Redox-regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H-site flexibility

Article abstract of DOI:10.1002/pro.3440

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X-ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH-binding site (G-site) and a hydrophobic co-substrate-binding site (H-site). At elevated H₂O₂ concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H-site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox-regulated enzyme.

Full text of DOI:10.1002/pro.3440

Article
Protein Science
DOI 10.1002/pro.3440
Redox regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9
induces Hꢀsite flexibility
Maria-Armineh Tossouniana,b,c, Khadija Wahnia,b,c, Inge Van Mollea,b,c, Didier Vertommend,
Leonardo Astolfi Rosadoa,b,c, and Joris Messensa,b,c,1
aVIBꢀVUB Center for Structural Biology, VIB, Bꢀ1050 Brussels, Belgium
bBrussels Center for Redox Biology, Bꢀ1050 Brussels, Belgium
cStructural Biology Brussels, Vrije Universiteit Brussel, Bꢀ1050 Brussels, Belgium
dde Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium
Running title: How plant Phi 9 GST survives oxidative stress
1To whom correspondence should be addressed: Joris Messens, VIBꢀVUB Center for Structural
Biology, Redox Signaling lab, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit
Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium, Phone: +32 2 6291992, Fax: +32 2
6291963; Eꢀmail: joris.messens@vibꢀvub.be
Number of manuscript pages (28), number of tables (4), number of figures (7), supplementary
material pages (5): materials and methods pages (1), number of tables (1) and figures (1)
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/pro.3440
© 2018 The Protein Society
Received: Feb 25, 2018; Revised: Mar 31, 2018; Accepted: Mar 01, 2018
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Page 2 of 44
Abstract
Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly
catalyse the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione
peroxidase. With Xꢀray crystallography, kinetics and thermodynamics, we studied the impact of
oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no
cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical
conserved GSHꢀbinding site (Gꢀsite) and a hydrophobic coꢀsubstrateꢀbinding site (Hꢀsite). At
elevated H2O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This
oxidation increases the flexibility of the Hꢀsite loop, which is reflected in lower activities for
hydrophobic substrates. Determination of the transition state thermodynamic parameters shows
that upon oxidation an increased enthalpic penalty is counterbalanced by a more favourable
entropic contribution. All in all, to guarantee functionality under oxidative stress conditions,
GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes
substrate of methionine sulfoxide reductases, making it a redox regulated enzyme.
Keywords: redox, methionine sulfoxide, Xꢀray structure, methionine sulfoxide reductase,
steadyꢀstate kinetics, thermodynamics
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Word statement: We report the impact of oxidation on the structure and on the active site
methionine residues of GSTF9 under hydrogen peroxide stress. Formation of methionine
sulfoxide causes an increased flexibility of the active site loop inducing a decrease in its
transferase activity, and a decrease of its glutathione peroxidase activity towards hydrophobic
substrates. We also show redox regulation of GSTF9 by methionine sulfoxide reductase enzymes,
which restores the GSTF9 activity.
Abbreviations: GST, glutathione transferase; GSTU23, glutathione transferase Tau23; GSTF9,
glutathione transferase Phi9; GSH, glutathione; GSOH, glutathione sulfenic acid; GSSG,
glutathione disulfide; GSO3, glutathione sulfonate; ROS, reactive oxygen species; MetꢀSO,
methionine sulfoxide; FOX assay, ferrous oxidationꢀxylenol orange assay AU, asymmetric unit;
;
CDNB, 2,4ꢀDinitrochlorobenzene; CuOOH, cumene hydroperoxide.
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Introduction
To understand the global effect of methionine oxidation in plants, we recently identified the
sulfoxome (proteome of oxidized methionines) in A. thaliana leaves exposed to high light stress,
and we mapped the modifications back to single amino acids, which is key for the understanding
of the possible consequences of this postꢀtranslational modification on protein function.1 We
found members of the Phi and Tau glutathione transferase (GST) families affected by methionine
oxidation.
