Tài liệu Báo cáo khoa học: ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin docx

ATP-dependent modulation and autophosphorylation
of rapeseed 2-Cys peroxiredoxin
Martin Aran
1
, Daniel Caporaletti
1
, Alejandro M. Senn
1
, Marı
´a
T. Tellez de In
˜
on
2
, Marı
´a
R. Girotti
1
,
Andrea S. Llera
1
and Ricardo A. Wolosiuk
1
1 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina
2 INGEBI-CONICET, Buenos Aires, Argentina
Rather than viewing reactive oxygen species (ROS) as
toxic by-products of aerobic metabolism we now
know them to be members of signaling networks
that modulate important physiological processes [1,2].
Germane to the homeostatic regulation of ROS con-
centrations, a large group of peroxidases devoid of

selenium- and heme-prosthetic groups, the peroxi-
redoxins (Prx) (EC 1.11.1.15), catalyze the reduction
of hydroperoxides and peroxinitrite [3–6]. The number
of subfamilies in this ubiquitous family of proteins
varies depending on the classification criteria used
but, in all cases, one subfamily encompasses polypep-
tides in which there is strict conservation of two cyste-
ine residues – the 2-Cys Prx [7–9]. The typical 2-Cys
Prx, found in prokaryotes and eukaryotes, is a head-
to-tail arranged homodimer in which one of the con-
served cysteines of the polypeptide is linked via an
intercatenary disulfide bond to the complementary
cysteine of the paired subunit. Crucial to the peroxi-
dase activity is the cysteine residue located at the
N-terminal region, ‘the peroxidatic cysteine’, whose
sulfhydryl group (-Cys-SH) turns into sulfenic acid
(-Cys-SOH) after reacting with a hydroperoxide
(ROOH). The sulfenate subsequently reacts with the
cysteine located in the C-terminal region of the paired
polypeptide, ‘the resolving cysteine’, forming a second
intermolecular disulfide linkage (-Cys-S-S-Cys-). Com-
pleting the peroxidatic cycle, one of the disulfides is
Keywords
2-Cys peroxiredoxin; ATP binding;
autophosphorylation; sulfinic-phosphoryl
anhydride; sulfonic-phosphoryl anhydride
Correspondence
R. A. Wolosiuk, Instituto Leloir, Patricias
Argentinas 435, C1405BWE Buenos Aires,
Argentina

Fax: +54 11 5238 7501
Tel: +54 11 5238 7500
E-mail:
(Received 12 November 2007, revised
14 January 2008, accepted 16 January
2008)
doi:10.1111/j.1742-4658.2008.06299.x
2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidas-
es that have been implicated in antioxidant defense and signal transduction.
Although their biochemical features have been extensively studied, little is
known about the mechanisms that link the redox activity and non-redox
processes. Here we report that the concerted action of a nucleoside triphos-
phate and Mg
2+
on rapeseed 2-Cys Prx reversibly impairs the peroxidase
activity and promotes the formation of high molecular mass species. Using
protein intrinsic fluorescence in the analysis of site-directed mutants, we
demonstrate that ATP quenches the emission intensity of Trp179, a residue
close to the conserved Cys175. More importantly, we found that ATP
facilitates the autophosphorylation of 2-Cys Prx when the protein is succes-
sively reduced with thiol-bearing compounds and oxidized with hydroper-
oxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the
phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl
[Prx-(Cys175)-SO
2
PO
3
2)
] and the sulfonic-phosphoryl [Prx-(Cys175)-SO
3

PO
3
2)
] anhydrides. Hence, the functional coupling between ATP and 2-Cys
Prx gives novel insights into not only the removal of reactive oxygen
species, but also mechanisms that link the energy status of the cell and the
oxidation of cysteine residues.
Abbreviations
2-Cys Prx, 2-Cys peroxiredoxin; ANS, 8-anilinonaphthalene-1-sulfonate; ROS, reactive oxygen species.
1450 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
cleaved back to thiols by the concerted action of cel-
lular reductants and protein-disulfide oxido-reductases.
Often regarded as a peroxidase, the additional func-
tion as a molecular chaperone, identified in human
and yeast 2-Cys Prx [10,11], exhibits a marked sensi-
tivity to a variety of compounds and conditions, such
as reductants and ROS. An imbalance between these
dual functions is probably associated with many
human pathologies, such as thyroid tumors, breast
and lung cancer, Alzheimer’s disease and neurodegen-
erative disorders [12].
By contrast to the extensive literature on the redox
modulation of different enzyme activities in chloro-
plasts and non-photosynthetic systems, mainly via
thiol–disulfide exchange [13–16], systematic efforts to
examine the opposite action of non-redox processes on
redox reactions are scarce. Most biochemical research
on 2-Cys Prx has studied the interplay between the
stimuli and the abundance of reductants and oxidants
(thermodynamic control), whereas the catalytic fea-

tures (kinetic control) have been less clear. Therefore,
the reactions leading to ROS generation and detoxifi-
cation have been elucidated, but little is known about
how oxidative stress is linked to non-redox processes
in the signaling networks that modulate cellular func-
tions. Studies addressing this issue have found signi-
ficant changes in the quaternary structure and dual
functions when human 2-Cys Prx is phosphorylated on
Thr90 by cyclin-dependent protein kinases, preferably
CDK1 (formerly Cdc2) [17,18]. A putative intermedi-
ate at the peroxidatic cysteine (-Cys-S(=O)-O-PO
3
2)
)
has recently been suggested in the multiple-step pro-
cess underlying sulfiredoxin-mediated reduction of
2-Cys Prx-SO
2
H, however, experimental evidence is lack-
ing [19–21]. As with many proteins, phosphorylation
of 2-Cys Prx via these two mechanisms requires the
participation of additional catalysts, i.e. protein kinase
and sulfiredoxin. Despite numerous studies showing
the close association between ATP and chaperone
activity [22], with the exception of serving as the phos-
phoryl donor for CDK1 and sulfiredoxin, the direct
interaction of a nucleotide with 2-Cys Prx has not been
previously addressed. Here, we report that the con-
certed action of a nucleoside triphosphate and Mg
2+

on rapeseed 2-Cys Prx impairs the peroxidase activity.
More importantly, MS studies show that the successive
action of a reductant and an oxidant makes the pro-
tein a recipient of the phosphoryl moiety in sulfonic
and sulfinic acid forms of Cys175. Hence, ATP has
emerged as both a novel regulator of 2-Cys Prx func-
tions and the phosphoryl donor for the autophospho-
rylation at the resolving cysteine.
Results
The concerted action of nucleotides and Mg
2+
modulates the peroxidase activity of 2-Cys Prx
A thorough inspection of the available X-ray structure
of human 2-Cys Prx (PDB entry: 1QMV) revealed that
the covalently linked homodimer creates a large inter-
nal cavity comprising segments of two polypeptides
(-Leu42–Pro46, Arg129–Ile133, Gln141–Asn146,
Gly151–Arg159-) and (-Pro54–Ile57, Cys175–Gly178-).
Because the size of the cavity (0.515 nm
3
) in silico eas-
ily docks a molecule of ATP (0.33 nm
3
), it was impor-
tant to know whether the functions of 2-Cys Prx were
sensitive to insertion of the nucleotide. As shown in
Fig. 1A, a nucleoside triphosphate in concert with
Mg
2+
lowered the peroxidase activity in a dose-depen-

dent manner, purine nucleotides being more potent
than pyrimidine derivatives. In particular, the response
of the peroxidase activity to increasing concentrations
of ATP exhibited three well-defined stages: (a) monot-
onous decay (I
0.5
= 0.25 mm), (b) stabilization at half
of the maximal activity from 0.9 to 1.2 mm, and (c) a
sharp decrease to undetectable levels beyond 1.5 mm
(I
0.5
= 1.40 mm). Interestingly, inhibition mediated by
the other purine nucleotide, GTP, was significantly
similar in the first two stages, but lacked the third.
Following these initial experiments, we investigated
whether other phosphorylated compounds and bivalent
cations exhibited similar capacity. In the presence of
2mm Mg
2+
, the rate of H
2
O
2
removal was inhibited
by 60, 5 and 5% when it was assayed with 2 mm
ADP, AMP or orthophosphate, respectively (not
shown). By contrast, Mg
2+
was the most efficient
cation in assisting nucleotide-dependent inhibition

