Báo cáo khoa học: Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress pdf

Identification and characterization of 1-Cys peroxiredoxin
from Sulfolobus solfataricus and its involvement in the
response to oxidative stress
Danila Limauro
1
, Emilia Pedone
2
, Luciano Pirone
2
and Simonetta Bartolucci
1
1 Dipartimento Biologia Strutturale e Funzionale, University of Naples ‘Federico II’, Complesso Universitario Monte S. Angelo, Naples,
Italy
2 Istituto di Biostrutture e Bioimmagini, C.N.R., Naples, Italy
Reactive oxygen species (ROS) are either generated
by incomplete oxygen reduction during respiration, or
by exposure to environmental factors such as light,
radiation or increased oxygen pressure. ROS, notably,
superoxide (O
2
•
)
) and hydroxyl radical (OH
•
), hydro-
gen peroxide (H
2
O
2
) and singlet oxygen (
1

O
2
) cause
damage to all major classes of biological macromole-
cules, leading to protein oxidation, lipid peroxidation,
DNA base modifications and strand breaks [1]. In
order to protect against toxic ROS, aerobic organisms
are equipped with a full array of defence mechanisms,
among which are antioxidative enzymes and antioxid-
ant molecules (e.g. superoxide dismutases, catalases,
peroxidases, thioredoxins, and glutathione) which are
developed by most cells [2–4]. Most aerobes have
multiple enzymes with overlapping ROS detoxification
pathways but different expression and regulation
times. For example, many bacteria induce catalase or
other protective enzyme expressions during the trans-
ition from the exponential to the stationary growth
phase, presumably as an adaptation to protect the
genome and other cellular components against oxida-
tion during a prolonged nongrowth phase. In recent
years, much attention has been given to peroxiredox-
ins (Prxs) [5–7], a new family of thiol-specific anti-
oxidant proteins. These include alkyl hydroperoxide
Keywords
Archaea; Sulfolobus solfataricus; oxidative
stress; ROS; peroxiredoxin
Correspondence
S. Bartolucci, Dipartimento di Biologia
Strutturale e Funzionale, Complesso
Universitario di Monte S. Angelo, Universita

`
di Napoli ‘Federico II’, Via Cinthia,
80126 Naples, Italy
Fax: +39 081679053
Tel: +39 081679052
E-mail:
(Received 9 November 2005, revised
14 December 2005, accepted 16 December
2005)
doi:10.1111/j.1742-4658.2006.05104.x
Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome data-
base of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Its
role in oxidative stress was investigated by transcriptional analysis of RNA
isolated from cultures that had been stressed with various oxidant agents.
Its specific involvement was confirmed by a considerable increase in the
bcp2 transcript following induction with H
2
O
2
. The 5¢ end of the transcript
was mapped by primer extension analysis and the promoter region was
characterized. bcp2 was cloned and expressed in Escherichia coli, the
recombinant enzyme was purified and the predicted molecular mass was
confirmed. Using dithiothreitol as an electron donor, this enzyme acts as a
catalyst in H
2
O
2
reduction and protects plasmid DNA from nicking by
the metal-catalysed oxidation system. Western blot analysis revealed that

the Bpc2 expression was induced as a cellular adaptation in response to the
addition of exogenous stressors. The results obtained indicate that Bcp2
plays an important role in the peroxide-scavaging system in S. solfataricus.
Mutagenesis studies have shown that the only cysteine, Cys
49
, present in
the Bcp2 sequence, is involved in the catalysis. Lastly, the presence of this
Cys in the sequence confirms that Bcp2 is the first archaeal 1-Cysteine per-
oxiredoxin (1-Cys Prx) so far identified.
Abbreviations
Bcp, Bacterioferritin comigratory protein; LB, Luria–Bertani; MCO, metal catalysed oxidation; Prx, peroxiredoxin; ROS, reactive oxygen
species.
FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 721
reductase, thiol-specific peroxidase, and bacterioferr-
itin comigratory protein (Bcp), which perform their
protective role in cells through antioxidant activity
(ROOH +2e
–
fiROH + H
2
O), i.e. by reducing and
detoxifying H
2
O
2
, peroxynitrite and numerous organic
hydroperoxides. They are ubiquitous enzymes found
in every domain of life such as eukarya, bacteria and
archaea. Prxs use a redox-active cysteine to reduce
peroxides. Based on the number of cysteinyl residues

involved in catalysis they can be divided into two
groups, 1-Cys and 2-Cys Prxs [8]. Structural and
mechanistic data actually support the division of
2-Cys Prxs into two subclasses, ‘typical’ and ‘atypical’
2-Cys Prxs. All three Prxs classes are found to have
in common an initial catalytic step, at which peroxid-
atic cysteine (Cys-S
p
H), generally near residue 50,
attacks the peroxide substrate and is oxidized to cys-
teine sulfenic acid (Cys-SOH). The second step of the
peroxidase reaction, the resolution of cysteine sulfenic
acid, varies according to the class considered. The
Cys-SOH of 1-Cys Prx, is presumably reduced by a
thiol-containing electron donor, although the physio-
logical partners have not ever been identified [9]. The
main characteristic of 2-Cys Prxs, the largest Prx
class, is their second redox-active cysteine, the resol-
ving cysteine (Cys-S
R
). In typical 2-Cys, S
p
at the N
terminus and S
R
at the C terminus of the protein
belong to different subunits and condense to form an
intersubunit disulfide bond; in atypical 2-Cys Prxs, S
p
and S

