Isolation and characterization of a thioredoxin-dependent
peroxidase from
Chlamydomonas reinhardtii
Aymeric Goyer
1
, Camilla Hasleka
Ê
s
2
, Myroslawa Miginiac-Maslow
1
, Uwe Klein
2
, Pierre Le Marechal
3
,
Jean-Pierre Jacquot
4
and Paulette Decottignies
3
1
Institut de Biotechnologie des Plantes, Universite
Â
Paris-Sud, Orsay Cedex, France;
2
Department of Biology,
Division of Molecular Biology, University of Oslo, Blindern, Oslo, Norway;
3
IBBMC, Universite
Â
Paris-Sud, Orsay Cedex,

Keywords: Chlamydomonas; peroxiredoxin; thioredoxin;
redox signaling; oxidative stress.
1
Peroxiredoxins (Prx) form a ubiquitous group of peroxid-
ases found in bacteria [1], yeast [2,3], animals [4], and, more
recently, in higher plants [5±7]. Prx can be classi®ed
according to the number of conserved cysteine residues:
the 2Cys-Prx subgroup, and 1Cys-Prx subgroup contain
two and one conserved cysteines, respectively. 2Cys-Prx
proteins are reduced by the AhpF protein in bacteria, and
by the thioredoxin/thioredoxin reductase system in yeast
and animals, w hile 1Cys-Prx may be r educed by a small
thiol molecule such as glutathione. R ecently, 1Cys-Prx has
been identi®ed i n y east and Arabi dopsis, and has been
shown to be thioredoxin-dependent and function in a
similar manne r to 2 Cys-Prx [ 8,9].
2Cys-Prx catalyzes, in vitro, the red uction of alkyl
hydroperoxide and h ydrogen peroxide. These enzymes exist
as homodimers. Each subunit contains the two conserved
cysteines that are essential residues for the reduction of
peroxides. The N-terminal cysteine is ®rst oxidized by a
peroxide to sulfenic acid (Cys-SOH), which rapidly reacts
with the C-terminal cysteine of the other s ubunit to form an
intermolecular disul®de [10]. In animals, yeast, a nd plants,
the disul®de is reduced via a thiol/disul®de redox inter-
change with reduced thioredoxin (Trx), thus regenerating an
active peroxidase.
The present study was aimed at setting up an af®nity
chromatography column for speci®c trapping of proteins
that react w ith Trx, b ased on their a bility to f orm mixed

Â
Paris-Sud, 91405
Orsay Cedex, France. Fax: + 33 1 69 15 34 23,
E-mail:
Abbreviations:Nbs
2
,5,5¢-dithiobis(2-nitrobenzoic acid); Prx, perox-
iredoxin; t-BOOH, tertiobutyl hydroperoxide; Trx, thioredoxin; ROS,
reactive oxygen species; NTR, NADPH-dependent thioredoxin
reductase; DCMU, 3-(3¢,4¢-dichlorophenyl)-1,1-dimethyl urea;
DBMIB, 3-methyl-6-isopropyl-p-benzoquinone.
(Received 13 July 2001, revised 29 October 2001, accepted 31 October
2001)
Eur. J. Biochem. 269, 272±282 (2002) Ó FEBS 2002
tubes at 32 °C. Cultures were kept in a 12-h light/dark
regime (light intensity 150 lmolám
)2
ás
)1
) and bubbled
with 2% CO
2
-enriched air. Cell density was adjusted daily
at the onset of light with fresh medium to  2 ´ 10
6
cellsámL
)1
.
Puri®cation of Ch-Prx1
Puri®cation of the native protein from Chlamydomonas

