Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: Kinetics, catalytic mechanism and oxidative inactivation

Article abstract of DOI:10.1042/BJ20111378

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 10⁶ M^-1·s^-1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu⁷⁹, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys⁴⁴-Cys⁹² disulfide bond and the pK_a of Cys⁴⁴ are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (K_peroxide and k_cat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe⁹⁰ is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite- scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins. The Authors Journal compilation

Full text of DOI:10.1042/BJ20111378

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Biochem. J. (2012) 442, 369–380 (Printed in Great Britain) doi:10.1042/BJ20111378
369
Hydroperoxide and peroxynitrite reductase activity of poplar
thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic
mechanism and oxidative inactivation
Benjamin SELLES*, Martin HUGO†‡, Madia TRUJILLO†‡, Vaibhav SRIVASTAVA§, Gunnar WINGSLE§, Jean-Pierre JACQUOT*,
Rafael RADI†‡ and Nicolas ROUHIER*1
*UMR1136 Nancy University-INRA, Interactions Arbres-Microorganismes, IFR 110, EFABA 54506 Vandoeuvre-le`s-Nancy cedex, France, †Departamento de Bioqu´ımica, Facultad de
Medicina, Universidad de la Repu´blica, 11800 Montevideo, Uruguay, ‡Centre for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Repu´blica, 11800
Montevideo, Uruguay, and §Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umea˚ Plant Science Center, 90183 Umea˚, Sweden
Gpxs (glutathione peroxidases) constitute a family of peroxidases,
including selenocysteine- or cysteine-containing isoforms
(SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione
or Trxs (thioredoxins) respectively. In the present paper we show
new data concerning the substrates of poplar Gpx5 and the
residues involved in its catalytic mechanism. The present study
establishes the capacity of this Cys-Gpx to reduce peroxynitrite
with a catalytic efficiency of 106 M− 1 · s− 1. In PtGpx5 (poplar
Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the
glutamine residue usually found in the Gpx catalytic tetrad,
is likely to be involved in substrate selectivity. Although the
redox midpoint potential of the Cys44–Cys92 disulfide bond and
the pKa of Cys44 are not modified in the E79Q variant, it
exhibited significantly improved kinetic parameters (Kperoxide and
kcat) with tert-butyl hydroperoxide. The characterization of the
monomeric Y151R variant demonstrated that PtGpx5 is not an
obligate homodimer. Also, we show that the conserved Phe90 is
important for Trx recognition and that Trx-mediated recycling
of PtGpx5 occurs via the formation of a transient disulfide
bond between the Trx catalytic cysteine residue and the Gpx5
resolving cysteine residue. Finally, we demonstrate that the con-
formational changes observed during the transition from the
reduced to the oxidized form of PtGpx5 are primarily determined
by the oxidation of the peroxidatic cysteine into sulfenic acid.
Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated
that the peroxidatic cysteine residue can be over-oxidized into
sulfinic or sulfonic acids. This suggests that some isoforms could
have dual functions potentially acting as hydrogen-peroxide- and
peroxynitrite-scavenging systems and/or as mediators of peroxide
signalling as proposed for 2-Cys peroxiredoxins.
Key words: glutathione peroxidase, over-oxidation, peroxynitrite,
redox property, site-directed mutagenesis, thiol peroxidase.
time they are toxic compounds leading to random or targeted
oxidation of DNA and proteins, or to lipid peroxidation with
associated potential drastic modifications of the physico-chemical
properties of these macromolecules [1,2]. Reactive species are
continuously produced by cells, but in particular conditions,
such as those occurring during environmental constraints, their
concentrations can increase dramatically and lead to conditions
of oxidative or nitro-oxidative stress. All organisms, and
particularly plants, have developed a wide range of detoxification
processes to tightly control cellular ROS and peroxynitrite
concentrations [4,5]. Among these scavenging enzymes are Tpxs
(thiol-dependent peroxidases), which use a reactive cysteine
residue to reduce various types of peroxides (from H2O2 to
phospholipid hydroperoxides and peroxynitrite). Five classes
of thiol peroxidases have been defined in plants, grouping
both the so-called Gpxs (glutathione peroxidases) and Prxs
(peroxiredoxins), the latter separated into 1-Cys Prx, 2-Cys Prx,
type II Prx and Prx Q subgroups [6]. Whatever the substrate
reduced, the regeneration of the sulfenic acid formed during
catalysis is dependent on thiol compounds, usually glutathione
INTRODUCTION
In plants, various metabolic pathways or physiological processes,
such as photosynthesis and respiration, produce ROS (reactive
oxygen species) in different subcellular compartments such as
mitochondria, chloroplasts or peroxisomes [1]. ROS can be
different species, such as singlet oxygen, hydroxy radicals,
superoxide radical anions (O2• − ) or hydrogen peroxide (H2O2).