(100%), whereas Ca
2+
(92%), Mn
2+
(85%) and Zn
2+
(65%) exhibited a varying capacity when the peroxi-
dase activity was assayed in the presence of fixed con-
centrations of both ATP (3 mm) and bivalent cations
(3 mm), (Fig. 1B). At this stage, the lack of peroxidase
activity might be attributed to an irreversible change in
2-Cys Prx triggered by the binding of ligands. Against
this possibility, totally inactive 2-Cys Prx, caused by
incubation with 3 mm ATP and 3 mm Mg
2+
, immedi-
ately recovered 75% of its original activity upon chela-
tion of Mg
2+
by the addition of 5 mm EGTA
(Fig. 1C). Clearly, the capacity of ATP to lower the
peroxidase activity in the absence of exogenous com-
ponents revealed a mechanism that is substantially
different from the inhibition brought about by the
phosphorylation of human 2-Cys Prx mediated by the
CDK1–cyclin B complex [17].
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1451
In addition to the peroxidase activity that functions
in the cellular defense against ROS, yeast and human

2-Cys Prx are molecular chaperones [10,11]. Therefore,
it was important to examine 2-Cys Prx beyond a single
activity and establish whether the regulation described
above previously had wider implications. We found
that the rapeseed orthologue efficiently prevents the
thermal aggregation of citrate synthase indicating that
the chaperone activity is likely to be a general function
of typical 2-Cys Prx (Fig. 1D). Remarkably, incorpo-
ration of increasing amounts of Mg
2+
into the incuba-
tion milieu led to a concomitant reduction in the
chaperone capacity which, at variance with the peroxi-
dase activity, was not affected by the presence of
2.5 mm ATP. These data provide the first evidence
that the dual functions of 2-Cys Prx can be differen-
tially regulated by ATP and Mg
2+
.
The interaction with ATP modifies structural
features of 2-Cys Prx
Given the essential role of ATP in the peroxidase
activity, we evaluated changes in the structure of
2-Cys Prx brought about by the concerted action of
the nucleotide and the bivalent cation. To accurately
determine the molecular mass of our 2-Cys Prx prepa-
rations, static light-scattering measurements were per-
formed, because this spectroscopic technique allows
direct estimation of the species in solution [23]. In
the absence of perturbants, the predominant form of

A
C
B
D
Fig. 1. Effect of nucleotides ⁄ Me
2+
on the functions of 2-Cys Prx. (A) Concerted action of nucleotides ⁄ Mg
2+
on the peroxidase activity. The
reaction, carried out in a solution supplemented with 3 l
M 2-Cys Prx, 2 mM MgCl
2
and the indicated nucleoside triphosphate, was started by
the addition of 0.13 m
M H
2
O
2
and the remnant of reduced dithiothreitol was estimated with Ellman’s reagent after 15 min. Data from seven
independent experiments were averaged and standard deviations were calculated. Control activity: 4.3 nmol H
2
O
2
reduced per min.
(B) Effect of ATP ⁄ Me
2+
on peroxidase activity. The assay was essentially similar to (A), except that the concentrations of ATP and the Me
2+
(Mg
2+

,Ca
2+
,Mn
2+
,Zn
2+
) were both fixed at 3 mM. (C) EGTA-mediated reversal of (ATP ⁄ Mg
2+
)-dependent inhibition of peroxidase activity.
2-Cys Prx (3 l
M) was incubated for 3 min with 2 mM ATP and 2 mM MgCl
2
. After the addition of EGTA to a final concentration of 5 mM, the
protein solution was further incubated for 5 min and the peroxidase activity was assayed as in (A). (D) Effect of ATP and Mg
2+
on the chap-
erone activity. 2-Cys Prx (5 l
M) was incubated in 25 mM Tris ⁄ HCl (pH 8) containing, as indicated, different concentrations of MgCl
2
and
2.5 m
M ATP. After 10 min at 25 °C followed by 10 min at 45 °C, the assay was started by the addition of citrate synthase and measured as
described in Experimental procedures [25].
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1452 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
rapeseed 2-Cys Prx (polypeptide: 22 316 kDa) had a
molecular mass of 260 kDa indicating that it was
essentially similar to counterparts from other sources,
wherein covalently linked dimers (a
2

) associate non-
covalently, forming doughnut-shaped decamers (a
2
)
5
(not shown) [24,25]. By contrast, size-exclusion chro-
matography revealed that protein dissolved in solu-
tions containing 3 mm ATP and 3 mm Mg
2+
exhibited
a 2550 kDa higher-order assembly that returned imme-
diately to the decameric state upon the removal of
ATP or Mg
2+
. It is noteworthy that low concentra-
tions of two well-known intracellular components
converted the rather stable decamer to higher-order
assemblies whose molecular mass approached that of
the dodecahedron [(a
2
)
5
]
12
observed recently in electron
microscopy preparations of the erythrocyte orthologue
treated with polyethylene glycol [26].
Data on the inhibition of peroxidase activity along
with the reversible oligomerization of 2-Cys Prx were
consistent with a specific binding of ATP ⁄ Mg to the

protein. In line with this prediction, positive and nega-
tive differences in absorbance appeared following incu-
bation of 2-Cys Prx with ATP in the absence and
presence of Mg
2+
, respectively (supplementary
Fig. S1). Although these experiments confirmed an
interaction between the nucleotide ⁄ Me
2+
couple and
the protein, the differential response could not be
attributed specifically to any of the interacting species.
Therefore, we turned our attention to fluorescence
emission spectroscopy which provides information
about the polarity of local environments surrounding
either extrinsic probes that bind to proteins or intrinsic
fluorophores buried in the protein interior. In a first
set of experiments, we relied on a biophysical probe
commonly used to study the characteristics of protein
surfaces, 8-anilinonaphthalene-1-sulfonate (ANS),
which, as expected, exhibited an emission maximum
wavelength at 512 nm that was not modified by the
presence of 3 mm ATP or 3 mm Mg
2+
(Fig. 2A). At
variance, reflecting the affinity of this extrinsic probe
towards exposed protein hydrophobic surfaces,
2-Cys Prx led to a marked enhancement of the emis-
sion intensity with a concurrent displacement of the
A