GST enzymes detoxify electrophilic substrates by glutathione (GSH) conjugation and by
peroxide scavenging.2,3 Particularly, they are involved in the detoxification of heavy metals4 and
herbicides.5 Some GST members deglutathionylate their substrates,6 and others are involved in
cell signaling by interacting with substrates that have an important signaling role.7 The A.
thaliana genome encodes for 55 GST enzymes, which are differently expressed and regulated
depending on the type of biotic and abiotic stress conditions.4 GST enzymes are subdivided into
different classes depending on their aminoꢀacid sequence, catalytic residues (Ser, Tyr or Cys),
and their tertiary structure.8,9 Although members of the plant GST classes have low primary
sequence identity, they share an overꢀall common structural fold.5,10,11
Most cytosolic GST enzymes are homoꢀdimeric and have an Nꢀterminal thioredoxinꢀlike domain,
which contains the glutathione (GSH) binding site (Gꢀsite) and a Cꢀterminal alphaꢀhelical
domain, which contains the hydrophobic substrateꢀbinding site (Hꢀsite).8 Upon GSH binding to
the Gꢀsite, the catalytic residue (Ser, Tyr or Cys), which is Ser for Phi and Tau class GSTs,
activates GSH (thiol) to GSꢀ (thiolate), which is then conjugated to an electrophilic substrate
located within the Hꢀsite via a thioꢀether bond.
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Some GST enzymes contain cysteines, located close to the active site, which are highly sensitive
to oxidation. For example, we recently showed that A. thaliana GSTU23 forms a reversible
disulfide that protects against oxidative damage.12 Other GST enzymes have no cysteines, like
some of the Phi class members, where methionine residues play a role in redox regulating their
transferase activity.13
Here, we present the impact of oxidation on the structure and function of GSTF9, an enzyme
with no cysteines. GSTF9 is constitutively expressed together with GSTF10, GSTU5 and
GSTU13, indicating its importance as housekeeping enzyme.14 Further, Horvath et al. showed
that an A. thaliana GSTF9 lossꢀofꢀfunction mutant has a decreased overall glutathione
peroxidase activity.15 We found that the oxidation of methionines located near or within the Gꢀ
and Hꢀsites of GSTF9 lead to increased flexibility of the Hꢀsite loop, which results in a decrease
in the affinity for the substrates, GSH and CDNB. Upon oxidation, GSTF9 glutathione
peroxidase activity decreases for the more hydrophobic peroxides.
Results
GSTF9 has a universal GST structural fold
This study focuses on the impact of oxidation on the structure and function of A. thaliana
glutathione transferase Phi 9 (GSTF9), which was identified within the list of the proteins with
an oxidized methionine, the A. thaliana sulfoxome.1 Reduced GSTF9 (GSTF9red) was purified to
homogeneity and crystallized using the hanging drop vapor diffusion method. We solved the
structure to a resolution of 2.34 Å (PDB code 6EZY) (Table I). As observed for other GSTs,
GSTF9red is a homodimer and forms trigonal crystals, with one GSTF9red dimer in the
asymmetric unit. Each monomer contains a thioredoxin domain followed by an αꢀhelical domain
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(Fig. 1). The thioredoxin domain contains the glutathione (GSH) binding site (Gꢀsite), while the
hydrophobic substrate binding site (Hꢀsite) is embedded within the Cꢀterminal helical bundle
domain. The Gꢀsite shows a high degree of structural conservation, while the conservation of the
Hꢀsite is rather limited.16 The catalytic Ser12 within the Gꢀsite is responsible for GSH activation
(Fig. 1). We observed a GSH molecule residing the Gꢀsite of the GSTF9red structure, while the
Hꢀsite is occupied by glutathione sulfenic acid (GSOH) (Fig.1). The presence of two GSH
molecules, or even a glutathione disulfide (GSSG) has been shown previously for the E. coli Nu
class of GST enzymes.17,18 Overlay of these structures with the GSTF9red structure shows that the
GSH molecules in YghU and YfcG are differently oriented.
Oxidation impacts transferase activity but not its H2O2 peroxidase activity
GSTF9 belongs to the Phi class of glutathione transferases, for which both glutathione
transferase and peroxidase activities have been described.14,19 To study the impact of oxidation
on transferase and peroxidase activities (Fig. 2), GSTF9red was oxidized with H2O2 (1:100 molar
ratio) to obtain GSTF9ox, followed by oxidant removal. The transferase activity of GSTF9 was
measured by monitoring the increase of absorbance at 340 nm for the conjugation of the thiol
group of GSH to the CDNB substrate in function of time. The initial velocities were plotted for
several conditions [Fig. 2(A)]. As previously reported by Jacques et al,1 we observed a 30%
decrease in initial velocity for GSTF9ox [Fig. 2(A)]. To study the influence of oxidation on the
glutathione peroxidase activity of GSTF9, we used a FOX assay [Fig. 2(B)]. Progress curves
[Fig. 2(B)] show that GSTF9red and GSTF9ox remove H2O2 with similar rates.