cysteine residue and has only its most reactive a ctive-site
cysteine left (Cys36). Site-directed mutagenesis of Trx h and
recombinant protein puri®cation was carried out as
described previously [12]. T o avoid the formation of thio-
redoxin dimers, the single-cysteine mutant thioredoxin was
pretreated with a 40-fold excess of 5,5¢-dithiobis(2-nitro-
benzoic acid) (Nbs
2
; Pierce) before coupling. The deriv-
atized thioredoxin was treated with dithiothreitol to
eliminate t he 5-mercapto-2-nitrobenzoate
3
adduct, and was
washed extensively to remove the dithiothreitol. After
loading on the column, the proteins were eluted with
15 mL of 1 m
M
dithiothreitol in 30 m
M
Tris/HCl, pH 7.9.
The e luted p roteins w ere d ialyzed against 30 m
M
Tris/HCl,
pH 7.9 and re-applied on the af®nity column. P roteins were
eluted with 15 mL of 1 m
M
dithiothreitol in 30 m
M
Tris/
HCl, pH 7.9, and analyzed by SDS/PAGE.

20 min at 4 °C, the s uspension w as centrifuged at 20 000 g
for 3 0 min to precipitate the nucleic acids. The supernatant
was subjected to 35±80% (w/v) ammonium sulfate frac-
tionation. After centrifugation at 20 000 g for 30 min, the
pellet w as re su spend ed i n 10 mL 3 0 m
M
Tris/HCl, pH 7.9,
dialyzed against 5 L o f 30 m
M
Tris/HCl, pH 7 .9, and the
Ch-Prx1 protein was puri®ed as described above on a C39S
Trx h af®nity column equilibrated with 3 0 m
M
Tris/HCl,
pH 7.9.
Polyacrylamide gel electrophoresis
Denaturating [4% ( w/v) SDS] electrophoresis was carried
out on 10% polyacrylamide gels. G els were st ained w ith
CoomassieBlue(2.5gáL
)1
).
Tryptic digestion, separation of the tryptic peptides,
analysis by sequencing and MALDI-TOF mass
spectrometry
Further puri®cation of Ch-Prx1 was achieved, prior to
digestion, by RP-HPLC o n a 4.6 ´ 25 cm Vydac C 4 (30 A
Ê
)
column. C h-Prx1 was eluted with a linear gradient from 2 8
to 70% acetonitrile in 0.1% tri¯uoroacetic acid over 30 min

RT-PCR
Ch-Prx1 cDNA was isolated by RT-PCR. In this approach,
primers used in cDNA synthesis w ere designed based on the
amino-acid sequence of N- and C-terminal peptides, a s
determined by the p rocedure described above. First-strand
cDNA was ®rst synthesized from total RNA with
M-MuLV reverse t ranscriptase (Life T echnologies). The
reaction mixture c ontained i n a volume of 20 lL: 0.9 lg
heat-denatured Chlamydomonas total RNA, 1 ´ RT buffer,
1m
M
dNTPs, 20 m
M
dithiothreitol, 100 U reverse tran-
scriptase (RT), and 1 l
M
of the degenerated reverse primer
5¢-GCGGATCCTTA(G/C)ACGGCGGCGAAGTAC
TCC-3¢. After reverse transcription for 30 min at 42 °C, the
®rst-strand cDNA was ampli®ed in a PCR performed under
the following conditions: 5 min at 96 °C; 39 cycles (94 °C
for 1 min, 64 °C for 2 min, and 72 °Cfor2min).In
addition to the above degenerated reverse primer two
direct primers 5¢-AACCATGGCCTCCCACGCCGAGA
AGCC(G/C)CTG-3¢ and 5¢-AACCATGGCCAGCCAC
Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 273
GCCGAGAAGCC(G/C)CTG-3¢ were used. PCR products
were separated b y electrophoresis on a 0.8% agarose g el.
A 600-bp fragment was puri®ed by using the nucleospin
extract k it (Macherey±N agel), then digested by NcoIand

