ThioredoxinCh1 of
Chlamydomonas reinhardtii
displays an unusual
resistance towardone-electron oxidation
Ce
´
cile Sicard-Roselli
1
, Ste
´
phane Lemaire
2
, Jean-Pierre Jacquot
3
, Vincent Favaudon
4
, Christophe Marchand
5
and Chantal Houe
´
e-Levin
1
1
Laboratoire de Chimie Physique and
2
Institut de Biotechnologie des Plantes, Universite
´
Paris XI, Orsay, France;
3
UMR 1136
Interaction Arbres Microorganismes INRA UHP, Universite
´
de Nancy I, Vandoeuvre, France;
4
U 612 INSERM, Institut Curie,
Centre Universitaire, Orsay, France;
5
Institut de Biochimie et Biophysique Mole
´
culaire et Cellulaire, CNRS UMR8619 and IFR46,
Universite
´
Paris XI, Orsay, France
To test thioredoxinresistance to oxidizing free radicals, we
have studied the one-electronoxidationof wild-type thio-
redoxin and of two forms with the point mutations D30A
and W35A, using azide radicals generated by c-ray or pulse
radiolysis. The oxidation patterns of wild-type thioredoxin
and D30A are similar. In these forms, Trp35 is the primary
target and is ÔrepairedÕ by one-electron reduction; first by
intramolecular electron transfer from tyrosine, and then
from other residues. Conversely, during oxidationof W35A,
Trp13 is poorly reactive. For all proteins, activity is con-
served showing anunusualresistancetoward oxidation.
Keywords: t hioredoxin; one-electron oxidation; radiolysis;
tryptophan35 oxidation.
Thioredoxins (Trx) are ubiquitous small proteins (100–120
amino acids) found in all living organisms from bacteria to
vertebrates [1]. T hese proteins, whose active site contains the
amino acid sequence -Cys-Gly-Pro-Cys-, exist either in an
oxidized form with an intramolecu lar disulfide bond (Trx-
S
2
) o r in a reduced form with two thiol functions [Trx-
(SH)
2
]. They are involved in the reduction of disufide bonds
and play a major role in the control of intracellular
reduction potential and defense against oxidative stress. In
addition, these proteins control t he release o f transcription
factors NFKB and AP-1, and thus their oxidation state i s
important in gene expression.
During aerobic life, amino acid residues in proteins are
subject to one-electronoxidation by reactive oxygen species,
in suc h a way that the efficiency of cell defense against
oxidative stress relies on the resistanceof Trx to oxidation.
Recently, Watson and Jones [2] showed that in cells both
nuclear and cytoplasmic type 1 thioredoxins (Trx1) are
relatively protected against oxidation and t hat the redox
state of the cysteine residues in Trx1 was a good marker of
oxidative stress. However, in addition to the cysteine
residues o f t he active site, o ther amino acids can be oxidized
by free radical p rocesses, which may induce modifications of
the enzymatic properties of Trx. In proteins, one-electron
oxidation is known to affect primarily Met, Tyr and Trp
residues [3]. T he major d egradation products r esulting from
such radical attack are dityrosine for tyrosine, N-formyl-
kynurenin for tryptophan and methionine sulfoxide for
methionine. Any of these transformations may affect the
function of the enzyme and thus the redox homeostasy.
The aim of this work was to determine the sensitivity of
Trx towardone-electron oxidation. Therefore we studied
the effect of overoxidation on Trx in its disulfide oxidized
form (Trx-S
2
) b y a zide radicals (N
3
_
) u sing pulse and
gamma radiolysis, and by measuring its enzymatic activity.
Pulse and gam ma ra diolysis are c omplementary techniques.