C
B
Fig. 2. Effect of the binding of ATP ⁄ Mg
2+
to 2-Cys Prx on the fluo-
rescence emission of extrinsic and intrinsic chromophores. (A) Sen-
sitivity of the extrinsic probe ANS. Binding of ANS to 2-Cys Prx
was performed for 10 min at 25 °C in solutions of 20 m
M Tris ⁄ HCl
(pH 7.8) containing 75 l
M ANS (e
350nm
: 5000 M
)1
Æcm
)1
), and, as
indicated, 10 l
M 2-Cys Prx, 3 mM ATP and 3 mM Mg
2+
. Protein
solutions were excited at 370 nm and emission spectra were
scanned from 410 to 600 nm. The spectral bandwidths were 5 nm.
(B) Quenching of tryptophan fluorescence. Equilibrium fluorescence
measurements were conducted increasing the concentrations of
ATP or ADP, as indicated, while keeping constant the concentration
of 2-Cys Prx (2 l
M) and Mg
2+
(2 mM). After correction for the inner

filter effect, data were fitted to the saturation curve equation using
nonlinear least-squares regression analyses. The difference in fluo-
rescence (DF) between 2-Cys Prx (F
o
) and 2-Cys Prx-ATP-Mg
2+
complex (F) at 340 nm was plotted according to Lehrer [28] (inset).
(C) Quenching of emission intensity in W88F and W179F 2-Cys Prx.
Fluorescence measurements were performed as described in (B),
except that W88F and W179F mutants replaced for the wild-type
2-Cys Prx.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1453
spectrum to a maximum at 480 nm. At this stage, the
addition of 3 mm ATP and 3 mm Mg
2+
did not shift
the maximum emission wavelength, but progressively
increased the emission intensity, indicating that the
nucleotide and the bivalent cation significantly
enhanced the proportion of protein hydrophobic
patches.
Although these experiments were informative regard-
ing the ability of 2-Cys Prx to interact with ATP, it
was imperative to determine the nucleotide binding
site. This information could be gained from the intrin-
sic fluorescence because the constituent polypeptide
held two conserved tryptophan residues that exhibited
a maximum emission wavelength centered at 343 nm,
suggesting a rather polar environment around the in-

dol side chains (supplementary Fig. S2A) [27]. Unfor-
tunately, the concentration of nucleotides in these
experiments never exceeded 0.2 mm because the intense
inner filter effect caused by the purine ring impaired
the excitation of tryptophan residues. Despite this limi-
tation, if ATP perturbs the environment of Trp88 or
Trp179 to some extent, the fluorescence emission
should show a shift in maximum wavelength or a
decrease in intensity when the nucleotide changes the
conformation of the protein or collides with the fluoro-
phore, respectively. Incorporation of ATP ⁄ Mg did not
shift the maximum wavelength but caused a marked
quenching of the emission intensity that was much less
pronounced with ADP ⁄ Mg (Fig. 2B). Stern–Volmer
analyses showed a pronounced downward curvature as
result of a heterogeneous response of intrinsic fluoro-
phores towards the quencher. In this context, if the
deviation from linearity reflected a fluorophore inac-
cessible to the nucleotide, the Stern–Volmer relationship
should become linear using the expression developed by
Lehrer [F
o
⁄ (F
o
)F)=1⁄ f
a
+(1⁄ {f
a
ÆK
SV

Æ[Q]})] [28,29].
As shown in Fig. 2B (inset), the straight line was con-
gruent with a unique tryptophan residue of 2-Cys Prx
accessible to ATP⁄ Mg (f
a
= 0.26; K
SV
= 9.7 · 10
)3
Æ
m
)1
). To unambiguously define the indol ring sensitive
to ATP ⁄ Mg, we examined the intrinsic emission fluo-
rescence in variants of 2-Cys Prx where Trp88 and
Trp179 were replaced conservatively by phenylalanine
via site-directed mutagenesis. The results in Fig. 2C
clearly illustrate that the marked reduction in emission
intensity caused by the quencher in W88F 2-Cys Prx
was similar to its wild-type counterpart, whereas the
W179F variant was insensitive to ATP ⁄ Mg. These
findings demonstrated that the ATP binds to 2-Cys
Prx close to Trp179 and, as a consequence, to the
resolving Cys175. In this study, two complementary
experiments indicated that the conservative replace-
ment of tryptophan residues did not lead to gross
alterations in the structure of 2-Cys Prx. First, the
emission spectrum of W88F 2-Cys Prx was similar to
its wild-type counterpart (k
max

= 343 nm), whereas
that of the W179F variant was slightly blue-shifted
(k
max
= 338 nm) (supplementary Fig. S2A). Second,
the catalytic capacity was not affected because neither
the basal nor the ATP-inhibited peroxidase activities
were appreciably different from wild-type 2-Cys Prx
(Fig. S2B).
The sequential action of reductants and oxidants
predisposes 2-Cys Prx to autophosphorylation
In considering whether the interaction with ATP ⁄ Mg
proceeded further to the specific phosphorylation of
2-Cys Prx, we noted that ten serine, one threonine and
two tyrosine residues appeared as putative sites for
phosphorylation (program netphos 2.0, Expassy).
Therefore, we conducted a phosphorylation assay in
which our preparation of rapeseed 2-Cys Prx was
first treated with reductants and oxidants, then sub-
sequently incubated with [c
32
P]ATP ⁄ Mg
2+
and finally
subjected to non-reducing SDS ⁄ PAGE (Fig. 3A).
In this successive in vitro reduction fi oxidation of
2-Cys Prx, we were compelled to use high concentra-
tions of cumene hydroperoxide in the oxidation step
because high concentrations of dithiothreitol, required
for the complete and fast cleavage of disulfide bonds,

remained in the solution. To our surprise, a 23 kDa-
labeled band appeared when the recombinant protein
was (a) incubated successively with 10 mm dithiothrei-
tol, 10 mm cumene hydroperoxide and [ c
32
P]ATP ⁄
Mg
2+
, (b) subjected to non-reducing SDS ⁄ PAGE, and
(c) characterized by Ponceau Red staining and autora-
diography (Fig. 3B). Although not shown, four control
experiments carried out under comparable conditions
were consistent with the specific covalent binding of
the phosphoryl moiety to 2-Cys Prx. First,
32
P-labeled
bands did not appear in the autoradiography when
chloroplast thioredoxin-m, chloroplast fructose-1,6-
bisphosphatase or a-lactalbumin were used in place
of 2-Cys Prx. Second, the autophosphorylation of
2-Cys Prx could not be attributed to artifacts linked to
the unspecific binding of the nucleotide, as neither the
presence of ADP, AMP or GTP, nor pulse and chase
experiments with 3 mm nonradioactive ATP affected
the incorporation of the
32
P-label into the protein.
Third, supporting the formation of a covalent link as
opposed to a protein highly resistant to SDS denatur-
ation [30], the radioactive label remained linked to

2-Cys Prx after boiling or digestion with trypsin but was
completely stripped from the protein by incubation
with alkaline phosphatase. Fourth, the requirement for
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1454 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
MgCl
2
was neither replaced nor affected by CaCl
2
or
MnCl
2
. Moreover, the requirement for the sequence
reduction fi oxidation was further supported by
experiments in which a compound generally used for
cleaving disulfide bonds (2-mercaptoethanol) partially
substituted for dithiothreitol and hydroperoxides
(H
2
O
2
, t-butyl hydroperoxide) and two structurally dif-
ferent quinones (2-hydroxy-1,4-naphthoquinone and
1,4-dihydroxyanthraquinone) were as efficient as cum-
ene hydroperoxide (supplementary Fig. S3).
The above quenching of Trp179 fluorescence by
ATP was of particular interest in characterizing the
autophosphorylation because the evolutionary conser-
vation of this residue in the 2-Cys Prx subfamily is
unknown. We therefore examined the ability of W88F