On the other hand, plants produce nitric oxide (NO) via several
mechanisms, but the best and unambiguously characterized
pathway so far is dependent on nitrite and requires nitrate
reductases [2]. Regardless of its origin, NO can lead to RNS
(reactive nitrogen species) formation, such as the oxidizing and
nitrating species peroxynitrite [the term peroxynitrite is used to
refer to both peroxynitrite anion (ONOO− ) and its protonated
form peroxynitrous acid (ONOOH, pKa = 6.6–6.8)], a peroxide
formed in v•iv−o from the diffusion-controlled reaction between
NO and O2
[3]. Some of these ROS and RNS are known
to be signalling molecules, being involved in apoptosis, cell
proliferation, or biotic and abiotic stress responses, but at the same
Abbreviations used: ABA, abscisic acid; ABI2, ABA INSENSITIVE 2; AP1, activator protein 1; COOH, cumene hydroperoxide; Cr, Chlamydomonas
reinhardtii; Dm, Drosophila melanogaster; DTPA, diethylenetriaminepenta-acetic acid; DTT. dithiothreitol; ESI, electrospray ionization; Gpx, glutathione
peroxidase; Grx, glutaredoxin; HRP, horseradish peroxidase; IAM, iodoacetamide; mBBr, monobromobimane; NBD-Cl, 4-chloro-7-nitro-2,1,3-
benzoxadiazole; PDT-bimane, (2-pyridyl)dithiobimane; Prx, peroxiredoxin; Pt, Populus trichocarpa; RNS, reactive nitrogen species; ROS, reactive oxygen
species; Tb, Trypanosoma brucei; t-BOOH, t-butyl hydroperoxide; Tc, Trypanosoma cruzi; TCA, trichloroacetic acid; Tpx, thiol-dependent peroxidase; Trx,
thioredoxin; WT, wild-type; Xf, Xylella fastidiosa.
1
To whom correspondence should be addressed (email nrouhier@scbiol.uhp-nancy.fr).
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370
B. Selles and others
or thiol-containing proteins such as Trxs (thioredoxins), Grxs
(glutaredoxins) or tryparedoxins. Whereas most, if not all, Tpxs
are able to reduce H2O2, the capacity to reduce peroxynitrite into
nitrite has only been reported for some mammalian, protozoa,
plant and bacterial members [5,7,8].
Peroxide and protein quantification
The concentration of H2O2 stock solutions was measured at
240 nm (ε240 = 43.6 M− 1 · cm− 1). The peroxynitrite concentration
was determined at alkaline pH at 302 nm (ε302 = 1670
M− 1 · cm− 1) [3]. Protein concentrations were measured spectro-
photometrically using a molar absorption coefficient at 280 nm of
20065 M− 1 · cm− 1 for PtGpx5 WT, the single cysteine mutants,
and the E79Q, D89K and F90E variants; 19940 M− 1 · cm− 1 for
the double cysteine mutant; and 18575 M− 1 · cm− 1 for PtGpx5
Y151R, as determined from their primary sequences (http://www.
expasy.ch/tools/protparam.html). The concentration of HRP
(horseradish peroxidase) was determined from its Soret band
absorption (ε403 = 1.02 × 105 M− 1 · cm− 1).