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G- and H-site methionines form a sulfoxide
In our previous study on GSTF9,1 we reported sulfur oxidation of Met35, which is located close
to the Gꢀsite (Fig. 1). We decided to determine whether the methionines (Met35, Met118,
Met123) located within or near the Gꢀ and Hꢀ sites of GSTF9 could influence its activity upon
hydrogen peroxide treatment (Fig. 1). Therefore, both GSTF9red and GSTF9ox samples were
trypsin or chymotrypsin digested, and the formation of methionine sulfoxide and sulfone was
analyzed by mass spectrometry. We found sulfoxide and sulfone formation on Met35 (Fig. 3),
which is located close to the Gꢀsite, and sulfoxide on Met118 and Met123, which are located
close to the Hꢀsite (Fig. 1). Aside from the active site methionines, also Met184, which is surface
exposed and located at the opposite side of the active site, was oxidized to methionine sulfoxide.
Taken its location at distance of more than 18 Å away from the active site Ser12 (Fig. 1), it is
highly unlikely that Met184 oxidation would directly influence the activity of GSTF9.
Oxidized GSTF9 is a methionine sulfoxide substrate
As methionine oxidation causes a decrease in the transferase activity of GSTF9 [Fig. 2(A)], we
wondered whether the transferase activity could be recovered by methionine sulfoxide reductase.
Therefore, we incubated 10 µM GSTF9ox with a 5 µM mixture of methionine sulfoxide reductase
A (MsrA) and B (MsrB) for 1 h at 25°C, and measured the initial velocity in the CNDBꢀGSH
conjugation assay. The drop in transferase activity seen for GSTF9ox was almost completely
recovered [Fig. 2(A)], which indicates that Msr enzymes regulate the activity of GSTF9 under
oxidative stress conditions.
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Oxidation has only a marginal effect on the specificity constants
Next, we wanted to understand the impact of oxidation on the kinetic parameters of GSTF9, and
this for both the Gꢀsite binding GSH substrate and the Hꢀsite binding CDNB substrate (Table II,
Fig. S1). From the MichaelisꢀMenten curves (Fig. S1) under respective substrate saturating
conditions, we determined KM and kcat values. GSTF9 oxidation increased the KM for CDNB
from 1.3 to 1.8 mM, but the kcat only slightly varies from 0.98 to 0.72 sꢀ1. For GSH, the KM
decreases 2ꢀfold from 131 µM to 71 µM, while the kcat only slightly varies from 0.85 to 0.5 sꢀ1.
The overall effect of oxidation on kcat/KM is only marginal as the specificity constants are within
the same range, ~102 Mꢀ1sꢀ1 for CDNB and ~103 Mꢀ1sꢀ1 for GSH (Table II).
GSTF9 oxidation induces H-site flexibility
To understand the impact of oxidation at the molecular level, we decided to solve the structure of
oxidized GSTF9 crystals. First, crystals were grown using GSTF9ox (GSTF9 oxidized using a
1:100 GSTF9:H2O2 ratio). The resulting GSTF9ox structure was solved to 2.2 Å resolution (Table
I, PDB code 6F05). Second, we exposed the GSTF9red crystals to the extreme oxidation
conditions of 0.5 M H2O2 and 1 mM NaOCl for 1 h (GSTF9H2O2+NaOCl, PDB code 6F01). While
GSTF9red (Fig. 1) formed trigonal crystals, the GSTF9ox formed triclinic crystals (Table I), with
10 copies of GSTF9 in the asymmetric unit (AU). Overlay of all the chains of GSTF9ox and
GSTF9H2O2+NaOCl clearly shows mobility of the Met35, Met118 and M123 side chains, and also
the electron density surrounding amino acids 120 to 127 is absent in all the 10 copies in the AU
[Fig. 4(A)]. Unfortunately, within the crystal structures of the oxidized GSTF9, GSTF9ox and
GSTF9H2O2+NaOCl, the side chain of Met35 was not always well defined, as it is localized on a
highly mobile loop between the β2ꢀstrand and theα2ꢀhelix (amino acids 34ꢀ43). In the
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structures of both oxidized GSTF9, GSTF9ox and GSTF9H2O2+NaOCl, the Met35, Met118, and
Met123 sulfurs do not show any sulfoxide or sulfone formation under the applied oxidation
conditions [Fig. 4(A)], which is different from what we observed in our mass spectrometric
results.