and W179F 2-Cys Prx to incorporate the
32
P-label
after successive incubations with dithiothreitol and
cumene hydroperoxide. As shown in Fig. 3C, the for-
mer variant was indistinguishable from wild-type
2-Cys Prx, whereas the latter was not functional. Near
Trp179, the resolving cysteine is an additional con-
served residue that can be predicted to interact with
ATP. Supporting this view, we estimated in modeling
work on 2-Cys Prx that the nitrogen atom in the indol
ring of Trp179 is located 1.571 and 0.401 nm from the
sulfur atoms of the peroxidatic and resolving cysteines,
respectively [31]. Taken together, the close proximity
to Trp179 and the requirement for sequential reduc-
tion fi oxidation raised the possibility that Cys175
was actively involved in incorporation of the phos-
phoryl moiety. Consistent with this, Fig. 3C shows
that a serine in place of Cys53 and Cys175 retained
and abrogated, respectively, the ability to incorporate
the
32
P-label into 2-Cys Prx. Notably, this active par-
ticipation of the resolving cysteine in the autophospho-
rylation uncovered a new function that departed
markedly from the known role in the peroxidase
activity.
Surprisingly, autophosphorylation of C53S
2-Cys Prx did not require successive incubation with
dithiothreitol and the hydroperoxide but it was extre-

mely sensitive to the addition of dithiothreitol
(Fig. 3D). Given that the sulfur atom in the cysteine
residues of proteins can adopt various oxidation
numbers, our preparations of C53S 2-Cys Prx may
have contained some proportion of spontaneously
oxidized Cys175. To evaluate this possibility, C53S
2-Cys Prx was digested with trypsin and the peptides
were examined by MS. A peak at m ⁄ z 2800.36
exhibited the expected mass of the intrapeptide span-
ning from residue 160 to residue 184 [-RflT
160
LQAL-
QYVQENPDEVCPAGWKPGEK
184
flS-], wherein the
sulfur atom of Cys175 was totally reduced (Fig. 4).
Of note, the presence of the sulfhydryl group at
Cys175 was confirmed in parallel experiments in
which MS studies were conducted with the adduct
formed between C53S 2-Cys Prx and iodoacetate
(not shown). Because the less intense peak at
m ⁄ z 2832.36 was consistent with the addition of two
A
CD
B
Fig. 3. Autophosphorylation of 2-Cys Prx. (A) Experimental outline.
(B) Requirement of reductants and oxidants. 2-Cys Prx was (a)
incubated successively with 10 m
M dithiothreitol (DTT), 10 mM
cumene hydroperoxide (CuOOH) and [c

32
P]ATP, (b) subjected to
non-reducing SDS ⁄ PAGE, and (c) transferred to nitrocellulose
membranes for protein estimation and autoradiography, as
described in Experimental procedures. (C) Role of conserved
tryptophan and cysteine residues. W88F, W179F, C53S and
C175S 2-Cys Prx were incubated, as indicated, with 10 m
M
dithiothreitol, 10 mM cumene hydroperoxide and [c
32
P]ATP prior to
non-reducing SDS ⁄ PAGE and autoradiography, as outlined in (A).
(D) Autophosphorylation of C53S 2-Cys Prx. C53S 2-Cys Prx was
incubated for 10 min only in the presence and absence of 10 m
M
dithiothreitol prior to the addition of [c
32
P]ATP, non-reducing
SDS ⁄ PAGE and autoradiography.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1455
oxygen atoms to the respective 160–184 tryptic pep-
tide, we further analyzed the sequence of informative
ions to confirm the presence of a sulfinic group at
Cys175. Accordingly, fragment ions from y1 to y9
showed the expected mass for residues spanning
from Lys184 to Pro176, whereas trapped ions
beyond y10 exhibited a mass shift of 32. The
unequivocal assignment of two oxygen atoms to
the Cys175 residue of C53S 2-Cys Prx revealed the

unsuspected formation of oxyacid groups at sulfur
atoms of the resolving cysteine.
ATP phosphorylates the sulfinic and sulfonic
forms of the Cys175 residue
Although the Tyr166 residue in the 160–184 peptide
appeared in silico to be one site for the incorporation
of a phosphoryl group (netphos 2.0, Expassy), we
found in separate experiments that autophosphoryla-
tion of the Y166F mutant was similar to wild-type
2-Cys Prx. Given the absence of another putative site,
we approached the localization of the phosphoryl moi-
ety by examining the mass spectra of proteolytic digests
obtained from 2-Cys Prx treated successively with
dithiothreitol fi cumene hydroperoxide fi ATP. To
locate any modification in the sequence of the 160–184
peptide, we relied on not only the difference in masses
(m ⁄ z value), but also the y-series, the complementary
b-series and the coincidence of stretches assigned to
identical signals from different experiments. In these
analyses, the peaks at m ⁄ z 2800.36 and 2832.36
reflected the expected mass of the 160–184 peptide
holding at Cys175 a sulfhydryl group and two addi-
tional oxygen atoms, respectively (Fig. 5A). As illus-
trated for the former signal and in consonance with
above results (see Fig. 4), the y- and b-ion series
obtained for selected trapped ions confirmed that the
sulfur atom of the resolving Cys175 bore a sulfhydryl
group. From the repertoire of less intense signals but
with low noise levels, two novel species at m ⁄ z 2934.36
and 2950.35 were particularly attractive because the

masses matched the monosodium adducts [M + Na]
+
of the phosphorylated 160–184 peptide bearing sulfinic
and sulfonic groups, respectively (Fig. 5B) [32–34]. As
illustrated for the latter signal, sequence informative
y-ions from m ⁄ z 0 to 970 were identical to those
obtained in the spectra of m ⁄ z 2800.36 (Fig. 5A) and
2832.36 (see Fig. 4), thus proving that they originated
from the 160–184 peptide. But more importantly, the
absence of ions from y10 to y19 and the presence of
Fig. 4. MS ⁄ MS spectra of the 160–184 tryptic peptide from C53S 2-Cys Prx. Expanded view of peaks at m ⁄ z 2800.36 and 2832.35 and the
fragmentation of the peak at m ⁄ z 2832.35. 2-Cys Prx was digested with trypsin and prepared for MALDI-TOF MS as described in Experi-
mental procedures. Data were first collected, smoothed and calculated the centroid using the software
FLEXANALYSIS, and then plotted in
GRAPHPAD PRISM. All labeled peaks were at least three times above background. The amino acid sequence of the 160–184 tryptic peptide
bearing the sulfinic group is displayed above the spectrum. The fragmentation patterns that generate ions y and b are illustrated along the
peptide sequence wherein (*) are fragment ions bearing –SO
2
H.
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1456 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
shifted ions from y10* to y17* revealed that the sul-
fonic form of Cys175 held the monosodium adduct of
one phosphoryl group (-SO
3
PO
3
2)
) thereby providing
the first direct evidence for the phosphorylation of an

oxyacid group at a cysteine residue. The diagnostic
value of MS profiles regarding mainly the selected
peaks was confirmed in a total of 17 independent
spectra obtained with different instruments and
samples.
Discussion
Over the last decade it has become apparent that
2-Cys Prx is a key component of signal transduction
A
B
Fig. 5. MS ⁄ MS spectra of the 160–184 tryptic peptide from 2-Cys Prx. Phosphorylated 2-Cys Prx was digested with trypsin and prepared
for MALDI-TOF MS as described in Experimental Procedures. Data were examined as described in Fig. 4. The amino acid sequence of the
160–184 tryptic peptide bearing unphosphorylated and phosphorylated cysteines are displayed above the spectrum. The fragmentation pat-
terns that generate ions y and b are illustrated along the peptide sequence wherein (*) are fragment ions bearing –SO
3
PO
3
HNa. (A)
Expanded view of peaks at m ⁄ z 2800.36 and 2832.35 and the fragmentation of the peak at m ⁄ z 2800.36. (B) Expanded view of peaks at
m ⁄ z 2934.36 and 2950.35 and the fragmentation of the peak at m ⁄ z 2950.35.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1457
pathways, ultimately controlling proteins involved in
diverse cellular processes, such as cell proliferation,
differentiation, apoptosis and photosynthesis [25,35–
38]. This study is the first to demonstrate that the
activities associated with 2-Cys Prx are regulated
directly by mechanisms sensitive to nucleotides and
bivalent cations in which the concerted action of
both compounds reversibly impairs the peroxidase