With regard to the Gpx class, they are usually monomeric or
tetrameric enzymes distributed into two major types, depending
on the presence of a selenocysteine (SeCys-Gpx) or cysteine
(Cys-Gpx) as the catalytic residue [9]. SeCys-Gpxs are present in
mammals or algae such as Chlamydomonas reinhardtii, whereas
Cys-Gpxs are found in non-vertebrate organisms, such as bacteria,
fungi, insects and plants. Despite being classified as Gpxs, most,
if not all, Cys-Gpxs are in fact regenerated by Trxs, but not, or
very poorly, by glutathione [6,10–12]. Cys-Gpxs usually contain
three conserved cysteine residues, but only two of these cysteine
residues are essential for activity [11]. The catalytic mechanism
employed by Cys-Gpxs is a two-step process. The first step
consists of a nucleophilic attack of the substrate by the most
N-terminal cysteine defined as the peroxidatic cysteine residue
(CysP), which is concomitant with the formation of a sulfenic
acid on this residue and with the release of a water or of an
alcohol molecule, depending on the nature of the substrate. Then,
the most C-terminal cysteine residue, named the resolving or
recycling cysteine (CysR) attacks the sulfenic acid moiety forming
an intramolecular disulfide bond [11,13–15]. The recycling of
the reduced active form usually proceeds via a dithiol/disulfide
exchange with Trxs. As observed for some other Tpxs, such
as 2-Cys Prxs and for some related enzymes belonging to the
methionine sulfoxide reductase protein family, the transition from
the reduced to the oxidized form in the dimeric poplar Gpx5
(termed PtGpx5; Pt is Populus trichocarpa) is accompanied by
an important conformational change [16–19]. Previous studies
on Gpxs have shown that three residues glutamate/glutamine,
tryptophan and asparagine, govern the reactivity of CysP, forming
a catalytic tetrad [13–15]. Although the residues are different, the
principle is very similar to Prxs, where a threonine and an arginine
residue participate in the activation of CysP, and a proline residue
stabilizes the active-site pocket [9,17].
Steady-state activity measurement
The Trx-dependent peroxidase activity of WT and mutated
PtGpx5 was measured using an NADPH-coupled spectropho-
tometric method at 25◦C as described previously [11]. The
assays were carried out in a total volume of 500 μl containing
TE buffer [30 mM Tris/HCl (pH 8.0) and 1 mM EDTA],
200 μM NADPH, 1 μM Arabidopsis thaliana NTRB (NADPH
thioredoxin reductase B), 25 μM poplar Trxh1, 200 nM PtGpx5
and various peroxide concentrations. The catalytic parameters for
one substrate have been obtained by varying its concentration at
saturating concentrations of the other substrate [typically between
1 and 100 μM for Trxh1, and between 40 μM and 12 mM for
the peroxides, either H2O2, t-BOOH (t-butyl hydroperoxide) and
COOH (cumene hydroperoxide)]. The decrease in absorbance was
followed at 340 nm. The peroxidase activity was determined after
subtracting the spontaneous reduction rate observed in the absence
of PtGpx5, and the number of micromoles of NADPH oxidized
per second per micromole of enzyme (i.e. turnover number,
s− 1) was calculated using the molar absorption coefficient of
NADPH (ε340 = 6230 M− 1 · cm− 1). Two or three independent
experiments were performed at each substrate concentration.
The kcat and Km values of PtGpx5 for Trxh1 or peroxides have
been calculated by non-linear regression using GraFit (Erithacus
Software). Inactivation of PtGpx5 and Arabidopsis 2-Cys Prx
(50 nM and 1.5 μM respectively) was evaluated under the same
conditions using H2O2 concentrations ranging from 50 μM to
2.5 mM by monitoring the mean rate of NADPH oxidation
between 30 s and 1 min after addition of thiol peroxidase.
In the present study, an in-depth biochemical and enzymatic
analysis has been performed on PtGpx5 to dissect different
steps of the catalytic and Trx-dependent recycling mechanisms.
This analysis has shed light on five important properties of
this Cys-Gpx: (i) it efficiently reduces peroxynitrites, (ii) it is
reduced by Trx both in monomeric and dimeric forms, (iii) the
conformational changes observed during the transition from
the reduced to the oxidized form are dependent on the oxidation
state of CysP, (iv) CysP is susceptible to over-oxidation into
sulfinic or sulfonic forms, and (v) an engineered enzyme, where
one of the residues of the catalytic tetrad, Glu79, is replaced by a
glutamine residue, is a better catalyst for the reduction of organic
hydroperoxides.