The first allows identification of transient radicals formed
with their absorption spectra, and the second is used to
oxidize protein solutions in greater quantity to perform
analysis of the degradation products. With radiolysis, very
specific radicals are generated quantitatively. Azide radicals
are powerful one-electron acceptors formed by the reaction
of N
3
–
with the OH
radicals produced during irradiation of
water solutions under N
2
O atmosphere [4]:
N
2
O þ e
À
aq
! OH
þ OH
À
þ N
2
ð1Þ
N
À
3
þ OH ! N
3
þ OH
À
ð2Þ
The reduction potential of N
3
_
is lower than that of OH
•
,and
the values are 1.3 V and 1.8 V vs. normal hydrogen
electrode at neutral pH, respectively. N evetheless, N
3
_
is
more selective and provides a simpler model of oxidation
than OH
radicals allowing the determination of the main
process of o xidation of OH
•
radicals, without all of the side-
effects. They are known to r eact first with aromatic residues
and m ost rapidly with tryptophan ( Reaction 3) [5,6]. Thus a
well-known kinetic scheme is expected for the one-electron
oxidation of proteins containing aromatic residues:
N
3
þ HTrp-XX-TyrOH ! Trp
-XX-TyrOH þ N
À
3
þ H
þ
ð3Þ
Trp
-XX-TyrOH ! HTrp-XX-TyrO
ð4Þ
Correspondence to C. Sicard-Roselli, Laboratoire de Chimie Physique,
CNRS UMR 8000, Baˆ t. 350, Universite
´
Paris XI, F-91405 Orsay
Cedex, France. Fax: +33 1 69 15 3 0 53, Tel.: +33 1 69 15 55 49,
E-mail: cecile.sicard@lcp.u-psud.fr
Abbreviations: Trx, thioredoxin; Trx1, type 1 thioredoxin; TyrOH,
tyrosine phenolic group; Ty rO
•
, tyrosinyl radical; WT, wild-type.
(Received 6 May 2004, revised 24 June 2004,
accepted7July2004)
Eur. J. Biochem. 271, 3481–3487 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04279.x
Trp
-XX-TyrOH ! HTrp-XX
-TyrOH ð5Þ
The tryptophan free radical Trp
•
resulting from Reaction 3
is then reduced by a t yrosine residue (Reaction 4) or by
various amino acids (Reac tion 5) by a n intramolecular
reaction. Thus, t he reaction ends up with dityrosine, among
other final compounds.
Trx possesses two tryptophan residues (positions 13 and
35). One of them (Trp35) is part of the conserved active
site sequence, Trp-Cys-Gly-(Ala/Pro)-Pro-Cys-(Lys/Arg).
Mutation of this residue affects the environment of the
two C ys residues [7] an d the protein biochemical activity [8].
Two t yrosine (positions 53 and 85) an d two methionine
residues (positions 41 and 79) are also present. Their role in
enzymatic a ctivity is not known. It is particularly interesting
to evaluate the sensitivity of Trp35 towards free radicals;
therefore in addition to the wild-type thioredoxin h from
the green alga Chlamydomonas reinhardtii, we oxidized the
mutant form W35A. The aspartic acid residue at position
30, which is highly conserved in Trx from different species
[9], is also crucial for general acid–base catalysis in the
reductive opening of the disulfide oxidized thioredoxin
[10,11], yet NMR studies have shown that the D30A mutant
has the same global fold as the wild-type (WT) protein. In
order to e valuate t he importance of this residue in oxidative
processes we also d etermined the reaction of the m utant
D30A with the N
3
_
radical.
Materials and methods
Proteins
Recombinant Trx h from the green alga C. reinhardtii was
purified from E. coli as described p reviously [12]. D30A and
W35A mutants were prepared, also as described previously
[13]. Samples for i rradiation were dialyzed several t imes
against phosphate buffer (final buffer: 20 m
M
phosphate,
100 m
M
NaN
3
, p H 7). Concentrations of the three forms of
Trx were adjusted to 7 7 l
M
(unless otherwise stated) u sing
absorbance and e
278
¼ 14 500
M
)1
Æcm
)1
for the WT a nd
D30A mutant, and e
278
¼ 8900
M
)1
Æcm
)1
for W35A.
Tryptophan
Tryptophan s olutions (500 l
M
) were prepared in 2 0 m
M
phosphate, pH 7, 500 m
M
NaN
3
buffer. Tryptophan solu-
tions containing tert-butanol contained t he same buffer
components, with the addition of 500 m
M
tert-butanol.
Gamma and pulse radiolysis experiments
Gamma radiolysis experiments were performed using a
panoramic
60
Co source (IL60PL, Cis-Biointernational,
Saclay, France). A Fricke dosimeter [4] w as used to
determine the dose rate.