R
belong to the same subunit and establish an
intrasubunit disulfide bond. The successive reduction
of 2-Cys Prxs involves a flavoprotein disulfide reduc-
tase and at least one additional protein or domain
with a CXXC motif, which is oxidized from the di-
thiol to the disulfide state during Prx reduction (e.g.
thioredoxin reductase and thioredoxin) (Fig. 1).
Two archaeal Prxs from Aeropyrum pernix APE2278
[10] and Pyrococcus horikoshii PH1217 [11,12] have
recently been characterized and subsumed within the
2-Cys family. APE2278 was found to have a hexadeca-
meric structure and it showed the ability to reduce
H
2
O
2
using the NADPH ⁄ thioredoxin reductase ⁄ thio-
redoxin system as electron donor partner [13]. Also
PH1217 has peroxidase activity but the electron donor
partner might be different from that found in A. pernix
because in the genome data base of P. horikoshii nei-
ther a homologue of A. pernix thioredoxin-like nor
other types of thioredoxins were found.
In this study we examined the involvement of the
peroxiredoxin Bcp2 in oxidative stress in the hyper-
thermophilic aerobic archaeon Sulfolobus solfataricus.
Furthermore, we report the cloning, the expression
and the characterization of the recombinant protein
rBcp2 in order to shed light on its role in the detoxifi-

cation process and on its catalytic mechanism.
Results
Identification of the bcp2 gene encoding putative
peroxiredoxin
The analysis of the complete sequenced genome of
S. solfataricus P2 [14] ( />projects/sulfolobus/) revealed four ORFs homologues
of Prxs and annotated as Bcp1 (SSO2071), Bcp2
(SSO2121), Bcp3 (SSO225) and Bcp4 (SSO2613). The
S. solfataricus Bcp which bears the greatest similarity
to other Prxs in the GenBank Database is Bcp2, which
encodes a putative protein of 215 amino acids with a
predicted molecular mass of 24744.79 Da and a theor-
etical pI of 6.85. The deduced amino acid sequence
shows 61% identity with the archaeal Prx (APE2278)
from the aerobic hyperthermophilic archaeon A. pernix
[10], 61% with the putative bacterial Prx (Q9WZR4)
derived from the hyperthermophilic bacterium Thermo-
toga maritima, 57% with the Prx (PH1217) from the
XH
2
X + H
2
O
1-Cys Prx
S
p
OH
1-Cys Prx
S
p

H
H
2
O
H
2
O
2
RSH
Flavoprotein disulfide
reductase
RSSR
2-Cys Prx
S
p
2-Cys Prx
S
p
H
H
2
O
H
2
O
2
S
R
H
2

O
2-Cys Prx
S
p
OH
S
R
H
B
S
R
H
A
Fig. 1. Peroxiredoxin mechanisms. (A) 1-Cys
Prx. (B) 2-Cys Prx. S
p
peroxidatic cysteine;
X unidentified electron donor; S
R
resolving
cysteine; RSH protein or domain with CXXC
motif (e.g. thioredoxin). In typical 2-Cys, S
p
at the N terminus and S
R
at the C terminus
belong to different subunits and condense
to form an intersubunit disulfide bond; in
atypical 2-Cys, S
p

at the N terminus and S
R
at C terminus, originate from the same
subunit.
Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al.
722 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS
anaerobic hyperthermophilic archaeon P. horikoshii
[11,12], all of which belong to the 2-Cys Prx family; in
addition, Bcp2 reveals 40% of identity with 1-Cys
Human PRDX6 (Fig. 2).
Following primary structure analysis, Bcp2 was clas-
sified as a 1-Cys Prx with only one conserved cysteine
residue (Cys
49
) in a consensus surrounding sequence
DFTPVCTTE which is also found both in prokaryotic
and eukaryotic Prxs. It is the first of all Prxs analysed
so far in archaea that has only one cysteine residue in
the sequence.
Transcriptional analysis of bcp2 under oxidative
stress and characterization of mRNA 5¢ end
In order to understand the involvement of bcp2 in oxi-
dative stress, the levels of bcp2 mRNA were assessed
after treatment of S. solfataricus cells with paraquat,
which was used to generate O
2
•
)
, with H
2

O
2
and tert-
butyl hydroperoxide as direct oxidants [15]. To estab-
lish the concentrations of agents whose effect can be
to slow down or otherwise affect growth, in the expo-
nential phase the cells were treated with varying
amounts of stressors (data not shown). Therefore, the
S. solfataricus P2 strain was grown until the early
exponential phase (0.3 OD
600 nm
) and then induced
with 0.05 mm H
2
O
2
, 0.1 mm paraquat or 0.05 mm tert-
butyl hydroperoxide for different periods of time
(Fig. 3). As shown, the addition of stressors to the cul-
tures inhibits growth without killing the cells.
The hybridizing band in the northern analysis
showed the expected size of about 680 bp indicating
that the gene is transcribed as a monocistronic mRNA.
When S. solfataricus cells were incubated with H
2
O
2
,
paraquat and tert-butyl hydroperoxide, the bcp2
Fig. 2. Multiple sequence alignment (CLUSTAL