Glutathione sulfonate (GSO3) inhibits the transferase activity
In addition to the high loop mobility close to the active site in both oxidized GSTF9 structures,
GSTF9H2O2+NaOCl and GSTF9ox, we observed GSH oxidized to glutathione sulfonate (GSO3)
within the Gꢀsite. GSO3 is bound in exactly the same orientation as GSH in the Gꢀsite [Fig. 4(B),
4(C)]. Both the GSH and GSO3 γꢀGlu moieties hydrogen bond to Glu65 and Ser66. The glycyl
moieties hydrogen bond to His39 and via a water molecule to Lys40. Where the GSH Cys sulfur
interacts with the Ser12 OH group [Fig. 1(B), 4(B)], the SO3 group of GSO3 makes hydrogen
bonds with the Ser12 OH group, as well as with the Ser12 backbone. Via a water molecule it also
interacts with the side chain of Tyr174 [Fig. 4(C)]. The GSH and GSO3 seem to be strongly
bound to the Gꢀsite as all the size exclusion chromatography attempts to remove the GSH from
GSTF9 were unsuccessful.
As a molecule of glutathione sulfonate (GSO3) resides in the active site of GSTF9ox and
GSTF9H2O2+NaOCl, we decided to test the effect of GSO3 on GSTF9 transferase activity. GSTF9
was incubated with increasing concentrations of GSO3 (0 to 300 µM), and the transferase activity
was measured. The increase in GSO3 concentration led to a decrease of the transferase activity
with an inhibition constant (Ki) of 34 µM [Fig. 5(A)]. Hence, formation of GSO3 during the
oxidation of GSTF9 could be another reason for the decrease in transferase and peroxidase
activity [Fig. 5(B)].
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GSTF9ox reduces hydrophobic peroxides less efficiently
Next, we decided to test whether increased Hꢀsite flexibility observed within GSTF9ox might be
the reason for the decreased transferase activity. Loss of a structured Hꢀsite under oxidative
stress [Fig. 4(A)] might affect the binding of hydrophobic substrates in the Hꢀsite of GSTF9. The
CDNB molecule used in the transferase assay is a hydrophobic molecule as it contains a benzene
ring that needs to be correctly oriented in the Hꢀsite for GSH transfer. Therefore, we decided to
test the GSTF9red and GSTF9ox peroxidase activity with two hydrophobic peroxides: cumene
hydroperoxide (with a benzene ring) and tertꢀbutyl hydroperoxide (with a more extended carbon
chain) (Fig. 6, Table III, SI). The results show a similar glutathione peroxidase activity for
GSTF9red and GSTF9ox towards H2O2 after 30 min (Fig. 6, Table SI). On the other hand, when
comparing the GSTF9red and GSTF9ox peroxide reduction after 30 min incubation with tertꢀbutyl
peroxide and cumene hydroperoxide, it became apparent that less peroxide is being consumed
for these more hydrophobic peroxides by GSTF9ox (Table III, SI). A clear trend was observed,
peroxidase activity towards more hydrophobic peroxides decreases for GSTF9ox compared to
GSTF9red (Fig. 6). The correlation between the concentration of peroxide consumed by GSTF9red
versus the hydrophobicity index (ALogP) of the peroxides corroborates the hypothesis that
GSTF9 oxidation impairs its activity towards hydrophobic substrates (Fig. 6).
GSTF9ox employs a thermodynamic compensatory system
To further understand the source of the increased structural flexibility observed upon GSTF9
oxidation, we decided to compare the thermodynamic activation parameters (ΔH#, ΔS# andΔ
G#) and the energy of activation (Ea) of oxidized and reduced GSTF9. Therefore, we determined
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the initial velocities of the transferase activity with CDNB at different temperatures. We derived
the thermodynamic parameters of the CDNB glutathione transferase reaction from the linear
Eyring and Ahrrenius plots and the respective equations (Table VI, Fig. 7). We observed an
enthalpyꢀentropy compensatory system where changes in enthalpy are balanced by changes in
entropy, leading to similar ΔG# values for GSTF9red and GSTF9ox.20 The energy of activation
(Ea) and enthalpy (ΔH#) values indicate a larger energetic barrier to be overcome by oxidized
GSTF9 compared to reduced GSTF9. The increased energy barrier is accompanied by a less
pronounced conformational change of GSTF9ox in order to reach the activated complex, leading
to an enthalpyꢀentropy compensatory mechanism.