Pulse radiolysis was performed using the linear electron
accelerator of the Curie Institute in Orsay [14]. The doses
per pulse (200 ns duration, 5–15 Gy) were calibrated from
the absorption o f the thio cyanate radical (SCNÞ
À
2
obtained
by radiolysis of thiocyanate ion solution in N
2
O-saturated
phosphate buffer { 10 m
M
KSCN, 10 m
M
phosphate, pH 7,
G[(SCNÞ
À
2
] ¼ 0.55 lmolÆJ
)1
, e
472
¼ 7580
M
)1
Æcm
)1
}[15].
All protein samples were prepared in 20 m
M
phosphate
buffer, pH 7, containing 100 m
M
NaN
3
and saturated with
N
2
O by flushing N
2
O gas for 1 h over the samples, avoiding
bubbling in the solution.
Absorption and fluorescence
Absorption spectra were recor ded at room temperature
with a PerkinElmer (k9) spectrophotometer. Fluorescence
spectra were recorded on a FL111 Spex fluorimeter.
Electrophoresis
SDS/PAGE was performed using a 12% (w/v) acrylamide/
bisacrylamide gel with a Tris/Tricine buffer [16]. Reductive
conditions were obtained by a dding 2-mercaptoethanol to
the protein. Proteins bands were stained with Coomassie
blue R-250.
HPLC analysis
HPLC was performed on a Beckman Gold 168 (Beckman
Coulter, Aulnay, France) with diode array detection. The
analytical column was a C4 r everse-phase column
(150 · 4.6 mm, 5 lm). The mobile phase eluants used were:
(A) 0.1% (v/v) trifluoroacetic acid in water; (B) 0.1% (v/v)
trifluoroacetic acid and 7 0% (v/v) CH
3
CN. G radient
elution used was 40–60% of B in 20 m in, 1 mLÆmi n
)1
flow
at room temperature.
Mass spectrometry
All spectra were acquired in positive-ion m ode on a Voyager
DE-STR MALDI-TOF mass spectrometer (Applied Bio-
systems, Courtaboeuf, France) equipp ed with a 337 nm
nitrogen laser. Determination of the molecular masses of
irradiated proteins was performed in linear mode (acceler-
ating voltage 25 kV, grid voltage 93%, guide wire 0.3%,
delay 600 ns) with external calibration.
Freeze-dried fractions obtained from HPLC purification
of irradiated Trx w ere d iluted with 15 lL o f 30% (v/v)
CH
3
CN, 0.3% ( v/v) trifluoroacetic acid. One m illilitre of the
solution was mixed with 4 lL of a saturated solution of
sinapinic acid in 30% (v/v) CH
3
CN, 0.3% (v/v) trifluoro-
acetic acid. F inally, 1.5 lL of t his premix w as deposited onto
the sample p late and a llowed to d ry at room temperature.
Activity measurements
The activity of Trx was measured using the reduction of
insulin [17]. One millilitre of t he following solution was
prepared: 1 00 m
M
phosphate, p H 7.1, 1 30 l
M
human
insulin (zinc form), 2 m
M
EDTA; to which 30 lLofa
Trx solution (77 l
M
Trx, 20 m
M
phosphate buffer, pH 7,
100 m
M
NaN
3
) was added t o obtain a final c oncentration of
2.5 l
M
of the protein. The experiment was started imme-
diately after the addition of 500 l
M
dithiothreitol and the
activity was monitored using the change in absorbance at
650 n m. A blank was made u sing the same conditions
without adding Trx. All these experiments were carried out
at 27 °C.
3482 C. Sicard-Roselli et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Results
Transients
Aqueous solutions of Trx ( 77 l
M
WT, W35A or D30A ,
in 20 m
M
phosphate buffer, 100 m
M
NaN
3
pH 7) were
irradiated by pulse radiolysis in the presence of NaN
3
under
an atmosphere of N
2
O. Under t hese conditions the N
3
_
radical was the only o xidant species generated by radiolysis.