heterologous primers, (5 ¢-GACTTCACCTTCGTGTGCC
CCACCGAG-3¢ and 5¢-GGGGTCGATGATGAACAG
GCCGCG-3¢), designed from the conserved 5¢ and
3¢ sequences of known 2Cys peroxiredoxin genes and
optimized for the codon usage of Chlamydomonas,were
used to amplify by PCR from genomic Chlamydomonas
DNA a fragment (  780 bp) of the Chlamydomonas
peroxiredoxin gene. The PCR fragment was cloned,
sequenced, and used as a probe to identify a Chlamydo-
monas BAC clone that contains the Ch -Prx1 gene sequence.
Two BAC clones that strongly hybridized to the probe on
the ® lter were am pli®ed and used to i solate the complete
Ch-Prx1 gene sequence using conventional S outhern ana-
lyses, subcloning, and sequencing techniques [ 13].
Sequencing of cDNA and genomic DNA
The BigDye T erminator Cycle Sequencing
5
Kit (Perkin-
Elmer) or the Thermo Sequenase Radiolab eled Terminator
Cycle Seq uencing Kit (United States Biochemicals) were
used to sequence the Ch-Prx1 cDNA and t he Ch-Prx1 gene,
respectiv ely.
RNA isolations
RNA for RT-PCR and for northern a nalyses was islolated
by alternative m ethods. I n t he ®rst method,  30 million
cells were pelleted by centrifugation (3000 g,5min).The
pellet was immediately resuspended in 1 mL TRIzol reagent
(Gibco BRL) and polysaccharides, membranes, and unlysed
cells were eliminated by centrifugation (12 000 g,10min).
The supernatant was treated as instructed by the supplier.

biotinylated single stranded template bound to magnetic
streptavidin-coated beads (Dynal) in a speci®c priming
reaction [18]. The speci®c primer used for the reaction was
the downstream primer used in the RT-PCR reaction. After
washing [17], the membrane was exposed t o X-ray ®lm with
an intensifying screen at )80 °Cfor 2days.
Antioxidant activity of the Ch-Prx1 protein
The antioxidant activity of the Ch-Prx1 protein was tested in
a DNA-cleavage assay modi®ed after [19,20]. Bluescript
plasmid DNA (2 lg) was exposed to a mixed function
oxidation system containing 0.32 m
M
dithiothreitol and
3 l
M
FeCl
3
. The reaction contained various amounts of
concentrated Ch-Prx1 p rotein (5±20 l
M
), and was initiated
30 min before addition of the DNA. Control reactions were
performed without Ch-Prx1 protein and with BSA
(400 lgámL
)1
). The reactions were stopped by adding
3.3 m
M
EDTA and analyzed on an agarose gel.
Thioredoxin-dependent peroxidase activity of Ch-Prx1

of Neuchaà t el, Switzerland). Thioredoxins m and h from
Chlamydomonas were puri®ed as described p reviously [22].
An alternative assay, avoiding t he need of a t hioredoxin
reductase, was also used, based on colorimetric determina-
tion of hydrogen peroxides or alkyl hydroperoxides with the
PeroXOquant kit (Pierce) following the supplier's recom-
mendations. Ch-Prx1 (43.8 l
M
) was incubated w ith 400 l
M
dithiothreitol, 16.6 l
M
Trx a nd 500 l
M
t-butyl hydroper-
oxide in 50 lLof30m
M
Tris/HCl, pH 7.9, buffer. The
quantity o f t-BOOH was measured on 5 lL a liquots added
to a spectrophotometer cuvette containing 500 lLof
PeroXOquant medium. The activity was estimated from
the decrease in absorbance at 595 nm.
274 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Isolation of a 2Cys-Prx by using a single cysteine
mutant of
Chlamydomonas
Trx h
In an attempt to isolate new T rx targets in Chlamydomonas,
a s trategy w as used based o n the formation o f stable mixed