Reduction of PtGpx5
PtGpx5 was reduced immediately before use by incubation with
1 mM DTT (dithiothreitol) for 30 min at 4◦C. Excess reductant
was removed by gel filtration using a HiTrap column (Amersham
Bioscience) and UV–visible detection at 280 nm. Samples were
extensively purged with argon once collected.
Pre-steady-state investigation of the oxidative part of the PtGpx5
catalytic cycle
EXPERIMENTAL
All pre-steady-state kinetic measurements were made using
a stopped-flow apparatus with a mixing time of 1.1 ms and
coupled with spectrophotometric and fluorimetric detection
(Applied Photophysics SV20). Experiments were performed
in 100 mM sodium phosphate buffer plus 0.1 mM DTPA
(diethylenetriaminepenta-acetic acid), at pH 7.4 and 25◦C, unless
otherwise indicated.
The kinetics of H2O2-mediated PtGpx5 oxidation was studied
by two pre-steady-state approaches. The first approach was
a direct determination that made use of the increase in
Cloning, production and purification of recombinant proteins
The cloning of PtGpx5 (POPTR_0001s09280) into pET-3d
has been described previously [11]. Several variants (E79Q,
D89K, F90E and Y151R) have been obtained by PCR-based
site-directed mutagenesis using two complementary mutagenic
primers (Supplementary Table S1 at http://www.BiochemJ.org/
bj/442/bj4420369add.htm). Culture and purification conditions
were similar to the procedures previously described for PtGpx5
WT (wild-type) [11].
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Catalytic mechanism of poplar Gpx5
371
PtGpx5 intrinsic tryptophan-dependent fluorescence intensity that
occurred upon enzyme oxidation. Reduced enzyme (2 μM) was
rapidly mixed with different concentrations of H2O2 in excess.
The time courses of total fluorescence increase (λex = 295 nm)
were followed and experimental curves were fitted to increasing
exponentials from which observed rate constants (kobs) were
determined. From the slope of the plot of kobs values against
H2O2 concentrations, the rate constant of H2O2-mediated PtGpx5
oxidation at pH 7.4 and 25◦C was determined. The second
approach was an indirect competition method which has been
described previously [20]. Briefly, we investigated the ability
of reduced PtGpx5 to inhibit HRP oxidation to compound I
After centrifugation (14000 g for 15 min at 20◦C), the pellets
were washed with 1 ml of 2% TCA. After another centrifugation
step (14000 g for 15 min at 20◦C), proteins were resuspended in
500 μl of 1 M Tris/HCl and 2% SDS for 30 min and their UV–
visible spectra were recorded on a Cary 50 spectrophotometer
(Varian).
ESI (electrospray ionization)–MS analysis of oxidized and reduced
PtGpx5
Solutions of PtGpx5 WT or C73/92S at 100 μM were either
reduced by 150 μM DTT or oxidized in the presence of a mixture
of 150 μM, 1.5 mM or 7.5 mM of both DTT and H2O2 for 5 min
at room temperature (20◦C). High-resolution ESI–MS spectra
of treated and untreated proteins were obtained on a Bruker
microTOF-Q spectrometer (Bruker Daltonik), equipped with an
Apollo II ESI source with ion funnel, operated in the negative-
ion mode. The concentrated sample (approximately 100 μl of
the 100 μM solution) in formic acid was injected at a flow rate
of 10–20 μl · min− 1. Data were analysed with the DataAnalysis
software (Bruker).
by H2O2, a reaction with a reported rate constant (kH2O2/HRP
)
of 1.7 × 107 M− 1 · s− 1 [21]. The time courses of compound
I formation from the reaction of 2 μM HRP and 0.65 μM
H2O2 in the absence or presence of 28.5 μM reduced PtGpx5
were followed at 398 nm. Experimental traces were fitted to
exponential curves and observed rate constants (kobs) of compound
I formation were obtained. Since kobs relates to the rate constant
of H2O2-mediated oxidation of HRP (kH2O2/HRP) and to the rate
constant of H2O2-mediated oxidation of PtGpx5 (kH2O2/PtGpx5
)
using kobs = kH2O2/HRP × [HRP] + kH2O2/PtGpx5 × [PtGpx5], both rate
Determination of the oligomerization state
constants were determined.