W
1.82) of Bcp2 from S. solfataricus and
Prxs from A. pernix (APE2278), T. maritima
(Q9WZR4), P. horikoshii (PH1217), and
human (PRDX6).
0
0,1
0,2
0,3
0,4
0,5
0,6
0246810
Time (h)
OD
m
n
0
0
6
Fig. 3. S. solfataricus P2 cultures treated with different oxidative
stress agents. Sulfolobus solfataricus P2 cultures were grown until
0.3 OD
600 nm
then the cultures were treated with 0.1 mM paraquat
(n), 0.05 m
M H
2
O
2

(m), 0.05 mM tert-butyl hydroperoxide(*), or con-
trol (r). The arrow indicates the OD
600 nm
value at which the anti-
oxidant agents were added.
D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus
FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 723
mRNA levels increased considerably (Fig. 4A, B and C ),
i.e. a 10-fold increase in transcriptional levels was
observed 15 min after the addition of H
2
O
2
, and a
fourfold increase within 30 min after paraquat treat-
ment; when the tert-butyl hydroperoxide was used, the
induction observed was less marked. To evaluate bcp2
expression in response to growth phases, the RNA
obtained from cultures harvested at 0.3, 0.6 and
1.0 OD
600 nm
corresponding to early, mid and station-
ary growth phases was analysed. The data obtained
suggest that bcp2 transcriptional levels were independ-
ent of the growth phase (Fig. 5).
Primer extension analysis was performed in order to
characterize the promoter region (Fig. 6). Figure 6B
shows the nucleotide sequence 5¢ of the upstream regu-
latory region of the bcp2 gene. A consensus sequence,
GGUG, with Shine–Dalgarno motifs of the Sulfolobus

species was observed upstream of the ATG start codon
[16]. The 5¢ end of the bcp2 transcript begins with
an adenine and maps 10 nucleotides upstream of the
ATG translation start codon. The presence of cis-act-
ing regulatory sequences typical of archaeal promoters
had been observed. These sequences are part of the
basal transcriptional apparatus, such as the TATA
box, centered at )27 from the transcriptional start site,
and the BRE motif, targets for the general transcrip-
tion factors TBP and TFB, respectively [17].
Purification and characterization of recombinant
Bcp2 (rBcp2)
In order to overproduce rBcp2, the gene was amplified
by PCR from S. solfataricus genomic DNA, as des-
cribed in Experimental procedures, and cloned into
pET-30c(+), rBcp2 was highly overexpressed in
Escherichia coli in soluble form, as a fusion with a
C-terminal eight-residue histidine tag (LEHHHHHH)
with a yield of 12.8% of homogeneous protein.
To purify the recombinant protein, the soluble frac-
tion (140 mg) of the cell extract was heated at 80 °C
for 15 min; this heat treatment removed about 40% of
E. coli proteins. rBcp2 was purified to homogeneity in
a two-stage process using affinity chromatography on
HisTrap HP and gel filtration on HiLoad Superdex 75
obtaining 30 mg and 18 mg, respectively. The
SDS ⁄ PAGE of the final preparation revealed a single
band with a molecular mass of 25 ± 1 kDa (Fig. 7).
The molecular mass of rBcp2, 25 678 Da, was deter-
mined using mass spectrometric analysis as reported in

Experimental procedures; the 131 shortfall compared
to the predicted 25 809 Da molecular mass suggested
the removal of the N-terminal methionine.
To assess the quaternary structure of the enzyme,
analytical gel filtration on PC 75 and Biosep-SEC-4000
of purified rBcp2 were performed. The protein was
eluted at a volume accounting for a monomeric struc-
ture, but it was observed that increasing protein
16S rRNA
bcp2 mRNA
0 15 30 45 60
CBA
min
H
2
O
2
0 15 30 45 60
paraquat
0 15 30 45 60
tert-butyl hydroperoxide
Fig. 4. Northern hybridization analysis of S. solfataricus P2 bcp2 transcripts: effect of oxidative stress agents. Cultures of S. solfataricus P2
were grown until the mid-exponential phase and treated with (A) 0.05 m
M H
2
O
2
(B) 0.1 mM paraquat (C) 0.05 mM tert-butyl hydroperoxide.
RNAs were obtained from cultures harvested at time shown. On the bottom 16S rRNA were reported as normalization.
A