Computer database searches based on the amino-acid
sequences of sequenced peptides revealed 75% identity
with a thioredoxin-dependent peroxidase (TPx), also named
peroxiredoxin, of barley (the BAS1 protein) and Arab idop-
sis. These proteins belong to the 2Cys-Prx group, because of
the p resence of two conserved cysteines [5,6]. Arabidopsis
BAS1 was s hown to be a chloroplastic protein. The identity
of our peptides with BAS1 and the presence of two cysteines
in alignment with the conserved cysteines of barley and
Arabidopsis BAS1 suggested that our 21-kDa protein also
belongs to this protein family, and could b e chlo roplastic.
We called the Chlamydomonas 21-kDa protein Ch-Prx1.
Cloning and sequences of
Ch-Prx1
cDNA
and the
Ch-Prx1
gene
In order to complete the sequence data for this new protein,
and to be able to make a thorough characterization of its
biochemical properties, we isolated the cDNA and
expressed it in E. coli to produce a pure recombinant
protein. We also isolated and sequenced the gene e ncod-
ing the 21-kDa polypeptide. Degenerate oligonucleotides
designed from the s equences of N- and C-terminal peptides
(P1 and P10) were us ed as primers to s ynthesize t he cDNA
of the coding sequence of t he mature Prx. The direct P CR
primers were synthesized with a 6-bp extension at their
5¢ ends (ATGGCC, encoding methionine and alanine) for
translation i nitiation and in frame cloning. The ampli®ed

4
2
0
.
1
3
0
4
3
6
7
9
7
21 kDa
Fig. 1. An alysis of elution products f rom the thioredoxin anity column
by red ucing SDS/PAGE. Protein extracts o f Chlamydomonas cu ltures
were app lied on a cytosolic Trx h C39S mutant anity column. The
elution was performed with d ithioth reitol.
Ó FEBS 2002 Peroxiredoxin from Chlamydomonas (Eur. J. Biochem. 269) 275
of the c oding region (Fig. 2). A chloroplast transit peptide
prediction program (
CHLOROP
) [27], predicted a putative
cleavage site between arginine 37 an d alanine 38, but the
mature protein starts at Ser39, as indicated by the peptide
sequencing (Fig. 2). It is possible that the protein c leaved
between positions 37 and 38 is further processed in the
chloroplast to the native form.
Southern blot analys is on genomic DNA digested with
ApaI ( an enzyme known t o c leave w ithin the Ch-Prx1 gene

BLAST
of peptide sequences of 21 kDa proteins in databases. Tryptic peptides were puri®ed by RP-HPLC and some of them were to tally or
partially analyzed by Edman sequencing (ááá) and/or by MALDI-TOF mass spectrometry (±±). The experimental masses (M + H)
+
were compared
with the c alculated masses ( indicated in brackets): P1, 1 681.71 (1681.89); P2 , 1853.42 (1853.93); P4, 297 2.26 (2972.51); P5, 163 1.07 (1630.87); P 6,
1393.66 (1393.73); P7, 2512.52 (2512.36). Accession numbers: barley BAS1, Z34917; Arabidopsis BAS1, X97910. The missing amino-acid stretches
and the transit peptide sequence were deduced from the cloned c DNA and gene sequences (accession numbe rs: AJ304856 for t he ge ne and
AJ304857 f or the cDNA). T he conserved r esidues are shade d in grey and the sequence of the putative t ransit peptide i s in i talics.
ApaI
EcoRI
12
10
4
1.7
1.4
kb
Fig. 3. Sou thern blot analysis of the Ch-prx1 gene. Ge nomic DNA
from CW15 Chlamydomonas strain was digested with ApaIorEcoRI,
size-fractionated on a 0.8% agar ose gel, and transferred to a Hybond
N
+
nylon m embrane. Th e ® lter was hybridized with the
32
P-radiola-
beled Ch-Prx1 c oding reg ion probe. M olecular siz e markers a re indi-
cated on t he left.
276 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
known t o p revent damage of DNA against R OS. ROS can
be produced by incubating dithiothreitol with Fe