Discussion
Glutathione transferase (GST) enzymes have an important role in the protection of plants against
different stress conditions including xenobiotic and peroxide stress.3,21 Understanding how these
enzymes function and how their activities are influenced under stress conditions is important to
understand how they survive extreme conditions. Prior to this study, two members of the GST
family, GSTU23 and GSTF9, were found present in the sulfoxome of A. thaliana leafs exposed
to high light stress,1 and as such sensitive to methionine oxidation. In a recent study, we have
investigated how the activity of GSTU23 is influenced under hydrogen peroxide stress.12 We
found that at lower H2O2 levels (100 µM), MetSO formation is the major postꢀtranslational
modification on GSTU23, which is recycled by methionine sulfoxide reductases (Msr). Here, we
focus on the impact of oxidation on GSTF9, an enzyme with no cysteines.
GSTF9 has no cysteines, but six methionines, three of which (Met35, Met118 and Met123) are
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located within or close to the Gꢀ and Hꢀsites (Fig. 1), and which could interfere with GSH or
hydrophobic substrate binding. We showed with mass spectrometry sulfoxidation of these three
methionines and how they influence the GSTF9 glutathione transferase activity [Fig. 2(A)] and
the glutathione peroxidase activity [Fig. 2(B), 6]. A similar case is observed in a study performed
by Lee et al.,13 where oxidation of methionines in A. thaliana GSTF2 and GSTF3 causes a
decrease in the GST transferase activity, and this activity could be recovered with methionine
sulfoxide reductase B7 (MsrB7). Important to note is that similar to GSTF9, both GSTF2 and
GSTF3 also contain no Cys residues.13 Similarly, we showed a decrease in glutathione
transferase activity of A. thaliana GSTU23 upon methionine oxidation, which is restored when
treated with MsrA and MsrB.12 Here, we show the importance of MsrA and MsrB in restoring
GSTF9 transferase activity [Fig 2(A)]. Treatment with the Msr enzymes recovers the transferase
activity of GSTF9ox up to 94%; however, 6% remains unrecovered. This might be due to the
presence of a methionine sulfone, as observed in the MS analysis, or the presence of GSO3,
which was observed in the active site of GSTF9ox structure [Fig. 4(C)].
The active site of GST enzymes consists of a highly conserved GSH binding site, also known as
the Gꢀsite, and a less conserved hydrophobic Hꢀsite, which coordinates the binding of the
hydrophobic coꢀsubstrates to which GSH is transferred.22 Mechanistically, Hꢀbonding of GSH
with a catalytic Ser residue in the Gꢀsite results in a drop of the sulfur pKa,23ꢀ25 stabilizing it as a
thiolate for a nucleophilic attack on an electrophilic coꢀsubstrate in the Hꢀsite.26 To study how
such oxidative modifications could interfere with the binding of the substrates to the two active
sites, we studied the steadyꢀstate kinetics by varying both substrates concentrations (GSH and
CDNB) (Table II, Fig. S1). The results show especially an increase in the KM for CDNB, and a
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decrease of the KM for GSH, with less variation in the kcat values (Table II). GSTF9 does not use
a KMꢀkcat compensatory mechanism to deal with increased H2O2 concentrations as observed for A.
thaliana GSTU23,12 or for cytosolic malate dehydrogenase 1 (MDH1).27
We clearly observed increased flexibility of the loop carrying Met35 (between β2 and α2) [Fig.
4(A)], located close to the Gꢀsite, and the loop carrying Met118 and Met123 (between α4 and
α5), located at the Hꢀsite [Fig. 4(A)]. Although methionine oxidation was observed with mass
spectrometry and we showed activity recovery with methionine sulfoxide reductases, methionine
sulfoxide formation was not observed in the structures, as electron density for the α4ꢀα5 loop is
missing in the oxidized structures. Oxidation of protein methionines, which lead to local
unfolding and impact the protein activity, have been reported in several other studies.28,29 For
example, the chaperoneꢀlike activity of A. thaliana chloroplast Hsp21 is influenced due to the
oxidation of its Nꢀterminal methionines (Met49, Met52, Met55 and Met59). Here, methionine
sulfoxide formation causes partial unfolding due to the local loss of the helical structure.28 For
GSTF9, it looks that methionine oxidation induces local flexibility of the loops between β2ꢀα2
and between α4ꢀα5, which could make these loops more accessible to the A. thaliana MsrA or
MsrB, and thus facilitating sulfoxide reduction.