B
16S rRNA
bcp2 mRNA
1 2 3
1 2 3
Fig. 5. Northern hybridization analysis of S. solfataricus P2 bcp2
transcripts at different growth phases. (A) RNA was extracted from
cultures harvested at 0.3 OD
600 nm
(1), at 0.6 OD
600 nm
(2), at
1.0 OD
600 nm
(3). (B) 16S rRNA levels were used as a control.
Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al.
724 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS
concentration (0.4–2 lgÆlL
)1
) or prolonged storage
time (24 h at 4 °C), produced changes in the quater-
nary structure with the appearance of the dimeric and
multimeric forms (data not shown).
Homogeneous rBcp2 was tested for its capacity to
serve as an antioxidant enzyme. One of the most widely
used test for detecting Prx activity is the ability to pro-
tect plasmids against the metal catalysed oxidation
(MCO) system (DTT ⁄ Fe
3+
⁄ O

2
); in the presence of an
electron donor, such as dithiothreitol (DTT), Fe
3+
catalyses the reduction of O
2
to H
2
O
2
, which is further
converted to OH
•
by the Fenton reaction [18]. The
MCO system causes damage to DNA by producing
OH
•
, which in turn can nick the intact supercoiled plas-
mid DNA [19] as shown in Fig. 8 (lane 4). The damage
was averted when rBcp2 was included in the reaction
mixture, showing that the enzyme is an active Prx and
can remove H
2
O
2
generated by the MCO system in vitro
(Fig. 8A). BSA was used as negative control.
The antioxidant activity of rBcp2 was then tested
for its ability to remove exogenously added H
2

O
2
in a
more quantitative in vitro spectrophotometric assay.
rBcp2 was capable of catalysing the removal of H
2
O
2
in a concentration-dependent manner using DTT as
the electron donor (Fig. 8B).
In order to characterize the thermophilicity of rBcp2,
peroxidase activity was investigated by measuring the
H
2
O
2
removal at increasing temperature. rBcp2 showed
maximum activity between 80 and 90 °C, which is in
the optimum temperature range for the growth of
A
B
Fig. 6. (A) Primer extension analysis and sequence of the S. solfataricus bcp2 gene. Total RNA was isolated from a culture of S. solfataricus
P2. Primer extension was carried out as described in Experimental procedures, and the products were separated by electrophoresis under
denaturing conditions alongside sequencing reactions with the same primer. (B) Nucleotide sequence of bcp2. The transcriptional start point
is shown by the bent arrow above the underlined boldface A nucleotide. The Shine–Dalgarno sequence is underlined by dotted line. A puta-
tive TATA box and BRE sequence are underlined. The ATG start codon is in bold and the TAA stop codon is marked by an asterisk.
D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus
FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 725
S. solfataricus. We also analysed the thermoresistance
of the enzyme by incubating rBcp2 for varying periods

of time at 80, 90, and 95 °C and then assaying the resid-
ual peroxidase activity. Following incubation for 6 h at
80 °C, the activity retained was 63%; the enzyme dis-
played a half-life of 3 h at 90 °C, while after 30 min at
95 °C the activity measured was 30% (Fig. 9).
Bcp2 expression in S. solfataricus
We investigated how the expression of Bcp2 was
induced by H
2
O
2
, paraquat and tert-butyl hydroper-
oxide. A polyclonal rBcp2-specific rabbit antiserum
was used to conduct a quantitative analysis of the
Bcp2 expression in S. solfataricus. The anti-Bcp2 anti-
serum was used in a western blot analysis on cytoplas-
mic extracts from cells harvested in the exponential
growth phase before and after addition of the stressor
agents at the identical concentrations utilized in the
northern analysis (Fig. 10). Twenty-five kDa signals
corresponding to Bcp2 were detected in noninduced
and induced cells. The increased amount of Bcp2 in
the cytoplasmic fraction of S. solfataricus correlated
with increased bcp2 mRNA level after treatment with
the stressors. In particular, the maximum Bcp2 expres-
sion ) a sevenfold increase compared to that monit-
ored for controls ) was observed after H
2
O
2

addition
as in the northern analysis.
Fig. 7. SDS ⁄ PAGE of different steps in the purification of rBcp2.
Lane 1, E. coli BL21-CodonPlus (DE3)-RIL ⁄ pETBcp2 cellular extract
not induced by IPTG; lane 2, E. coli BL21-CodonPlus (DE3)-
RIL ⁄ pETBcp2 induced by 1 m
M isopropyl-thio-b-d-galactopyrano-
side; lane 3, heat-treated sample; lane 4, molecular weight
markers; lane 5, sample after affinity chromatography; lane 6, sam-
ple after size-exclusion chromatography.
A
B
Fig. 8. (A) rBcp2 assayed as antioxidant enzyme: DNA cleavage protection assay performed by rBcp2. Supercoiled pUC19 plasmid was
exposed to the MCO system (DTT ⁄ Fe
3+
⁄ O
2
) alone and with different rBcp2 concentrations. Nicked form (NF) and supercoiled form (SF) of
pUC19 are indicated on the left by arrows. (B) rBcp2 was assayed for its ability to remove H
2
O
2
in an in vitro assay system in the presence
(r) and absence (d) of DTT. Peroxidase activity was measured at 80 °C using the ferrithiocyanate complex as described in experimental
procedures. The nonenzymatic removal of H
2
O
2
by heat was performed in parallel.
Fig. 9. Thermoresistance was measured as residual peroxidase