towards H
2
O
2
or t-BOOH was e xamined indirectly by
measuring the oxidation r ate of NADPH (followe d by the
decrease in A
340
) in t he presence of NTR and thioredoxin.
Pure recombinant proteins expressed i n Escherichia coli
were used in this test: NTR from A. thaliana,Trxhfrom
Chlamydomonas and C h-Prx1. Figure 7A shows a tim e-
course of NADPH oxidation with either H
2
O
2
or t-BOOH
as substrates. C learly, C h-Prx1 was equally ef®cient with
both, and the reaction required all three protein components
(Ch-Prx1, Trx h and NTR). The speci®c activity of the
recombinant e nzyme w as identical to the speci®c activity of
the native protein puri®ed fro m Chlamydomonas (dat a not
shown). The rate of NADPH oxidation was very weak
when Trx m from Chlamydomonas was used (data not
shown), probably b ecause o f t he weaker af® n ity of Ar abid-
opsis NTR for Trx m [21]. Therefore, the ability of Trx m
from Chla mydomonas to donate protons to Ch-Prx1 w as
measured directly by following the degradation of t-BOOH,
A
A340nm

2
O
2
(e)
or t-BOOH (j). Controls: m in us T RX ( m), minus PRX (r), minus
peroxide (s), minus NTR (q). (B) Peroxidase activity w ith various
thioredoxins. T he concentration of t-BOOH was measured colori-
metrically and expressed a s a percentage of the i nitial c oncentration.
Thioredoxins were reduced with dithiothreitol. Chlamydomonas Trx h
(m)orTrxm(j), spinach T rx f (d)orTrxm(r). Control w ithout
Trx (n).
SK DNA
20
µ
M
Ch
-Prx1
5
µ
M
Ch
-Prx1
C
OC
S
Fig. 6. Prote ction of DNA against free r adical attac k by the recombi-
nant Ch-Prx1 protein. Plasmid Bluescript SK DNA was incub ated for
30 min in a thiol-MFO system containing 3.0 l
M
Fe

4
3
6
7
9
7
k
D
a
Fig. 5. Ana lysis by SDS/PAGE of the monomer/dimer shift of recom-
binant Ch-Prx1. The protein was e ith er r educed with 2-mercapto-
ethanol, or not, a s indicated.
278 A. Goyer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
using the peroXOquant kit (see Experimental procedures),
in the presence of dithiothreitol as an electron donor to Trx.
The r ate of d isappearance of t-BOOH was i dentical with
TrxmandTrxhfromChlamydomonas used at the same
concentration ( Fig. 7B). When Trx was omitted, the r ate of
disappearance of t-BOOH was negligible, proving that
dithiothreitol alone, used at low concentration, cannot
signi®cantly act ivate Ch-Prx1. In order to d etermine
whether d ifferent chloroplastic T rx differ in their abilities
to function with Ch-Prx1, we compared the e f®ciencies of
two Trx isoforms f and m from spinach, b ecause no T rx f
from Chlamydomonas has been isolated until now. Both
were active with Ch-Prx1, but while Trx f was as ef®cient as
Trx h and m from Chlam ydomonas, T rx m from spinach
showed a lower ef®ciency.
Regulation of
Ch-Prx1

cells bubbled with pure o xygen for 90 min in the dark
(Fig. 8C, lane 2). This shows that oxidative s tress, directly or
indirectly, affects Ch-Prx1 gene expression.
Taken together, the results support the notion that the
redox state and/or the concentration of reactive oxygen
species in the chloroplast play a role in regulating the level of
transcripts of the Ch-Prx1 gene in Chlam ydomonas.
DISCUSSION
The mixed disul®de approach as a tool to isolate new
thioredoxin targets
The formation of stable mixed-disul®de cross -linked com-
plexes has been used p reviously to determine interactions
between target enzymes, such as phosphoribulokinase or
NADP-malate dehydrogenase, and thioredoxins [12,29].
This approach also provid ed evidence for conformational
changes occurring in the structure of thioredoxin reductase
upon interaction with its substrate thioredoxin [30]. U sing
the mixed disul®de approach for isolation of Trx target
proteins in vivo is dif®cult because of the limited stability o f
Trx-target complexes in c ells in which reductants t hat split
disul®des, are abundant. To o vercome this dif®culty,
thioredoxin-de®cient yeast or E. coli mutant strains have
been used to express a single-cysteine m utant thioredoxin
allowing the i solation of thioredoxin targets in y east [8] and
Fig. 8. Ch -Prx1 gene expression. (A) Levels of Ch-Prx1 transcripts in cells growing in 12-h light/dark cycles. Total RNA was extracted in two hours
intervals from division-synchronized Chlamydomonas cells kept in a 12- h light/dark regime. RNA samples were processed as described in Materials
and methods. RNA gel blots were hybridized to the radiolabeled Ch-Prx1 cDNA probe. Numbers ab ove the lan es indicate the time a t which the
RNA samples were take n. (B) I nduction of Ch-Prx1 gene expression in Chlamydomonas by light in the abse nce an d in th e prese nce of DCMU or
DBMIB. Cultures were grown in continuous light and R NA samples were analyzed for Ch-Prx1 transcript levels by northern analysis. Lane 1, cells
taken after 16 h in the dark; lane 2, cells taken 3 h a fter the start of the light period; lane 3, cells taken a fter 3 h in the light in the presence of 20 lM