The absorption s pectra of protein free radicals are shown
in Fig. 1. Comparison of Fig. 1A and B (WT and D30A)
shows that both p roteins yield a similar behavior. The spe c-
trum obtained 50 ls after the pulse exhibited a broad
absorption band at 510 n m, characteristic of the
tryptophanyl radical (Trp
•
) and a narrow peak at 410 nm
reminiscent of a tyrosinyl radical (TyrO
•
) [18]. The second-
order r ate c onstants o f formation o f the Trp
•
radical,
determined under pseudo fi rst-order conditions, were o f the
same order of magnitude for both proteins (Table 1). The
intensity of the 410 nm peak subsequently increased at the
expense of the 510 nm band and reached a pseudo-plateau
after approximately 300 ls. The rate constants for th is
reaction measured at 510 and 410 nm were very close to
each other, suggesting quantitative oxidationof TyrOH by
Trp
•
, as for other p roteins. However, the intramolecular r ate
constants of charge transfer d iffered b y a factor of t wo
between WT Trx and the D30A mutant (Table 1).
The Trp
•
and TyrO
•
yields were estimated from the
magnitudes of the absorbance changes at 510 and 410 nm,
assuming that the extinction coefficients of protein bound
radicals are the same as those for free amino acids or
peptides, namely, for Trp
•
1800 and 300 mol
)1
ÆLÆcm
)1
at
510 and 410 nm, respectively; for TyrO
•
, 70 and 2600
mol
)1
ÆLÆcm
)1
at 510 and 410 nm respectiv ely [18,19]. Thus,
the stoichiometry N
À
3
/Trp
•
was estimated to be equal to
1 : 1 and the percentage of transfer to around 60% in both
compounds. This transfer is not total, as some tryptophan
radical persists at the end of the reaction (approximately
300 ls after the pulse).
Figure 1C shows the absorption spectrum of the W35A
mutant form of Trx obtained 50 and 300 ls after the pulse.
Among the transients t hat were formed by o xidation of
W35A, the Trp
•
510 n m broad band was not detected
anymore; instead, a peak at 420 nm and weak bands at 390
and 480 nm appeared. The peak at 420 nm indicates that
tyrosine could be oxidized directly by N
3
radicals. The rate
constant of formation was determined under p seudo first-
order c onditions (Table 1) and was substantially lower than
in other proteins.
The bands at 390 and 480 nm might belong to a
methionyl residue. In general, N
3
radicals are unable to
perform oxidization of methionine becau se the o ne-electron
reduction potential of methionine is higher than that of N
3
.
However it w as shown that interaction with other r esidues
and particularly c arbonyl groups, may lower t he methionine
redox potential considerably [20]. Here t he methionine
radical would app ear as a n S-O complex with an a bsorption
spectrum peaking around 390 nm [21] and/or interacting
with another sulfur atom, giving an absorption spectrum
with a maximum at 480 nm. Both types of radicals can e xist
simultaneously in the same molecule [20]. Alternatively the
480 nm band might be assigned to a Trp
•
radical according
to Joshi and Mukherjee [22,23]. These authors oxidized
tryptophan by CCl
3
O
2
radicals and observed a blue-shift
from 510 nm to 480 nm, which t hey a scribed t o a change in
polarity of the environment. However, this blue-shift was
formerly interpreted by Packer et al. as an a dduct o f
Cl
3
COO
•
on the C2 or C3 of the indole ring of tryptophan
[24]. I n o rder to properly assign the 480 nm b and, we
investigated the effect o f the solvent polarity o n the
absorption of a Trp radical. N
3
-induced pulse radiolys is
oxidation of tryptophan was performed in phosphate buffer
solution in the presence of tert -butanol (500 m
M
NaN
3
,
500 m
M
tert-butanol). The presence of tert-butanol induced
a b roadening and a red-shift of the absorption f rom 510 nm
to 540 nm (not shown). Therefore, assigning the 480 nm
band to Trp
•
appears to be unlikely.
Analysis of final compounds
Three analytical methods were used to gain insight into the
nature o f the oxidized forms o f T rx generated by c-ray
radiolysis up to 900 Gy.
Fig. 1. Differential absorption spectra of WT, D30A and W35 Trx
protein free radicals obtained by pulse r adiolytically generated N
3
•
after
50 and 30 0 ls. Proteins were 77 l
M
throughout. The optical path was
2 cm. (A) WT Trx; (B) D30A; (C) W35A. The dose was 6.5 Gy.
Ó FEBS 2004 One-electronoxidationresistanceofthioredoxinCh1 (Eur. J. Biochem. 271) 3483
Firstly, reducing and nonreducing SDS/PAGE analysis
was carried out on the three forms of oxidized Trx. WT Trx
(Fig. 2) and W35A exhibit a single band corresponding to
the mass (12 kDa) expected from intact Trx. For D30A,
two new higher molecular mass bands are generated in a
dose-dependent manner, suggesting t he formation of aggre-
gates.