activity with 50 lgÆmL
)1
of rBcp2 after incubation for different
times at 80 °C(n), 90 °C(r), 95 °C(m).
Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al.
726 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS
Role of the conserved cysteine residue in rBcp2
To investigate the catalytic role of the Cys residue we
constructed a mutant enzyme in which the cysteine at
position 49 was replaced by serine (C49S). The mutant
Bcp2 protein was expressed in E. coli BL21-CodonPlus
(DE3)-RIL cells and purified from the soluble fraction
of bacterial cells as described in Experimental proce-
dures. The yield of C49S was the same of that
obtained for the wild-type protein.
The activity of C49S was tested by peroxidase
(Fig. 11A) and DNA cleavage protection (Fig. 11B)
assays and compared to that of the wild-type protein.
In both cases the mutant showed no peroxidase or
plasmid DNA protection activity. These findings indi-
cate that Cys
49
is required for the proceeding of the
enzymatic reaction and that Cys
49
-SOH can be conver-
ted back to Cys-SH using DTT, and that Cys
49
-SH is
responsible for scavenging the OH

•
induced by the
MCO system.
Discussion
The natural environment in which S. solfataricus
lives is strongly oxidative, in addition ROS can be
generated by naturally occurring phenomena such as
ultraviolet irradiation of water, autoxidations and
aeration turbulence. Consequently, to survive in this
harsh habitat S. solfataricus should have developed
antioxidant enzymes and molecules that protect it
from ROS. At the present time the investigation of
response to oxidative stress in S. solfataricus is at an
initial stage and has been focused mainly on the
superoxide dismutase (Fe-SOD) (EC 1.15.1.1) [20,21].
This enzyme represents the primary defense against
O
2
•
)
as suggested by its ubiquitous location in the
membrane and in the cytoplasm [22], by its constitu-
tive level [21] and by the long half-life (2 h) of the
mRNA [23]. The dismutation of O
2
•
)
by SOD deve-
lops H
2

O
2
that can go freely through the membrane
CBA
Fig. 10. Bcp2 expression in S. solfataricus cells in response to H
2
O
2
(A), paraquat (B) and tert-butyl hydroperoxide (C). Twenty micrograms
of cytoplasmic proteins extracted from nonexposed and exposed culture for 30, 60 and 120 min were analysed by western blot with anti-
rBcp2 IgG.
AB
Fig. 11. Effect on peroxidase activity of replacement of Cys
49
of rBcp2 with serine. (A) At different enzyme concentrations the peroxidase
activity was assayed as previously reported. H
2
O
2
removal by rBcp2 (r) and C49S (m) was measured over a range of concentrations (0–
100 lgÆmL
)1
). (B) Effect on protection against DNA cleavage of the replacement of Cys
49
of rBcp2 with serine. Lane 1, pUC19; lane 2,
pUC19 and 10 m
M DTT; lane 3, pUC19 and 3 lM FeCl
3
; lane 4, pUC19, 10 mM DTT, and 3 lM FeCl
3

; lane 5, pUC19, 10 mM DTT, 3 lM FeCl
3
,
and 50 lgÆmL
)1
rBcp2; lane 6, pUC19, 10 mM DTT, 3 lM FeCl
3
, and 50 lgÆmL
)1
C49S.
D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus
FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 727
in the cell, where it must be scavenged to prevent
damage of biological molecules.
In S. solfataricus the peroxide detoxification system
has not yet been studied. Genome analysis has shown
the absence of putative catalases and the presence of
four putative Bcps proteins: Bcp1, Bcp2, Bcp3, Bcp4
whose roles should be clarified in detail and could play
a key role in the detoxification processes.
In this study we examined the role of Bcp2 in order
to increase the knowledge of the enzymatic activity
involved in the oxidative stress in S. solfataricus.
To detect differences in the response to various
agents we induced oxidative stress with paraquat,
an O
2
•
)
generating compound, H

2
O
2
and tert-butyl
hydroperoxide, an alkyl hydroperoxide. Our results
show that compounds acting both indirectly and
directly as oxidants can induce transcription of bcp2.
Data reveal that transcription of bcp2 in S. solfataricus
is upregulated by the various stressors, and the differ-
ent kinetics in response to these agents imply that sev-
eral regulatory mechanisms or at least variations on
the same mechanism could be involved in controlling
the expression of bcp2. Western analysis performed
after treatment with H
2
O
2
showed a slower and more
slight increase in protein level than mRNA level. This
could imply that post-transcriptional processes, such as
lower rate of translational or protein instability caused
by oxidative stress conditions, are important in deter-
mining the level of Bcp2 protein. Similar results are
observed for genes and related proteins involved in
oxidative stress in other microrganisms [24]. Moreover,
the basal level of bcp2 transcript in the early and
mid-exponential, and the stationary phase of growth
suggests that bcp2 is not involved in the control of
endogenous peroxides that are produced during aero-
bic respiration.