activity, whereas only Mg
2+
lowers the chaperone
capacity [10,11]. In addition to the differential regula-
tion of the dual functions, inhibition of the peroxi-
dase activity is highly specific because, of the
nucleotides presented here, purine derivatives are
markedly more effective than pyrimidine bases. Given
that nucleotides do not participate directly in the
reduction of hydroperoxides, it follows that the
observed loss of activity is almost certainly due to a
local effect on the structure of the protein (see
below). These findings are important for understand-
ing the fundamental question of how 2-Cys Prx uti-
lizes non-redox compounds to regulate the associated
functions and, in so doing, to cope with situations of
oxidative stress. This extremely rapid and reversible
association with low molecular mass compounds
devoid of redox capacity may have wide applicability
because we recently reported that 2-Cys Prx in con-
certed action with fructose-1,6-bisphosphate and
Ca
2+
stimulates the activity of chloroplast fructose-
1,6-bisphosphatase [25].
2-Cys Prx is an obligate homodimer (a
2
) whose con-
version to doughnut-shaped (a
2

)
5
decamer is redox
sensitive [24]. Apropos, oxidants drive the human and
yeast orthologues from lower molecular mass forms to
higher molecular mass complexes and, in so doing,
impair the peroxidase activity and enhance the chaper-
one capacity [10,11]. Although the transition of
2-Cys Prx among oligomers with different molecular
masses may be conceptually adequate for the regula-
tion of associated functions [24,27], the unprecedented
ATP-mediated oligomerization is beyond the scope of
this study and it will be reported elsewhere. However,
spectroscopic studies of 2-Cys Prx variants clearly dis-
cerned the role of ATP. UV-differential spectropho-
tometry and fluorescence emission of the extrinsic
probe ANS initially revealed that the protein interacts
directly with ATP, and further exploration of the
intrinsic fluorescence emission in site-directed mutants
unambiguously assigned the binding site close to
Trp179. In the crystal structure of human 2-Cys Prx,
this region encloses a cavity large enough to hold nu-
cleotides in which the tryptophan residue homologous
to rapeseed Trp179 is located far from the peroxidatic
Cys53 and close to the resolving Cys175 [31]. Given
that (a) the mechanism of peroxidase activity includes
the formation of an intercatenary disulfide bond link-
ing the peroxidatic cysteine with the resolving counter-
part [3] and (b) ATP locates near the latter (this
study), it is reasonable to suggest that the reversible

binding of ATP ⁄ Mg
2+
halts the catalytic cycle via ste-
ric perturbation of the resolving cysteine. However, we
can not exclude the possibility that the reduction of
hydroperoxides is inhibited by an allosteric effect of
ATP ⁄ Mg
2+
on the peroxidatic cysteine. Although fur-
ther studies are required to clarify this issue, our data
definitively identify the region surrounding the resolv-
ing cysteine of typical 2-Cys Prx as the target for
nucleotides.
The main outcome of our study is, however, the
importance of oxyacid groups at the resolving Cys175
for the in vitro autophosphorylation of 2-Cys Prx. A
combination of evidence from the lack of a similar
capacity in other proteins to the behavior of site-direc-
ted mutants clearly dismiss the possibility that trace
quantities of contaminating bacterial kinases may co-
purify with the recombinant protein [38]. The finding
that the successive addition of a reductant and an oxi-
dant promotes incorporation of the c-phosphoryl moi-
ety of ATP indicates that, like other events mediated
by 2-Cys Prx, autophosphorylation depends on a spe-
cific redox state. The 23 kDa subunit contains two
cysteines conserved throughout evolution, and analyses
of site-directed mutants show that Cys175 holds the
unique reactive thiol involved in autophosphorylation.
Moreover, MS detection of over-oxidized sulfur atoms

at the resolving cysteine led us to conclude that the
sulfonic and sulfinic forms are necessary for linking
the phosphoryl moiety to the protein. Further exami-
nation of the oxidative step reveals that the autophos-
phorylation proceeds in redox environments milder
than those induced by harsh oxidants. Indeed, the
midpoint redox potentials of 2-hydroxy-1,4-naphtho-
quinone (E
m7
= )0.15 V) and 1,4-dihydroxy-9,10-
anthraquinone (E
m7
= )0.18 V) are much lower than
H
2
O
2
(E
m7
= )1.76 V) which is usually used in studies
of ROS [1,2,4]. These data uncover the capacity of the
rapeseed resolving Cys175 for the oxidation to sulfinic
acid, a process that clearly departs from similar sulfur
chemistry at the peroxidatic cysteine [19,20,39,40].
Two lines of research have examined the phos-
phorylation of 2-Cys Prx. First, it has been shown
that several cyclin-dependent protein kinases promote
in vitro the specific phosphorylation of human
2-Cys Prx at a threonine residue homologous to
Thr91 in the rapeseed orthologue [17]. Second,

the finding that the thiol of mammalian sulfiredoxin
[Srx-SH] recruits the c-phosphoryl moiety of ATP
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1458 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
yielding a thiophosphate [Srx-S-PO
3
2)
] led to the
proposal that sulfiredoxin subsequently transfers the
phosphoryl group to the sulfinic form of the peroxid-
atic cysteine in human PrxI [-Cys-S(=O)-OH] [19–21].
At this stage, the sulfinic–phosphoric mixed anhydride
[-Cys-S(=O)-O-PO
3
2)
] would be cleaved by a thiol
reductant [R-S-H] yielding a disulfide-S-monoxide
[-Cys-S(=O)-S-R] that would be finally reduced back
to thiol [-Cys-SH]. In this context, the strategy of our
phosphorylation of 2-Cys Prx diverges markedly from
previous studies in two important aspects: neither
requires a complementary catalyst, like cyclin-depen-
dent kinases or sulfiredoxin, nor proceeds via Thr91
or the peroxidatic cysteine. Indeed, our data provide
entry into a previously unsuspected mechanism by
which the successive reductionfioxidation of 2-Cys
Prx generates oxyacid groups at Cys175 for the subse-
quent formation of the sulfinic-phosphoryl [-(Cys175)-
SO
2