The oligomerization state of WT and mutated Gpxs was
analysed by size-exclusion chromatography on a Superose 12
HR 10/30 column connected to an FPLC system (Pharmacia Fine
Chemicals). Proteins (200 μl) at a concentration of 10 mg · ml− 1
were loaded on to the column equilibrated in 50 mM Tris/HCl
(pH 8.0) containing 150 mM NaCl and 1 mM EDTA. The flow
rate was fixed at 0.25 ml · min− 1 and detection was recorded at
280 nm. The column was calibrated by loading 100 μl of bovine
milk lactoserum [24].
The kinetics of peroxynitrite-mediated PtGpx5 oxidation was
also investigated by two pre-steady-state approaches. First, we
followed the initial rate of peroxynitrite decomposition in the
absence or presence of reduced PtGpx5 at 310 nm as reported
previously [22]. However, the reaction rate was too fast to be
accurately followed by this methodology. Therefore we used
a competition approach, by determining the inhibitor effect
of increasing reduced PtGpx5 concentrations on peroxynitrite-
mediated HRP oxidation to compound I in a similar way as
performed with H2O2. From the plot of kobs against PtGpx5
concentrations, kONOOH/HRP and kONOOH/PtGpx5 were determined.
Determination of the midpoint redox potential
Oxidation/reduction titrations, using the fluorescence of
adducts formed between protein thiol groups and mBBr
(monobromobimane), were carried out at room temperature in
500 μl reaction mixtures containing 100 μg of protein in 100 mM
Hepes/NaOH buffer (pH 7.0) and defined mixtures of oxidized
and reduced DTT to set the ambient potential (Eh) with an overall
concentration in DTT of 2 mM [11].
Pre-steady-state investigation of the reductive part of the PtGpx5
catalytic cycle
The kinetics of the reactions of oxidized PtGpx5 with GSH,
poplar thioredoxin h1 (Trxh1) and h5 (Trxh5) were investigated
by a direct fluorescence approach, where reduced PtGpx5
was firstly oxidized by treatment with an almost equimolar
H2O2 concentration (∼0.8:1) followed by mixing with different
concentrations of reductant in excess. The time courses of total
fluorescence intensity decrease (λex = 295 nm) were fitted to
exponential curves and kobs values of fluorescence change were
plotted against the reductant concentration. From the slopes of
the latter plots, the rate constants of the PtGpx5 reaction by GSH,
Trxh1 or Trxh5 at pH 7.4 and 25◦C were determined.
pKa of PtGpx5 sulfhydryl groups
Reactions were started by the addition of PDT-bimane [(2-
pyridyl)dithiobimane] to a final concentration of 25 μM into
a cuvette containing 10 μM pre-reduced proteins in 500 μl of
sodium citrate or phosphate buffer ranging from pH 3.0 to 8.0,
following a procedure described in [25]. The reaction of PDT-
bimane with cysteine residues forms pyridine-2-thione, which
has a maximum absorption wavelength at 343 nm.
Detection of sulfenic acid with NBD-Cl (4-chloro-7-nitro-2,1,
3-benzoxadiazole)
Circular dichroism
NBD-Cl was used to detect the formation of sulfenic acids on
purified proteins after treatment with H2O2 [23]. Stock solutions
of NBD-Cl (100 mM) were prepared in DMSO and stored in
the dark. The recombinant proteins were reduced by incubation
with 5 mM DTT for 1 h at 25◦C. After reduction, proteins were
subjected to successive dilution/concentration steps in TE buffer
by ultrafiltration in order to remove excess DTT. Then, 50 μM
reduced proteins were oxidized by incubation in 500 μl reaction
mixtures with various concentrations of H2O2, from 250 μM
to 5 mM. NDB-Cl (250 μM) was added after 1 min and the
reaction mixture was incubated in the dark for 1 h at 25◦C. The
proteins were separated from excess NBD-Cl by precipitation
for 15 min on ice with 10% (v/v) TCA (trichloroacetic acid).
CD spectra of the different enzymes [25 μM in 10 mM phosphate
buffer (pH 7.1)], either reduced by DTT or oxidized by an almost
equimolar H2O2 concentration, were recorded at 25◦C in a quartz
cuvette (1-mm pathlength) between 200 and 250 nm, with a
bandwidth of 10 nm using a Jobin–Yvon CD6 spectropolarimeter.
The mean residue molar ellipticity [θ] was expressed in
deg · cm2 · dmol− 1
.