In contrast with Prxs discovered in the aerobic
hyperthermophilic archaeon A. pernix and the anaer-
obic hyperthermothilic archaeon P. horikoshii, the ana-
lysis of the primary structure of Bcp2 shows only one
cysteine (Cys
49
). This residue is positioned inside the
DFTPVCTTE sequence in the N-terminal region of
the protein that is conserved both in 1-Cys and 2-Cys
classes of Prxs. Site-directed mutagenesis showed that
Cys
49
is required for peroxidase activity. Both func-
tional data and analysis of homologous sequences sup-
ported the classification of Bcp2 in the family of
peroxiredoxin in the 1-Cys Prx class and Bcp2 could
be considered the first ancient Prx developed in the
early stages of evolution.
The results indicate that Bcp2 displays peroxidase
activity with a temperature optimum between 80 and
90 °C which is the temperature range for growth of
S. solfataricus. The enzyme appears to be less thermo-
stable (60% of activity after 15 min at 90 °C) than the
Prx of P. horikoshii that retains full activity on heating
at 90 °C for 20 min; this difference reflects the differ-
ence in the optimum growth temperature between the
two organisms. The enzyme can function at 37 °Cas
verified by the protection of DNA in MCO system and
by removal of H
2

O
2
(data not shown) but it has the
maximum activity in the range 80–90 °C. The peroxi-
dase activity of Bcp2 is DTT dependent, suggesting a
mechanism in which Cys
49
residue is firstly oxidized by
H
2
O
2
and successively reduced by DTT that could be
the electron donor partner in vitro. The physiological
partner has not yet been found. Recently, a thioredox-
in reductase has been characterized in S. solfataricus;
the presence in the genome of two thioredoxins and
two other thioredoxin reductases suggest their involve-
ment as physiological partners as electron donor.
These speculations require experimental evidence and
studies are underway in our laboratory.
Finally size-exclusion chromatography has shown
that the protein can shift from a monomer to a multi-
meric form depending on the protein concentration
and the temperature. On the basis of the data repor-
ted in the literature on the structure of Prxs [25] ionic
interactions play an important role in oligomerization;
further analyses are in procress to define completely
the quaternary structure of the enzyme.
Experimental procedures

Strains, media and growth conditions
Sulfolobus solfataricus P2 strain liquid cultures were grown
aerobically at 80 °C in mineral medium supplemented with
0.1% Bacto
TM
yeast extract (Becton, Dickinson and
Company, Franklin Lakes, NJ, USA), 0.1% tryptone
(Oxoid, Basingstoke, Hampshire, UK) and 0.2% sucrose
(TYS medium) in an orbital shaker. Oxidative stresses
were created by adding H
2
O
2
, paraquat, or tert-butyl hydro-
peroxide at a final concentration of 0.05 mm, 0.1 mm and
0.05 mm, respectively, to S. solfataricus cultures in early
exponential growth phase (0.3 OD
600 nm
).
Escherichia coli Top F¢10 was used as a general host for
DNA manipulation, E. coli XL1-Blue (Invitrogen SRL,
Milan, Italy) was used to transform the mutagenesis
products. Escherichia coli BL21-CodonPlus(DE3)-RIL
(Stratagene, La Jolla, CA, USA) was used for expression of
the recombinant Bcp2. These strains were cultivated in
Luria–Bertani (LB) medium at 37 ° C. When necessary
100 lgÆmL
)1
ampicillin, or 50 lgÆmL
)1

kanamycin and
33 lgÆmL
)1
chloramphenicol were added to the medium to
maintain plasmids.
Antioxidant activity of Bcp2 in S. solfataricus D. Limauro et al.
728 FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS
RNA extraction
Sulfolobus solfataricus cultures, grown to early exponential
phase, were stressed with H
2
O
2,
paraquat or tert -butyl
hydroperoxide. Aliquots were collected at different times by
centrifugation at 5000 g for 10 min at 4 °C. Total RNA
was extracted by the guanidinium isothiocyanate method as
described in Sambrook et al. [26]. The integrity and concen-
tration of total RNA were verified by electrophoretic analy-
sis by separating total RNA on 1% agarose gel containing
formaldehyde.
Northern hybridization
Northern blot analysis was used to quantify the amount of
bcp-2 mRNA in different stress conditions and to determine
the size of the specific transcript. Genomic DNA from S. sol-
fataricus P2 was used as a template for PCR amplification
of bcp2 using HF Taq DNA polymerase (Roche Applied
Science, Monza, Italy) and the following primers: forward
primer (the inserted Nde I restriction site is underlined)
5¢-CTAGGTGAA