PO
3
2)
] and sulfonic-phosphoryl [-(Cys175)-SO
3
PO
3
2)
] anhydrides (Scheme 1). Related to this, the
mechanism by which dithiothreitol alone dramatically
abrogates the autophosphorylation remains unknown.
Does the reductant impair the process (a) before
incorporation of the phosphoryl moiety by remov-
ing the oxyacid groups or (b) after formation of
-(Cys175)-S(=O)
1–2
-O-PO
3
2)
by cleaving the mixed
anhydride? The answer to these questions will reveal
whether the oxyacid group itself at Cys175 or the
sulfi(o)nic-phosphoric anhydride are endowed with an
unusual reactivity to reductants.
Although many studies have concentrated on the
events underlying phosphorylation of the peroxidatic
cysteine of 2-Cys Prx [19–21], we know of none that
addressed the resolving cysteine. In almost all typical
2-Cys Prx, the function of the latter residue was hith-
ertho confined to participating in the formation of an

intercatenary disulfide bond with the sulfenic acid of
the peroxidatic cysteine. Moreover, in line with the
current paradigm on the mechanism for the reduction
of hydroperoxides [8], the resolving cysteines of try-
paredoxin peroxidase and AhpC from Trypanosoma
brucei brucei and Salmonella typhymurium have been
identified as targets in the reduction of the disulfide
bond for the reactivation of peroxidase activity
[41,42]. Against this background, we put forward a
new scenario wherein ATP interacts actively with
2-Cys Prx and, in so doing, modifies the quaternary
structure and associated functions. Moreover, the
unusual phosphorylation of Cys175 oxyacid groups
brings together the redox chemistry of the sulfur atom
and the phosphorylating capacity of ATP, thereby
providing a versatile mechanism wherein Cys175
appears as dual sensor able to perceive changes in the
redox and energy status of the cell. By virtue of the
flexibility of using redox and nonredox chemistries at
a single cysteine residue, the possibilities to process a
wide spectrum of stimuli into different cellular
responses greatly extend the prevalent view circum-
scribed to redox transformations of sulfhydryl groups
[43].
Experimental procedures
Materials
Recombinant rapeseed 2-Cys Prx was prepared as described
previously [25]. Biochemicals were purchased from Sigma-
Aldrich (St Louis, MO, USA).
Construction of 2-Cys Prx mutants

C-terminal hexahistidine-tagged variants of 2-Cys Prx were
generated by the PCR megaprimer method using, in the
Scheme 1. Reaction scheme for the autophosphorylation of
2-Cys Prx. A reductant cleaves the intercatenary disulfide bond
yielding the reduced form of the sulfur atom at Cys175 (reaction 1).
The subsequent oxidation transforms the sulfhydryl group into the
sulfinic and sulfonic species (reaction 2). The reactivities of these
groups and the close proximity to the ATP binding site facilitate the
incorporation of the c–phosphoryl moiety (reaction 3) linking in con-
sequence redox and nonredox chemistries. Eventually, a phospha-
tase facilitates the hydrolysis of the phosphoryl group and returns
Cys175 to the oxidized state.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1459
first round of amplification, the cDNA of rapeseed
2-Cys Prx cloned in pET-22b(+) vector as template, 5¢-
TAATACGACTCACTATAGG-3¢ [for the T7 promoter of
pET-22b(+) vector] as 5¢-primer and 5¢-TCTCCGTAGG
GGAGACAAAAGT-3¢,5¢-ATCCCGCGGGGGAAACCT
CATC-3¢ and 5¢-CTGTTTGGAC
GAACGCAAGATG-3¢
as 3¢-primers for C53S, C175S and W88F variants, respec-
tively (mutated codons are underlined) [44]. The amplified
DNA and 5¢-GCCAGTTATTGCTCAGCGG-3¢ [for the
T7 terminator of pET-22b(+) vector] were used as 5¢- and
3¢-primers, respectively, in the second round of DNA
amplification. To construct the W179F variant, the muta-
tion was similarly introduced by PCR using first the 5¢-pri-
mer 5¢-GGGATTCAAGCCTGGGGAGAAATC-3¢ and
the 3¢-flanking T7 terminator and subsequently the 3¢-mega-

primer and the 5¢-flanking for the T7 promoter. After
amplification, all DNA fragments were cloned at the
XbaI ⁄ XhoI restriction sites of the pET-22b(+) vector and
mutations were confirmed by DNA sequentiation.
Protein purification
JM109 (kDE3) Escherichia coli cells harboring 2-Cys Prx
expression plasmids were grown at 37 °C in Luria–Bertani
medium supplemented with 100 lgÆmL
)1
ampicillin. After
induction with 0.6 mm isopropyl b-d-thiogalactoside, bacte-
ria were harvested by centrifugation (3000 g, 10 min),
washed with 20 mm Tris ⁄ HCl (pH 8.0), resuspended in the
same buffer containing 0.5 m NaCl and subjected to sonica-
tion. After centrifugation (20 000 g, 30 min), the superna-
tant fraction was loaded onto a Ni
2+
-iminodiacetate–
Sepharose column that was washed successively with
20 mm Tris⁄ HCl (pH 8.0) containing 20 and 100 mm imid-
azole. The fusion protein released in the latter elution was
dialyzed against and stored in 20 mm Tris ⁄ HCl (pH 8.0). A
molar extinction coefficient of 23.555 m
)1
Æcm
)1
at 280 nm
was used to estimate the protein content of homogeneous
2-Cys Prx preparations.
Assay of peroxidase activity

The peroxidase assay was performed at 25 °C by follow-
ing the consumption of either H
2
O
2
or dithiothreitol.
In the former assay, 0.130 mm H
2
O
2
were added to a
solution containing 100 mm Tris ⁄ HCl (pH 7.0), 10 mm
dithiothreitol and 3 l m 2-Cys Prx, supplemented with
nucleotides and bivalent cations when appropriate (final
volume: 1 mL). At selected times, aliquots were withdrawn
to estimate the remaining H
2
O
2
via the oxidation of Fe
2+
in the presence of xylenol orange [45]. Alternatively, the
catalytic reaction was carried out similarly (final volume:
0.5 mL), except that the concentration of dithiothreitol
was 0.5 m m and the remaining dithiothreitol was mea-
sured spectrophotometrically at 420 nm after the reaction
with the Ellman’s reagent [46].
Assay of chaperone activity
The chaperone activity was estimated essentially by the cit-
rate synthase assay [47]. 2-Cys Prx (3–10 lm) dissolved in

0.4 mL of 25 mm Tris ⁄ HCl (pH 8) was incubated for
10 min at 25 °C and subsequently for 10 min at 45 °C. The
assay was started by the addition of porcine heart citrate
synthase to a final concentration of 150 nm. Thermal aggre-
gation of citrate synthase was measured for 60 min by
monitoring light scattering at 360 nm in a Jasco FP 770
spectrofluorometer (excitation and emission light-paths: 0.2
and 1 cm, respectively).
Static light scattering measurements
The average molecular mass of 2-Cys Prx was determined
at 25 °C on a Precision Detectors PD2010 light-scattering
instrument connected in tandem to a Sephadex G-50 col-
umn and a LKB 2142 differential refractometer. 2-Cys Prx
(43 lm) was incubated for 5 min in 50 mm Tris ⁄ HCl
(pH 7.8) in the presence and absence of 3 mm ATP ⁄ 3mm
Mg
2+
and subsequently applied to a Sephadex G-50 col-
umn which had been equilibrated beforehand with the incu-
bation solution. The 90° light scattering and refractive
index signals of the eluting material were transferred to a
PC and analyzed with the discovery32 software supplied
by the manufacturer. The 90° light scattering detector was
calibrated using bovine serum albumin (66.5 kDa) as a
standard. Molecular masses were determined from the ratio
of the two detectors, light-scattering and refractive index,
using the Rayleigh-Debye-Gans light-scattering model for
dilute polymer solutions.
Intrinsic fluorescence measurements
The steady-state fluorescence of 2 lm 2-Cys Prx in 20 mm