In-gel digestion and MS analysis of the PtGpx5–Trxh1 heterodimer
The formation of heterodimers between PtGpx5 and poplar Trxh1
C42S was induced by incubating a pre-reduced Trxh1 C42S
and native (oxidized) PtGpx5 at 1 μg/μl in 50 μl of 50 mM
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372
B. Selles and others
Table 1 Steady-state kinetic parameters of mutated or WT PtGpx5 and comparison with PtGpx3 and CrGpxH
Values for the different peroxides were obtained using PtTrxh1 as the electron donor as described in the Experimental. Values for PtGpx3 and CrGpxH have been extracted from [11,27].
H2O2
t-BOOH
COOH
Trxh1
kcat/Km (M− 1 · s− 1
(× 104)
)
kcat/Km (M− 1 · s− 1
(× 104)
)
kcat/Km (M− 1 · s− 1
(× 104)
)
Peroxide
Km (μM)
kcat (s− 1
)
Km (mM)
1.41
kcat (s− 1
)
Km (μM)
kcat (s− 1
)
Km (μM)
PtGpx3
CrGpxH
PtGpx5
PtGpx5 E79Q
PtGpx5 D89K
PtGpx5 Y151R 18 0.5
PtGpx5 F90E
545
11
7.4 0.2
4
2
9.1
11.3 0.7
0.64
1.6 0.6
0.3 0.03
14.7 3.7
0.2 0.06
0.08 0.006
0.1 0.05
239
63 11
81 19
54
95 17
739 24
60 2.3
13
8.9 0.4
6
5.3
14.1 3.3
7.8 1.4
26.9 5.1
5.5 0.57
0.5 0.08
9.9 0.33
12
–
5.2 0.7
3.9 2.6
4.2 1.1
3.7 0.5
25.4 2.8
+
+
+
+
+
+
+
+
+
54
5
13.7 0.49
0.73 0.11
−
+
−
−
+
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
32 0.12
56 0.9
80 2.5
0.13 13.3 0.6
1.91 0.2
6
9
9
6
5
0.86
0.3
−
−
−
−
+
−
+
−
+
−
+
−
−
−
+
−
−
−
+
+
+
+
+
+
+
+
13 2.4
24.0 1.4
11.7 2.4
0.01 24.9 0.81
0.058 0.007
1.5
2.3
2.8
0.6
6
14 3.8
−
−
−
−
−
−
+
−
−
−
+
+
+
+
+
+
+
+
9
5
8
0.9
4.383 1.8
5
4
6
1.2
−
−
+
−
+
−
−
−
−
−
−
+
−
+
−
−
−
+
+
+
+
+
+
+
7.366 1.44
0.52
0.03
−
−
−
−
−
−
−
+
+
+
+
+
+
+
60 6.8
1.4
13.4 2.04
4.78 1.4
−
−
−
−
−
−
−
Tris/HCl (pH 8.0) for 1 h at room temperature. Pre-reduced Trxh1
C42S was obtained after reduction of 500 μM protein using
10 mM DTT for 30 min and desalting on a Sephadex G-25
column. Approximately 7.5 μg of each protein was loaded on
to non-reducing SDS/PAGE. The gel band corresponding to the
PtGpx5–Trxh1 heterodimer was excized, washed and alkylated
by IAM (iodoacetamide) in the presence or absence of DTT.
Finally, it was digested in-gel with sequencing grade modified
trypsin (Promega/SDS Biosciences) or chymotrypsin (Roche).
The resulting peptides were extracted, dried and dissolved in
0.1% formic acid for mass spectrometric analysis.
Peptide analysis was performed by reverse-phase liquid
chromatography ESI–MS/MS (tandem MS) using a nanoAC-
QUITY UPLC (ultra-performance liquid chromatography)
system coupled to a QTOF (quadrupole time-of-flight) mass
spectrometer (Q-TOF Ultima, Waters Corporation). Raw data
were processed by ProteinLynx Global Server (version 2.2.5)
software for database searches. Proteins were identified by an in-
house Mascot server and the Mascot Daemon application (version
2.1.6; Matrix Science, http://www.matrixscience.com) using the
Populus protein database. The following settings were used for
the database search: trypsin- or chymotrypsin-specific digestion
with one missed cleavage allowed, carbamidomethylated
cysteine, oxidized methionine, dioxidized and trioxidized cysteine
in variable mode, peptide tolerance of 80 p.p.m., and fragment
tolerance of 0.1 Da. Peptides with Mascot ion scores exceeding
the threshold for statistical significance (P < 0.05) were selected.