CATATGAGTGAGGAAAGAATTCC-3¢
and the reverse primer 5¢-GGAGCTGGATTAATG
CTC
GAGTCTCCTATTAG-3¢ (the inserted XhoI restriction site
is underlined). The PCR products obtained were purified
from agarose gel, and the NdeI–XhoI fragment was labelled
with a
32
P(dATP) and with Random primed DNA labelling
kit (Roche).
Primer extension
Primer extension analysis was carried out with avian myelo-
blastosis virus reverse transcriptase (Roche) as described
in Limauro et al. [27] using the synthetic oligonucleotide
5¢-CATCAGGTAGTTTTATCCTGCC-3¢ complementary
to nucleotides 1801–1822 of DB source AE006819. The
sequencing reaction of the corresponding DNA fragment
cloned, which had been primed with the same synthetic
oligonucleotide, was used as a marker to locate the prod-
ucts on 6% urea ⁄ polyacrylamide gel.
Construction and expression of recombinant
protein
Genomic DNA of S. solfataricus was prepared as described
by Arnold et al. [28]. bcp2 was amplified by PCR using
chromosomal DNA as template and the same two primers
as used to generate the northern blot probe. Amplification
by PCR was carried out at 94 °C for 1 min, 45 °C for
1 min, and 72 °C for 1 min, for 35 cycles using HF Taq
DNA polymerase (Roche). The PCR product was purified
with QIAquick PCR purification kit (Quiagen Spa, Milan,

Italy) and cloned in pGEMTeasy vector (Promega Italia
srl, Milan, Italy). The nucleotide sequence of the inserted
gene was determined to ensure that no mutations were pre-
sent in the gene. Then, the NdeI–XhoI fragment was cloned
into pET-30c(+) (Novagen, Darmstadt, Germany) giving
the recombinant plasmid pETBcp2 that was used to trans-
form the E. coli BL21-CodonPlus (DE3)-RIL.
Purification of the recombinant Bcp2 protein
(rBcp2)
BL21-CodonPlus (DE3)-RIL ⁄ pETBcp2 cells were grown at
37 °C in 1000 mL LB medium supplemented with kanamycin
and chloramphenicol to an OD
600
of 1 was reached. Induc-
tion was carried out by the addition of 1 mm isopropyl-
thio-b-d-galactopyranoside to the medium and growth was
continued for 12 h. Cells were harvested by centrifugation,
suspended in 20 mm Tris ⁄ HCl pH 8.0 and distrupted by
ultrasonication (Sonicator Ultrasonic liquid processor; Heat
System Ultrasonics Inc., Plainview, NY, USA). The suspen-
sion was ultracentrifugated at 160 000 g for 30 min. The
crude extract obtained was heated at 80 °C for 15 min, and
the denaturated proteins were removed by centrifugation
(15 000 g for 30 min). The extract was concentrated (Am-
icon, Millipore Corp., Bedford, MA) and loaded on a Hi-
sTrap HP (Amersham Biosciences Europe GmbH, Milan,
Italy) equilibrated with 50 m m Tris ⁄ HCl pH 8.0 ⁄ 0.3 m NaCl
(buffer A). After the column was washed with buffer A
containing 20 mm imidazole, proteins were eluted with the
same buffer A supplemented with 250 mm imidazole. The

active fractions were pooled and dialyzed against 20 mm
Tris ⁄ HCl pH 8.0 ⁄ 1mm DTT. The concentrated sample was
applied to HiLoad Superdex 75 column (1.6 cm · 60 cm,
Amersham) connected to an FPLC system (Amersham) and
eluted with 50 mm Tris ⁄ HCl pH 8.0 ⁄ 0.2 m KCl at a flow rate
of 1 mLÆmin
)1
. The active fractions were pooled, concentra-
ted and extensively dialysed against 20 mm Tris ⁄ HCl pH 8.0.
Determination of quaternary structure
The molecular mass of the protein was determined by
gel-filtration chromatography on a Superdex 75 PC
(0.3 cm · 3.2 cm) and a Biosep-SEC-S4000 (30 cm · 0.78
cm, Phenomenex Inc., St Torrance, CA, USA) connected to
AKTA system (Amersham). Protein was eluted with buffer
50 mm Tris ⁄ HCl pH 8.0 ⁄ 0.2 m KCl at a flow rate of
0.04 mLÆmin
)1
and with 20 mm NaPO
4
pH 7.2 at a flow
rate of 0.5 mLÆ min
)1
. b-Amylase (200 kDa), alcohol dehy-
drogenase (150 kDa), BSA (65.4 kDa), ovalbumin
(48.9 kDa), chymotrypsinogen (22.8 kDa) and the RNA-
se A (15.6 kDa) were used as molecular weight standards.
Analytical methods for protein characterization
Protein concentration was determined using BSA as the
standard [29]. Protein homogeneity was estimated by

D. Limauro et al. Antioxidant activity of Bcp2 in S. solfataricus
FEBS Journal 273 (2006) 721–731 ª 2006 The Authors Journal compilation ª 2006 FEBS 729
SDS ⁄ PAGE [30] using a 12.5% (w ⁄ v) acrylamide resolving
gel and a 5% acrylamide stacking gel. Samples were heated
at 100 °C for 5 min in 2% SDS ⁄ 2% 2-mercaptoethanol
and run in comparison with molecular weight standards.
Gels were stained with the Coomassie blue.
The molecular mass of the protein was also estimated
using electrospray MS recorded on a Bio-Q triple quadrupole
instrument (Thermofinnigan, San Jose, CA, USA). Samples
were dissolved in 1% (v ⁄ v) acetic acid ⁄ 50% (v ⁄ v) acetonitrile
and injected into the ion source at a flow rate of 10 lLÆmin
)1
using a Phoenix syringe pump. Spectra were collected and
elaborated using MASSLYNX software provided by the
manufacturer. Calibration of the mass spectrometer was
performed with horse heart myoglobin (16.9 kDa).
Assay of peroxidase activity rBcp2 was tested for its
ability to remove H
2
O
2
in an in vitro assay system as fol-
lows. The reaction was started adding H
2
O
2
at a final
concentration of 0.2 mm to the reaction mixture contain-
ing 50 mm Hepes pH 7.0 ⁄ 10 mm DTT in the presence of

