ions could be replaced by selenate ions [20]. The stimulation by sulfate ions was also reported for trithionate hydrolase, another periplasmic enzyme involved in sulfur metabolism of acidithiobacilli [35,36]. Meulenberg et al.[36]suggested two reasons for the stimulatory effect. First, the high concentration of ammonium sulfate prevented the enzyme from precipitating under low ionic strength conditions. Second, the enzyme needed a high sulfate concentration for its activity. In the present work, precipitation of the enzyme at low ionic strength was not observed. HPLC analysis of reaction products demonstrated the presence of thiosulfate and pentathionate. In further work, the stoichiometry of the reaction needs to be determined. Neither trithionate nor hexathionate was detected. Efforts to determine elemental sulfur following the method of Hazeu et al. [17] were not successful possibly because the concentration of S° was below the level of detection. Sulfate formation was qualitatively detected with barium chloride method [32]. HPLC analysis of the products of tetrathionate hydrolysis revealed a different pathway in presence of ammonium sulfate: neither thiosulfate nor pentathionate was detected in the presence of 1.5 M ammonium sulfate and elemental sulfur was formed in the reaction mixture. While the proper stoichiometry of tetrathionate hydrolase reaction from A. caldus requires further studies, it may be expressed in light of the present study as: 2S 4 O 2À 6 þ H 2 O ! S 2 O 2À 3 þ S 5 O 2À 6 þ SO 2À 4 þ 2H þ ð4Þ Tetrathionate hydrolysis in A. caldus as in other acidithio- bacilli yields mixed products, and chemical interaction between thiosulfate, polythionates and possibly sulfur makes it extremely difficult to assign a specific role for the enzyme in the sulfur oxidation pathway. The 14 amino acid sequence of the N-terminus may be used to design degenerative primers for amplifying the gene(s) for this enzyme for cloning and molecular characterization. Acknowledgements This work was supported by Teknikbrostiftelsen i Umea ˚ ,Umea ˚ , Sweden. The authors thank Olli H. Tuovinen for the fruitful critical reading of the article. 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Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus Zhanna Bugaytsova* and E. Bo¨ rje Lindstro¨m Department. profile of tetrathionate hydrolase from A. caldus. Theassaymixture(50 m M formate buffer, pH 3.0 and 1 m M potassium tetrathionate) was preincubated at indicated