combining two mutations to generate protein variants in which
only CysP (C73/92S) or CysR (C44/73S) remained. In addition,
with the aim of dissecting some steps of the catalytic cycle, we
have generated other mutated proteins for residues presumably
involved in dimerization (Y151R), in Trx recognition (F90E), in
the reactivity of CysP (E79Q) and in the instability of α2-helix
(D89K).
Substrate specificities of PtGpx5
Using activity measurements achieved under steady-state
conditions, we have first confirmed that, as previously reported
for some plant Gpxs, the recombinant form of PtGpx5 cannot
appreciably reduce peroxides using glutathione as a reductant
(results not shown) [10,11,26]. The kinetic parameters of
PtGpx5 have thus been determined using a NADPH/NTR/Trxh1
reduction system and various peroxides. The catalytic efficiency
(kcat/Kperoxide) of PtGpx5 towards these peroxides is quite variable
with values of 133, 78 and 3 × 103 M− 1 · s− 1, for H2O2, COOH and
t-BOOH respectively (Table 1). The marked difference observed
with t-BOOH (a difference of a factor 25 to 45 compared with the
other peroxides tested) essentially correlates with a difference
in the apparent substrate affinity, with Km values of 32 and
81 μM for H2O2 and COOH, and 1.9 mM for t-BOOH. The
comparison of these values with those determined previously for
two different Cys-Gpxs from photosynthetic organisms, PtGpx3
and CrGpxH (Cr is Chlamydomonas reinhardtii), indicates that
all three enzymes have very similar properties, t-BOOH being the
poorest substrate in all cases (Table 1) [11,27].
RESULTS
We took advantage of the fact that, similarly to PtGpx3,
PtGpx5 exhibits fluorescence changes between the reduced and
the oxidized state, to study the kinetic of H2O2 reduction
independently of the presence of the recycling system. Addition
of excess H2O2 to reduced PtGpx5 caused a rapid increase
in its intrinsic fluorescence intensity that did not occur when
PtGpx5 C44S, a variant mutated on CysP, was used (Figure 1A,
inset). Addition of increasing concentrations of H2O2 led to a
linear dose-dependent increase of the observed rate constants of
PtGpx5 intrinsic fluorescence change (Figure 1A), indicating that
the bimolecular reaction between the enzyme and the oxidant
was the rate-limiting step of the process leading to the change
observed in tryptophan fluorescence. From the slope of the
We previously determined the involvement of two cysteine
residues (Cys44 and Cys92, formerly classified as Cys107 and Cys155
when including the plastidial targeting sequence) out of the three
conserved cysteine residues in the catalytic mechanism of plant
Cys-Gpxs using PtGpx3 as a model [11]. Moreover, we have
been able to solve the three-dimensional structure of PtGpx5, a
paralogue isoform, in both reduced and oxidized forms, which
confirmed first that Cys44 and Cys92 of PtGpx5 are the CysP and
CysR respectively, and which indicated that a large conformational
change, i.e. unwinding of the beginning of the α1-helix and of
the whole α2-helix, occurs in order to bring the two distant
cysteine residues together to form an intramolecular disulfide
bond [18]. This change in oxidation state is also associated with
a modification of the intrinsic fluorescence of the protein and
with a shift in migration in non-reducing SDS/PAGE [11]. In
the present study, we have generated different mutated PtGpx5
proteins, substituting first individually all cysteine residues by
serine residues (Gpx5 C44S, C73S and C92S variants), and then
4
− 1
plot, a second-order rate constant of (5 1) × 10 M · s− 1 at
+
−
pH 7.4 and 25◦C was determined. These results were confirmed
by measuring the inhibition of HRP compound I formation by
PtGpx5. Indeed, reduced PtGpx5 (28.5 μM) caused only a
marginal decrease in the yield of HRP (2 μM) oxidation to
compound I by H2O2 (0.65 μM) and a slight increase in the kobs
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The Authors Journal compilation 2012 Biochemical Society