different concentrations of Bcp2 in a final volume of
0.1 mL. The reaction was incubated at 80 °C for 1 min
and stopped by the addition of 0.9 mL trichloroacetic
acid (10% w ⁄ v) [19]. Peroxidase activity was determined
from the amount of peroxide remaining, which was detec-
ted by measurement of the purple-coloured ferrithiocya-
nate complex developed after the addition of 0.2 mL
10 mm Fe(NH
4
)
2
(SO
4
)
2
and 0.1 mL 2.5 n KSCN using
H
2
O
2
as standard. The amount of ferrithiocyanate com-
plex was determined by absorbance at 490 nm. The per-
centage of H
2
O
2
removed was calculated on the basis of
the change in A
490
obtained with Bcp2 relative to that

obtained without Bcp2.
Thermophilicity and thermoresistance
Thermophilicity was evaluated in the temperature range
50–90 °C by measuring peroxidase activity by the ferrithio-
cyanate method. rBcp2 thermoresistance was estimated by
measuring the residual peroxidase activity at 80 °C for
1 min after heat treatment at 80 °C, 90 °C, 95 °C for differ-
ent times.
DNA cleavage assay by the MCO system
The ability of Bcp2 to protect DNA from oxidative nick-
ing by OH
•
was determined as described previously [19].
A reaction mixture of 50 lL included 3 lm FeCl
3
,10mm
DTT for the thiol MCO system, 100 mm Hepes pH 7.0,
different concentrations of rBcp2 or BSA as a negative
control. The reaction was initiated incubating the mixture
for 40 min at 37 °C before adding 2 lg plasmid pUC19
and developed for an additional 4 h at the same tempera-
ture. DNA bands were evaluated on a 0.8% (w ⁄ v)
agarose gel after staining with ethidium bromide
(5 lgÆmL
)1
).
Site-directed mutagenesis
The mutant rBcp2 C49S was obtained by following the pro-
tocol outlined in the QuickChange II site directed mutagen-
esis kit (Stratagene) using primers complementary to the

coding and noncoding template sequence (pET30Bcp2) con-
taining a double mismatch. To generate the C49S mutant,
the forward primer 5 ¢-GATTTCACACCGGTG
AGCA
CTACGGAGTTCTAC-3¢ and a complementary reverse pri-
mer were used (the underlined letter indicates the base pair
mismatch). The reaction (50 lL) contained 50 ng template
DNA (pET30Bcp2), 125 ng each primer, 200 lm dNTP and
2.5 U Pfu Ultra HF DNA polymerase. Twelve cycles of
95 °C for 30 s, 55 °C for 1 min and 68 °C for 6 min were
carried out in a Mini Cycler followed by one cycle at 4 °C
for 2 min. To digest methylated template, each reaction
mixture was treated with 10 U DnpIat37°C for 1 h. Muta-
genesis products were transformed into XL-1 Blue cells. Sin-
gle colonies were selected on LB plates containing
kanamicin, and isolated plasmid DNA was sequenced
throughout the coding region at Primm (DNA sequencing
service Naples, Italy). The plasmid pETBcp2Mut containing
the mutation inserted was used to transform BL21-Codon-
Plus (DE3)-RIL competent cells. C49S was expressed and
purified with the same procedure reported above for rBcp2.
Western blot analysis
Sulfolobus solfataricus was stimulated during early exponen-
tial growth with 0.05 mm H
2
O
2
, 0.1 mm paraquat or
0.05 mm tert-butyl hydroperoxide for 30, 60, and 120 min.
Cells were harvested by centrifugation, suspended in 20 mm

Tris ⁄ HCl pH 8.0 and the crude extracts were prepared by so-
nication, disrupting the cells with 3 · 1-min pulses at 4 °Cat
20 Hz and ultracentrifugated at 160 000 g for 30 min at 4 °C.
Following SDS ⁄ PAGE of homogenates, proteins were
electrophoretically transferred to polyvinylidene difluoride
membranes. The membranes were blocked for 1 h with 5%
milk powder, 0.1% Tween in TBS (20 mm Tris ⁄ HCl
pH 7.5, 0.9% NaCl) and then incubated with rBcp2-specific
rabbit antibodies (Igtech, Paestum, Salerno, Italy) for 2 h,
followed by peroxidase-conjugated secondary antibodies
for 1 h. Antibodies to rBcp2 were detected by enhanced
chemiluminescence using ECL Plus western blotting Detec-
tion system (Amersham Biosciences).
Acknowledgements
This work was supported by grants from MIUR
(PRIN 2002).
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