Tris ⁄ HCl (pH 7.8) containing 2 mm MgCl
2
was measured
at 25 °C in a Jasco FP 770 spectrofluorometer. Tryptophan
emission spectra were taken from 305 to 370 nm using an
excitation wavelength of 295 nm and excitation and emis-
sion bandwidths of 4 and 5 nm, respectively. To minimize
the inner filter effect, aliquots from a 0.1 m ATP stock
solution were sequentially added to a final concentration of
0.2 mm. After correction for the inner filter effect and aver-
aging three successive emission spectra, we analyzed the
data using the Stern–Volmer equation which describes colli-
sional quenching processes, (F
o
⁄ F) ) 1=K
SV
.[Q], where
F
o
and F are the fluorescence intensity in the absence and
the presence of quencher, respectively, K
SV
is the apparent
quenching constant and [Q] is the concentration of the
quencher (i.e. ATP). When the Stern–Volmer analyses
were not linear, we relied on the equation described by
Lehrer for two populations of fluorophores [28,29],
F
o
⁄ (F

o
) F)=1⁄ f
a
+(1⁄ {f
a
.K
SV
.[Q]}), where f
a
is a quen-
ching factor due to the presence of multiple fluorophores
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1460 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
in different environments. Nonlinear least-squares regres-
sion analyses were performed with the program graphpad
prism.
Autophosphorylation of 2-Cys Prx
Protein phosphorylation was carried out at 25 °Cin
0.04 mL of 50 mm Tris ⁄ HCl (pH 7.9) containing 10 lm
2-Cys Prx. The protein was incubated for 10 min at each
stage with (a) 10 mm dithiothreitol, (b) 10 mm cumene
hydroperoxide and (c) 0.1 mm (2.5 lCi) [c
32
P]ATP plus
3mm MgCl
2
. After the addition of SDS⁄ PAGE sample
buffer, proteins were transferred to nitrocellulose mem-
branes and detected by staining with Ponceau Red followed
by autoradiography. In control experiments, dithiothreitol

was replaced with other reductants (2-mercaptoethanol,
reduced glutathione) and cumene hydroperoxide with other
oxidants (H
2
O
2
, t-butyl hydroperoxide, quinones).
MS analyses
2-Cys Prx (10 lm) was (a) incubated for 10 min with
10 mm dithiothreitol and subsequently with 10 mm cumene
hydroperoxide, (b) dialyzed to remove the reductant and
oxidant, (c) incubated for 10 min with 0.1 mm ATP, and
(d) concentrated in a SpeedVac centrifuge. After addition
of ammonium bicarbonate (pH 8.5) and urea to a final con-
centration of 0.1 and 2 m, respectively, 2-Cys Prx was
digested at 37 °C for 2 h with trypsin (2-Cys Prx ⁄ trypsin
100 : 1, w ⁄ w). A matrix of a-cyano-4-hydroxy-cinnamic
acid (5 mgÆmL
)1
in 50% acetonitrile containing 0.1% tri-
fluoroacetic acid) was mixed with the sample in a ratio of
8 : 2, spotted onto the Anchor-Chip plate and air dried.
Peptide spectra over a mass range of m ⁄ z 500–3500 and
MS ⁄ MS spectra of select ions were collected on an Ultra-
flex II MALDI-TOF ⁄ TOF (Bruker Daltonik, Bremen,
Germany). Peak identification and monoisotopic peptide
mass assignation were automatically performed using the
software flexanalysis (Bruker Daltonik). The spectra
obtained were interpreted using the FindPept tool (http://
ca.expasy.org/tools/findpept.html) and the MS-Product of

ProteinProspector ( />4.0.7/html/msprod.htm).
Acknowledgements
We acknowledge Gaston Mayol for technical help.
The assistance of Dr Silvia Moreno de Colonna, Dr
Gonzalo Prat Gay and Dr Leonardo Alonso with the
mass spectroscopy is gratefully appreciated. This study
was supported by grants from the Agencia Nacional
de Promocio
´
n Cientı
´
fica y Tecnolo
´
gica and the Uni-
versidad de Buenos Aires, and doctoral fellowships
from the Consejo Nacional de Investigaciones Cientı
´
fi-
cas y Te
´
cnicas (MA, DC, AS and MRG). MTI, ASL
and RAW are Established Investigators of the latter
institution.
References
1 Toledano MB, Delaunay A, Monceau L & Tacnet F
(2004) Microbial H
2
O
2
sensors as archetypical redox

signaling modules. Trends Biochem Sci 29, 351–357.
2 Rhee SG, Kang SW, Jeong W, Chang TS, Yang KS &
Woo HA (2005) Intracellular messenger function of
hydrogen peroxide and its regulation by peroxiredoxins.
Curr Opin Cell Biol 17, 183–189.
3 Wood ZA, Schroder E, Harris JR & Poole LB (2003)
Structure, mechanism and regulation of peroxiredoxins.
Trends Biochem Sci 28, 32–40.
4 Peskin AV, Low FM, Paton LN, Maghzal GJ, Hamp-
ton MB & Winterbourn CC (2007) The high reactivity
of peroxiredoxin 2 with H
2
O
2
is not reflected in its reac-
tion with other oxidants and thiol reagents. J Biol Chem
282, 11885–11892.
5 Radyuk SN, Sohal RS & Orr WC (2003) Thioredoxin
peroxidases can foster cytoprotection or cell death in
response to different stressors: over- and under-expres-
sion of thioredoxin peroxidase in Drosophila cells.
Biochem J 371, 743–752.
6 Klughammer B, Baier M & Dietz KJ (1998) Inactiva-
tion by gene disruption of 2-Cys-peroxiredoxin in Syn-
echocystis sp. PCC 6803 leads to increased stress
sensitivity. Physiol Plant 104, 699–706.
7 Hofmann B, Hecht HJ & Flohe L (2002) Peroxiredox-
ins. Biol Chem 383, 347–364.
8 Copley SD, Novak WRP & Babbitt PC (2004) Diver-
gence of function in the thioredoxin fold suprafamily:

evidence for evolution of peroxiredoxins from a thio-
redoxin-like ancestor. Biochemistry 43, 13981–13995.
9 Noguera-Mazon V, Krimm I, Walker O & Lancelin JM
(2006) Protein–protein interactions within peroxiredoxin
systems. Photosynth Res 89, 277–290.
10 Moon JC, Hah YS, Kim WY, Jung BG, Jang HH, Lee
JR, Kim SY, Lee YM, Jeon MK, Kim CW et al. (2005)
Oxidative stress-dependent structural and functional
switching of a human 2-Cys peroxiredoxin isotype II
that enhances HeLa cell resistance to H
2
O
2
-induced cell
death. J Biol Chem 280, 28775–28784.
11 Jang HH, Lee KO, Chi YH, Jung BG, Park SK, Park
JH, Lee JR, Lee SS, Moon JC, Yun JW et al. (2004)
Two enzymes in one; two yeast peroxiredoxins display
oxidative stress-dependent switching from a peroxidase
to a molecular chaperone function. Cell 117, 625–635.
12 Kang SW, Rhee SG, Chang TS, Jeong W & Choi MH
(2005) 2-Cys peroxiredoxin function in intracellular sig-
nal transduction: therapeutic implications. Trends Mol
Med 11, 571–578.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1461
13 Mora-Garcia S, Stolowicz F & Wolosiuk RA (2005)
Redox signal transduction in plant metabolism. In Con-
trol of Primary Metabolism in Plants. Annual Plant
Reviews (Plaxton W & McManus M, eds), vol. 22,

pp. 150–186.
14 Bald D, Noji H, Yoshida M, Hirono-Hara Y &
Hisabori T (2001) Redox regulation of the rotation of
F1-ATP synthase. J Biol Chem 276, 39505–39507.
15 Wu G, Ortiz-Flores G, Ortiz-Lopez A & Ort DR (2007)
A point mutation in atpC1 raises the redox potential of
the Arabidopsis chloroplast ATP synthase c-subunit
regulatory disulfide above the range of thioredoxin
modulation. J Biol Chem 282, 36782–36789.
16 Xu SZ, Sukumar P, Zeng F, Li J, Jairaman A, English
A, Naylor J, Ciurtin C, Majeed Y, Milligan CJ et al.
(2008) TRPC channel activation by extracellular
thioredoxin. Nature 451, 69–73.
17 Chang TS, Jeong W, Choi SY, Yu S, Kang SW & Rhee
SG (2002) Regulation of peroxiredoxin I activity by
Cdc2-mediated phosphorylation. J Biol Chem 277,
25370–25376.
18 Jang HH, Kim SY, Park SK, Jeon HS, Lee YM, Jung
JH, Lee SY, Chae HB, Jung YJ, Lee KO et al. (2006)
Phosphorylation and concomitant structural changes in
human 2-Cys peroxiredoxin isotype I differentially regu-
late its peroxidase and molecular chaperone functions.
FEBS Lett 580, 351–355.
19 Biteau B, Labarre J & Toledano MB (2003)
ATP-dependent reduction of cysteine-sulphinic acid by
S. cerevisiae sulphiredoxin. Nature 425, 980–984.
20 Jonsson TJ, Murray MS, Johnson LC, Poole LB &
Lowther WT (2005) Structural basis for the retroreduc-
tion of inactivated peroxiredoxins by human sulfi-
redoxin. Biochemistry 44, 8634–8642.