Secondly, UV absorption spectra of the three forms of
Trx were recorded before and after one-electron oxidation,
as tyrosine dimers can be evidenced with an absorption at
315 nm coupled to a fluorescence band at 410 nm. A new
315 nm a bsorption band i ncreased with dose for t he W35A
mutant only. In addition to absorption, fluorescence
analysis of the three forms of Trx after radiolytic oxidation
was performed. Excitation at 315 nm induced a broad
fluorescence band at 410 n m for the W35A mutant (Fig. 3)
while no new signal could be seen for WT and D30A Trx.
Thirdly, liquid chromatography was performed to isolate
the degradation products of WT Trx, D30A and W35A
after oxidation with a dose of 100 Gy. For each form, the
chromatograms were very similar and showed the forma-
tion of a s ingle major product. The y ield of formation of t his
product (W35A, 47 nmolÆJ
)1
; WT, 35 nmolÆJ
)1
; D 30A,
40 nmolÆJ
)1
) w as calculated using the area of each peak
assuming that the sum of both peak areas represents 100%.
The product was isolated and analyzed using mass spectr-
ometry. For D30A and WT Trx, no difference between the
mass of the intact protein and the oxidized one could be
detected (Table 2). I n the case of W35A, a small increase o f
themass(<40Da)wasevidenced.
Enzymatic activity
Enzymatic activity of the three Trx was measured before
and after exposure to 100 Gy (Fig. 4). Firstly, unirradiated
D30A had the highest activity, compared to the WT and
W35A forms. Secondly, as expected, the activity of W35A
compared to that of WT Trx was reduced by a factor of
approximately two [25]. T hirdly, a ctivity of D 30A was
weakly modified only after irradiation w ith 100 Gy, while
that of WT and of W35A increased by a factor of 1.5 and
1.2, respectively (Table 3).
Discussion
As already observed for several proteins or p eptides [6,26–
29], in WT and D30A the N
3
radical first oxidizes a
tryptophan residue that is subsequently reduced in the
course of intramolecular charge transfer to a tyrosine
residue (Reactions 3 and 4). The rate constants of
Reaction 3 are in th e same range as for tryptophan residues
in other proteins (Table 1). The reaction between N
3
radicals and the Trp residue is stoichiometric, as reported
for other proteins [6]. Mutating Asp30 by replacement with
an alanine did not affect this reaction. Because the 510 nm
band is missing in W35A, we suggest that in the case of WT
and D30A, Trp35 is the residue oxidized by N
3
.
Evolution of the transient absorption spectra indicates
that Trp3 5 i s p artly ÔrepairedÕ by a t yrosine residue acting as
a one-electron donor. Trx contains two tyrosines. From the
known structure of WT and D30A Trx [30], the distances
Table 1. Rate c onstants of the reaction of N
3
•
with WT Trx , D30A an d W35A. These values a re compared to those proposed for other peptides o r
proteins. Data are an average of values g iven in [29,30,43]. The distance is taken from Figs 4 and 5 of [43].
k
(Reaction 3)
(mol
)1
ÆLÆs
)1
)
Intramolecular rate constants (s
)1
)
k
reaction
Trp-Tyr distance of the couple
involved (A
˚
)
WT Trx (1.1 ± 0.2) · 10
9
(9.9 ± 0.4) · 10
3
18.4 (Trp35-Tyr85)
D30A (1.2 ± 0.3) · 10
9
(4.4 ± 1.7) · 10
3
18.7 (Trp35-Tyr85)
W35A (0.46 ± 0.01) · 10
9
Hen egg white lysozyme (7.9 ± 0.8) · 10
8
120 ± 10 [6] 14 (Trp62/63-Tyr53)
Trp-(Pro)
3
-Tyr % 5 · 10
9
[31,43]
a
(1.5–2.3) · 10
3
[29,31,43] % 8 [43]
Trp-(Pro)
4
-Tyr % 5 · 10
9
[31,43]
a
5.13 · 10
2
[43] % 11 [43]
Trp-(Pro)
5
-Tyr % 5 · 10
9
[31,43]
a
3.05 · 10
2
[43] % 14 [43]
a
Authors gave only the order of magnitude of this rate constant.