Numerous glutathione transferase enzymes can also act as hydroxyl peroxidases but with rate
constants much lower30,31 than those for established glutathione peroxidases and
peroxiredoxins.32,33 While oxidation has been shown to decrease the H2O2 peroxidase activity of
GSTU23,12 our results show that the GSTF9 glutathione peroxidase activity is resistant to
oxidation. However, the glutathione peroxidase activity of GSTF9ox is here more impaired for
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hydrophobic peroxides (Table III, SI, Fig. 6). This suggests that the loss of a structured
hydrophobic Hꢀsite upon oxidation of GSTF9 forms the basis for the loss in peroxidase activity
towards more hydrophobic peroxides.
To further understand the observed changes in kinetic efficiency of GSTF9, we studied the
transition state thermodynamic parameters of reduced and oxidized GSTF9 (Fig. 7, Table VI).
To study the transition state of GSTF9red and GSTF9ox, thermodynamic parameters were
calculated and the data of the activated complex show a compensatory system with different
enthalpy and entropy values for oxidized and reduced GSTF9 (Table VI). Generally, when
comparing both forms, the overall spontaneity of the E:S# formation is similar (reflected by
similar ΔG# values), but a different intermediate transition state along the reaction coordinates
might get temporally populated. The Ea and ΔH# are a result of the sum of interatomic
interactions that undergo rearrangement during the E:S activation step. These interatomic
interactions are hydrogen bonds and van der Waals interactions between enzyme and
substrates.34 A positive difference of 2.3 kcal/mol was found when the absolute value of ΔH# of
the oxidized GSTF9 was subtracted from the ΔH# value obtained for its reduced counterpart.
The less pronounced energetic barrier for GSTF9red indicates that the number of interactions that
needs to be rearranged in the reduced form is smaller than in the oxidized form (Table VI),
leading to the observed energetic difference. Subsequently, the E:S complex formed by the
reduced form seems to be less pronounced and more prone to rearrangement in order to achieve
the transition state. Entropy is the measure of the number of microstates available for a specific
species and is more widely described as the degree of disorder. The generation of disorder when
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E:S is converting to E:S# comes from bond rearrangement and changes in solvation.35 The value
obtained for Δ(TΔS#)oxꢀred indicates that GSTF9red undergoes a larger conformational change in
order to reach the transition state when compared with GSTF9ox.36 The more positive TΔS# and
positive ΔH# indicates that the increased flexibility of the loops between β2ꢀα2 and between
α4ꢀα5 is a positive feature, and to achieve the transition state a larger number of interatomic
interactions need to be rearranged. Accordingly, in order to maintain its enzymatic activity under
oxidative stress, GSTF9 seems to follow a structural compensatory system. The best scenario is
composed by an increase in flexibility in the regions directly involved in catalysis and an
increase in rigidity in all the other areas, however, GSTF9 oxidation increased the flexibility
globally, leading to a neglectable Δ(ΔG#)oxꢀred value.36
In conclusion, we showed that methionine oxidation near the two active sites causes local loopꢀ
flexibility. As a consequence, oxidized GSTF9 becomes substrate for methionine sulfoxide
reductases. By thermodynamic and structural compensatory mechanisms, GSTF9 has evolved to
become an enzyme that is able to catalyze detoxification reactions under oxidative conditions.
Materials and methods
GSTF9 purification and oxidation – Protein expression, purification and oxidation were
performed as described by Jacques et al.4 Prior to oxidation, GSTF9 was reduced with 10 mM
DTT at RT for 30 min followed by gel filtration using a Superdex75 HR 10/30 size exclusion
chromatography (SEC) column equilibrated in 1xPBS. The collected sample was incubated with
1:100 molar ratio of protein to H2O2 for 135 min at 10°C, as performed by Jacques et al.4 Micro
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®
BioꢀSpin Chromatography Column (BIOꢀRAD) equilibrated with 1xPBS was used to remove
excess of H2O2.