21 Jeong W, Park SJ, Chang TS, Lee DY & Rhee SG
(2006) Molecular mechanism of the reduction of cyste-
ine sulfinic acid of peroxiredoxin to cysteine by mam-
malian sulfiredoxin. J Biol Chem 281, 14400–14407.
22 Horovitz A & Willison KR (2005) Allosteric regulation
of chaperonins. Curr Opin Struct Biol 15, 646–651.
23 Gast K & Modler AJ (2005) Studying protein folding
and aggregation by laser light scattering. In Protein
Folding Handbook, Vol. 2 (Buchner J & Kiefhaber T,
eds), pp. 673–736. Wiley-VCH, Weinheim.
24 Wood ZA, Poole LB, Hantgan RR & Karplus PA
(2002) Dimers to doughnuts: redox-sensitive oligomeri-
zation of 2-cysteine peroxiredoxins. Biochemistry 41,
5493–5504.
25 Caporaletti D, D’Alessio AC, Rodriguez-Suarez RJ,
Senn AM, Duek PD & Wolosiuk RA (2007) Non-
reductive modulation of chloroplast fructose-1,6-
bisphosphatase by 2-Cys peroxiredoxin. Biochem
Biophys Res Commun 355 , 722–727.
26 Meissner U, Schroder E, Scheffler D, Martin AG &
Harris JR (2007) Formation, TEM study and 3D recon-
struction of the human erythrocyte peroxiredoxin-2
dodecahedral higher-order assembly. Micron 38 , 29–39.
27 Konig J, Lotte K, Plessow R, Brockhinke A, Baier M
& Dietz KJ (2003) Reaction mechanism of plant 2-Cys
peroxiredoxin. Role of the C terminus and the quater-
nary structure. J Biol Chem 278, 24409–24420.
28 Lehrer SS (1971) Solute perturbation of protein fluores-
cence. The quenching of the tryptophyl fluorescence
of model compounds and of lysozyme by iodide ion.

Biochemistry 10, 3254–3263.
29 Eftink MR & Ghiron CA (1976) Exposure of trypto-
phanyl residues in proteins. Quantitative determination
by fluorescence quenching studies. Biochemistry 15,
672–680.
30 Manning M & Colon W (2004) Structural basis of
protein kinetic stability: resistance to sodium dodecyl
sulfate suggests a central role for rigidity and a bias
toward b-sheet structure. Biochemistry 43, 11248–11254.
31 Schroder E, Littlechild JA, Lebedev AA, Errington N,
Vagin AA & Isupov MN (2000) Crystal structure of
decameric 2-Cys peroxiredoxin from human erythro-
cytes at 1.7 A
˚
resolution. Struct Fold Des 8, 605–615.
32 Chen SL, Huddleston MJ, Shou W, Deshaies RJ,
Annan RS & Carr SA (2002) Mass spectrometry-based
methods for phosphorylation site mapping of hyper-
phosphorylated proteins applied to Net1, a regulator of
exit from mitosis in yeast. Mol Cell Proteomics 1, 186–
196.
33 Dormeyer W, Ott M & Schno
¨
lzer M (2005) Probing
lysine acetylation in proteins: strategies, limitations, and
pitfalls of in vitro acetyltransferase assays. Mol Cell
Proteomics 4, 1226–1239.
34 Ham BM, Jacob JT & Cole RB (2005) MALDI-TOF
MS of phosphorylated lipids in biological fluids using
immobilized metal affinity chromatography and a solid

ionic crystal matrix. Anal Chem 77, 4439–4447.
35 Dietz KJ (2003) Plant peroxiredoxins. Annu Rev Plant
Biol 54, 93–107.
36 Geourgiou G & Masip L (2003) An overoxidation jour-
ney with a return ticket. Science 300, 592–594.
37 Neumann CA, Krause DS, Carman CV, Das S, Dubey
DP, Abraham JL, Bronson RT, Fujiwara Y, Orkin SH
& vanEtten RA (2003) Essential role for the peroxire-
doxin Prdx1 in erythrocyte antioxidant defence and
tumor suppression. Nature 424, 561–565.
38 Bakal CJ & Davies JE (2000) No longer an exclusive
club: eukaryotic signalling domains in bacteria. Trends
Cell Biol 10, 32–38.
39 Woo HA, Jeong W, Chang TS, Park KJ, Park SJ, Yang
JS & Rhee SG (2005) Reduction of cysteine sulfinic acid
by sulfiredoxin is specific to 2-Cys peroxiredoxins.
J Biol Chem 280, 3125–3128.
40 Rey P, Becuwe N, Barrault MB, Rumeau D, Havaux
M, Biteau B & Toledano MB (2007) The Arabidopsis
thaliana sulfiredoxin is a plastidic cysteine–sulfinic acid
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1462 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
reductase involved in the photooxidative stress
response. Plant J 49, 505–514.
41 Budde H, Flohe L, Hofmann B & Nimtz M (2003) Ver-
ification of the interaction of a tryparedoxin peroxidase
with tryparedoxin by ESI-MS ⁄ MS. Biol Chem 384,
1305–1309.
42 Jonsson TJ, Ellis HR & Poole LB (2007) Cysteine reac-
tivity and thiol-disulfide interchange pathways in AhpF

and AhpC of the bacterial alkyl hydroperoxide reduc-
tase system. Biochemistry 46, 5709–5721.
43 Cooper CE, Patel R, Brookes PS & Darley-Usmar VM
(2002) Nanotransducers in cellular redox signaling:
modification of thiols by reactive oxygen and nitrogen
species. Trends Biochem Sci 27, 289–492.
44 Sarkar G & Sommer S (1990) The ‘megaprimer’ method
of site-directed mutagenesis. BioTechniques 8, 404–407.
45 Wolff SP (1994) Ferrous ion oxidation in the presence
of ferric ion indicator xylenol orange for measurement
of hydroperoxides. Methods Enzymol 233, 182–189.
46 Riddles PW, Blakeley RL & Zerner B (1983) Reassess-
ment of Ellman’s reagent. Methods Enzymol 91, 49–60.
47 Studer S, Obrist M, Lentze N & Naberhaus F (2002) A
critical motif for oligomerization and chaperone activity
of bacterial a-heat shock proteins. Eur J Biochem 269,
3578–3586.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Effect ATP-Mg
2+
on UV differential spectra
of rapeseed 2-Cys Prx.
Fig. S2. W88F and W179F 2-Cys Prx.
Fig. S3. Autophosphorylation of 2-Cys Prx.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1463