Fig. 2. SDS/PAGE analysis in the presence of 2-mercaptoethanol (12% acrylamide gel) of Trx after c-ray ir radiation (up to 900 Gy). Left : WT Trx;
middle: W 35A; right: D30A. For ea ch lane, 2 lg of protein was used. The molecular mass standards used for W35A and D30A are shown on the
gels. For D30A, the arrows point out two new bands increasing with the dose.
3484 C. Sicard-Roselli et al.(Eur. J. Biochem. 271) Ó FEBS 2004
between Trp35 and Tyr (53 and 85) were calculated at 29.8
and 18.4 A
˚
, respectively (Table 1). We therefore propose
that th e tyrosine residue involved in the i ntramolecular
Trp
•
fi Tyr transfer i s Tyr85. These distances are much
larger than in the other proteins or peptides investigated to
date (Table 1), y et the rate constant of this step is also much
higher [5,6,31]. It is currently agr eed that the rate constant of
long-range intramolecular electron transfer decays expo-
nentially with the donor–acceptor distance [32,33] and that
this dependence would be the same for all proteins and
peptides [34]. O ur results c learly demonstrate t hat this
correlation does not apply for Trx. For D 30A, the
intramolecular rate constant is reduced by a factor of two
(Table 1) compared with that for the WT protein although
the distances between Trp35 and the tyrosine residues are
the same. Sakata et al. [35] suggested that a change in
orientation of the donor/acceptor residues could induce a
slower rate constant. Whether this effect would be due to
electrostatic changes s uch as d ipole moment d ifferent
orientation or to modifications of the structure of the
solvation layer is not known. Here, no significant difference
between the Tyr and Trp residues orientation could be
demonstrated by superimposing their respective crystallo-
graphic structures. Asp30 has an important role i n driving
the hydrogen bond network linking its carboxylic group to
the active s ite. Changes in t he kinetics of intramolecular
electron t ransfer b y mutation of Asp30 could thus b e a
consequence of hydrogen bond rearrangement at the active
site.
Weak bands at 390 and 480 nm, but no 510 nm band
that could be assigned to Trp, was observed in the case of
W35A (Fig. 1 C). Several explanations were proposed for
the band at 480 nm. Such a blue-shift was observed and
assigned to Trp
•
radical i n c asein a nd bovine serum albumin
as these proteins w ere transferred to a s olvent of low
polarity inducing changes in the protein environment and
conformation [22,23]. Under our conditions, a decrease in
solvent po larity by addition of tert-butanol did not produce
any shift of the T rp
•
absorption ban d. W e t hus propose that
the bands at 390 and 480 nm could be related to oxidation
of methionine residues. Indeed, Trx possesses two methio-
nine residues at positions 41 and 79. Met79 is close to the
carbonyl function of Phe31 (less than 4 A
˚
). Hence its
reduction potential could be lower than that of Met41 which
is in a polar environment with solvent access [ 20] allowing
Met79 o xidation by N
3
. T he end product would be a
MetS
+
radical, which, in interaction with the oxygen atom
of the carbonyl function, would lead to Met S–O radical
absorbing at 390 nm [36]. In addition Met79 is at 5.2 A
˚
from the sulfur atom of Cys39 and c ould form a Met S–S
+
radical absorbing at 480 nm.
Final products are different for t he three p roteins.
Aggregation was observed only for D30A, for doses above
100 Gy. This aggregation was als o seen with electrophoresis
under reducing conditions, w hich excludes t he formation o f
a disulfide bond . Surprisingly, aggregation did not correlate
with the appearance of 315 nm absorption/420 nm fluores-
cence bands, a s could b e e xpected for c ovalent t yrosine
dimerization [37,38]. Therefore, dityrosine is unlikely to be
formed. We propose that the polypeptide chain can also
take par t in the one-electron processes. For example, in
lysozymes oxidationof tryptophan residues leads to poly-
peptide bond cleavage [6]. Also, in hen egg white lysozyme,
one-electron reduction of the 6-127 disulfide bond leads to
peptide bond cleavage [39]. W e therefore propose hydrogen
Table 2. Mass analysis of intact and oxidized thioredoxin after separ-
ation using liquid chromatography.