Structural and functional characteristics of Ch-PRX1
The amino-acid sequence of Ch-Prx1 shares highest identity
with the BAS1 protein of Brassica, spinach, barley and
A. thaliana,PR1ofPhaseolus and MHF
9
of A. thaliana.
These proteins b elong to the 2Cys-Prx subfamily. A ll plan t
2Cys-Prx proteins, except BAS1 of barley, the complete
cDNA of which has not been isolated, contain putative
chloroplast-targeting sequences. C h-Prx1 is likely to be a
chloroplastic protein because it is synthesized as a precursor
protein containing a short transit peptide that is predicted to
be cleaved at a conserved s ite. The homologous BAS1
protein of Ara bidopsis wasshowntobeimportedinto
isolated plastids after post-translational modi®cation [6].
Like other 2Cys-Prx enzymes previously described i n
yeast, mammals and plants, Ch-Prx1 d isplayed antioxidant
and peroxidase activities. The enzyme could reduce hydro-
gen peroxide as well as alkyl hydrogen peroxide and exerted
a s trong protective effect against DNA degradation by f ree
radicals of oxygen. More extensive biochemical character-
izations, including K
m
determinations, are needed to better
de®ne the substrate speci®city of Ch-Prx1.
Peroxiredoxin and thioredoxin speci®city
There is i ncreasing evidence for a role o f Trx in coping with
oxidative stress. A m utant strain o f yeast Sacc haromyces
cerevisiae in which both Trx genes were disrupted has been
found to be particularly sensitive to hydrogen peroxide [8]

and defence against oxidative stress
Light is an important environmental factor inducing,
directly and indirectly, the production of ROS. ROS is
known to impair photosynthesis by damaging chloroplast
structures such as the D1 protein, LHCII, the chloroplast
ATPase an d r ibulose 1 ,5-bisphosphate carboxylase/oxygen-
ase (RubisCO) [35]. In Arabidopsis, peroxiredoxins have
been found to protect chloroplast structures from damage
by ROS [35]. Regulation of Ch-Prx1 gene expression may
be controlled either d irectly by ROS, e.g. by H
2
O
2
,whichis
known for its role in signal transduction [36], or indirectly by
sensors of redox conditions i n the chloroplast, e.g. ascorbate
[37]. We f ound that transcript levels of the Ch-Prx1 gene
markedly increased in illuminated cells (Fig. 8A,B) but a lso
upon bubb ling cultures w ith 100% oxygen in t he dark
(Fig. 8 C), conditions in which p roduction of ROS is likely
to be high. Blocking noncyclic photosynthetic e lectron ¯ow
with DCMU or DBMI B
10
inhibited the accu mulation of
Ch-Prx1 transcripts in the light (Fig. 8B), suggesting an
in¯uence o f the photosynthetic electron ¯ow on Ch-Prx1
gene expression. The redox state of plastoquinone, known
to regulate the expression of s ome genes [38,39], does not
seem to be the r esponsible for this r egulation, because both
DCMU and DBMIB exert an inhibitory effect. The

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