Glutathione transferase activity assay – The assay was performed as described by Habig et
al.37 Briefly, a UVꢀvisible Varian Cary 100 spectrophotometer was used to measure the GSTF9
catalysed conjugation of GSH onto CDNB at A340. The reaction mixture was composed of 0.5
mM GSH and 0.2 µM GSTF9 in 250 mM MOPS pH 6.5 and 150 mM NaCl buffer. The assay
was initiated by the addition of 3.8 mM CDNB to a final reaction volume of 500 µL.
To determine the influence of Msr incubation on the GSTF9ox transferase activity, a 2:1 molar
ratio of GSTF9ox (10 µM) was incubated with Corynebacterium diphtheriae MsrA38 or MsrB12
(5 µM) for 1 h at 25°C in a 1xPBS buffer containing 0.5 mM DTT. As a control, GSTF9red was
also incubated with MsrA, MsrB and DTT. The samples were diluted and 10 µL was transferred
to the assay mixture (500 µL), where the final concentrations were: 0.2 µM GSTF9, 0.1 µM Msr
and 10 µM DTT. A sample with only GSTF9ox and DTT was included as a control. Prior to the
incubation of GSTF9 with Msr enzymes, both MsrA and MsrB were incubated with 20 mM DTT
for 30 min at room temperature. Following reduction, DTT was removed on a Micro BioꢀSpin®
Chromatography Column (BIOꢀRAD) equilibrated with 1xPBS.
To determine the kinetic parameters of GSTF9, at varying concentrations of GSH (0 to 750 µM)
or CDNB (0 to 4.5 mM) the initial velocities were measured and the MichaelisꢀMenten equation
1 was used to fit these data and obtain the kinetic parameters kcat and KM. For varying [GSH], the
CDNB concentration used was 3.8 mM and for varying [CDNB], 0.5 mM GSH was used. Two
independent replicates were measured for each substrate concentration. In equation 1, v
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represents the initial velocity, Vmax the maximum velocity, KM the Michaelis constant and [S] the
substrate concentration.
ꢅ ꢇ
ꢆ
ꢁ
ꢂꢃꢄ
ꢀ = ꢈ
equation 1
ꢊꢅꢆꢇ
ꢉ
GSTF9 FOX assay with GSH and GSO3 – Ferrous oxidationꢀxylenol orange (FOX) assay was
used to monitor the peroxidase activity of GSTF9.39 The assay was performed as described by
Tossounian et al.12 Briefly, the reaction mixture included 50 µM GSTF9red or GSTF9ox, 1 mM
GSH and 150 µM H2O2 in 100 mM potassium phosphate buffer at pH 6.4, which was incubated
at room temperature (RT). At three time points (0, 15 and 30 min), 10 µL of the sample was
mixed with 490 µL of the FOX assay mixture and incubated in the dark. After 30 min,
SpectraMax340PC spectrophotometer (Molecular Devices) was used to measure the absorption
at 560 nm. In order to determine the effect of GSO3 on the glutathione peroxidase activity of
GSTF9, the same procedure was followed with the addition of 0.5 mM GSO3. As background,
the reaction mixture without enzyme was used. A reaction mixture containing 1 mM GSO3 was
also used as a control. Glutathione peroxidase activity of GSTF9 was also tested by replacing
H2O2 (200 µM) with two hydrophobic peroxides, cumene (200 µM) or tertꢀbutyl hydroperoxide
(200 µM). Three independent measurements were performed and the data representing the
∆[peroxide]oxꢀred versus ALogP value of H2O2 (ꢀ0.18), tertꢀbutyl hydroperoxide (0.79) and
CuOOH (2.02) was plotted using GraphPad prism7. The AlogP values were obtained from
ChEMBL.
GSTF9-GSO3 inhibition assays - To determine the GSTF9 inhibition by GSO3, the
SpectraMax340PC spectrophotometer (Molecular Devices) was used, where increasing
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Page 18 of 44
concentrations of GSO3 (0, 25, 50, 100, 200, 300, 400 and 500 µM) were added to the reaction
mixture containing 0.4 µM reduced GSTF9, 0.5 mM GSH and 3.8 mM CDNB in 250 mM
MOPS pH 6.5 and 150 mM NaCl buffer. The reaction started by the addition of CDNB. As
background, the reaction mixture without enzyme was used. The inhibition constant for the
inactivation of GSTF9 by GSO3 was determined from the plot of the initial velocities versus the
GSO3 concentration (equation 2). For all data, two independent measurements were performed
and GraphPad prism7 was used to plot the data. In equation 2, vi represents the initial velocity, Ki
the inhibition constant, V the maximum velocity and [GSO3], the concentration of inhibitor.
