Báo cáo khóa học: Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus pot

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:
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
() 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. J.B. thanks Mark Dopson and Siv Sa
¨
a
¨
ffor
practical help, Arunas Leipus for help in protein chromatography and
Henrik Larsson for helpful assistance with HPLC.
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