Intact protein Oxidized protein
WT Trx 11711 ± 10 11713 ± 10
D30A 11677 ± 10 11676 ± 10
W35A 11638 ± 10 11606 ± 10
Table 3. Activity of the different forms of Trx before and after oxida-
tion.
Thioredoxin
Non irradiated
DA
650
Æmg
)1
Æmin
)1
Irradiated 100 Gy
DA
650
Æmg
)1
Æmin
)1
WT 0.79 0.97
W35A 0.44 0.67
D30A 1.60 1.58
Fig. 3. Fluorescence s pe ctrum of W35A and
D30A (inset) (77 l
M
,20m
M
phosphate buffer
pH 7) recorded at room temperature with
excitation a t 31 5 nm. Doses for W35A: 0 Gy;
403 Gy and 646 Gy. Doses for D30A (inset):
0 Gy and 100 Gy.
Ó FEBS 2004 One-electronoxidationresistanceofthioredoxinCh1 (Eur. J. Biochem. 271) 3485
loss from a Ca atom followed by aggregation through the
carbon-carbon bond. This process occurs easily in polymers
[4] and glycine r esidues are good candidates [40]. In
thioredoxin, oxidized forms other than aggregates are
formed. Oxidationof tyrosine residues in aqueous solution
and in t he presence of oxygen by OH radicals leads to
formation of 3 ,4-dihydroxyphenylalanine. However, th is
requires oxygen (which is absent in our system) and
therefore, we can exclude the formation of 3,4-dihydroxy-
phenylalanine in the three forms of Trx after radiolytic
oxidation.
No aggregate was formed from the W35A mutant but a
new fluorescent product appeared after oxidation. As this
420 nm fluorescence band is not due to dimerization, it
could arise from a d egradation product of the Trp13
residue. Mass spectrometry indicates an increase of mass
lower than 4 0 Da. This and fl uore scence experiments
suggest t hat one-electron o xidation of W35A could produce
N-fo rmylkynurenin at position 1 3. It would mean that
Trp13 is involved in t he final step of the one-electron
oxidation of W35A by azide.
Although many examples of enzyme i nactivation have
already been reported (reviewed in [41,42]), no inactivation
of Trx was shown to result from oxidation. This means t hat
no amino acid involved i n insulin reduction activity is
irreversibly affected by oxidation and that the tertiary
structure is not altered to a large extent. Moreover, an
opposite effect is observed for W35A, i.e. this Trx is found
to be more active after a 100 Gy irradiation.
Conclusion
The use of azide r adicals g enerated by pulse radiolysis
allowed us to determine the reactivity of WT and two
mutant forms, D30A and W35A, of T rx toward
one-electron o xidants. We were particularly interested in
the reactivity of the Trp35 residue, as this is h ighly
conserved in the active site and may be a part of t he
defence o f livin g organisms toward reactive oxygen
species. We show h ere that oxidationof Trx (WT and
D30A) with N
3
occurs first at the Trp35 residue. The Trp
•
radical s ubsequently undergoes intramolecular reduction
by a tyrosine residue. The tyrosine residue involved in this
transfer is probably Tyr85. Such intramolecular electron
transfer thus protects Trp35, and hence the enzyme’s
biological activity, i n cases of oxidative stress. When
Trp35 is absent, the other Trp residue (Trp13) may be
oxidized, however, indirectly through tyrosine and/or
methionine oxidation followed by intramolecular electron
transfer. This suggests that protection against oxidation is
not due to the accessibility of s ensitive residues to free
radicals, but rather to some kind of ÔrepairÕ through long
range intramolecular electron transfer. The main point is
that no degradation of Trx was observed. It means that in
the case ofoxidation stress, if thioredoxin reductase is
active, oxidized thioredoxin can be r ecycled and the
defenses of the cells are not affected.
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Ó FEBS 2004 One-electronoxidationresistanceofthioredoxinCh1 (Eur. J. Biochem. 271) 3487
. Thioredoxin Ch1 of
Chlamydomonas reinhardtii
displays an unusual
resistance toward one-electron oxidation
Ce
´
cile Sicard-Roselli
1
, Ste
´
phane. Gy.
Ó FEBS 2004 One-electron oxidation resistance of thioredoxin Ch1 (Eur. J. Biochem. 271) 3483
Firstly, reducing and nonreducing SDS/PAGE analysis
was carried