ꢈ ∗ꢍ
ꢌ
vꢋ = ꢈ ꢊꢅꢎꢏꢐ ꢇ + A
equation 2
ꢑ
ꢌ
GSTF9 thermodynamic parameter determination - GSTF9 thermodynamic parameters were
assessed by measuring the initial velocity variation in function of temperature. The reactions
were carried out at 17°C, 20°C, 25°C, 30°C and 37°C in quartz cuvettes using a UVꢀvisible
Varian Cary 100 spectrophotometer and were monitored for 180 s with GSTF9 (reduced or
oxidized) final concentration 0.2 µM, 0.5 mM GSH and 3.8 mM CDNB in 250 mM MOPS pH
6.5 and 150 mM NaCl buffer. The values presented correspond to two replicates. Subsequently,
the thermodynamic parameters were obtained fitting the data to Ahrrenius (equation 3) and
Eyring (equation 4) approaches. The Gibbs free energy of activation (ΔG#) was calculated using
equation 5. In equation 3, Ea represents the reaction activation energy, R, the gas constant (8.314
J.molꢀ1Kꢀ1) and A, the product of the collision frequency (Z). For equation 4, kB represents the
Boltzmann constant (1.3805x10ꢀ23 J.Kꢀ1), h, the Planck’s constant (6.6256x10ꢀ34 J.s). For
equation 5, ΔH# and ΔS# represent the enthalpy and entropy variation of activation.
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ꢓ
ꢃ
ꢕ
lnꢒ = − ꢔ ꢖ + lnA
equation 3
#
#
∆ꢚ
ꢔ
ꢕ
ꢖ
∆ꢆ
ꢔ
ꢘ
ꢛ
ln ꢗꢘꢖꢙ = −
+
+ ln ꢗ ꢜ ꢙ
equation 4
equation 5
ΔG# = ΔH# ꢀ TΔS#
Mass spectrometric analysis of Met oxidation - For the identification of oxidized Met residues
(methionine sulfoxide and methionine sulfone), GSTF9red and GSTF9ox, were analyzed by mass
spectrometry as described by Tossounian et al.12
Acknowledgements: This work was made possible thanks to financial support from (i) Vlaams
Instituut voor Biotechnologie (VIB) (ii) Omics@VIB Marie Curie COFUND fellowship to IVM,
(iii) FWO PhD fellowship granted to MAT (iv) Fonds voor Wetenschappelijk Onderzoek
Vlaanderen (FWO project G0D7914N: “Sulfenomics: oxidatieve schakelaars in planten. Hoe
zwavelhoudende planteneiwitten via 'agressieve' zuurstof praten”) granted to JM, (v) the
equipment grant HERC16 from the Hercules foundation granted to JM, (vi) the Vrije Universiteit
Brussel Strategic Research Programme (SRP34) granted to JM, and the FWO Excellence of
Science project G0G6918N (EOS ID 30829584) granted to JM.
We thank the beamline scientist at the Diamond Light Source beamline IO4, for their technical
support. Furthermore, we thank Kristof Moonens from VIBꢀVUB Center for Structural Biology
for Xꢀray data collection on the GSTF9 crystals. We thank Frank Van Breusegem from Ghent
University, Belgium for giving us At-gstF9 plasmid.
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Author contributions: GSTF9 purification was performed by MAT, KW and IVM.
Biochemical, kinetic and thermodynamic assays were performed by MAT. Crystallization was
performed by KW and IVM, and Xꢀray crystal structures were solved by IVM. Mass
spectrometry and data analysis was performed by DV. MAT, IVM, LAR and JM wrote the
manuscript. JM supervised all aspects of the project.
Accession codes: The structures of GSTF9red (PDB 6EZY), GSTF9H2O2+NaOCl (PDB 6F01),
GSTF9ox (PDB 6F05), have been deposited in the Protein Data Bank.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents
of this article.
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Figure legends
Figure 1. Crystal structure of reduced GSTF9. (A) The Xꢀray crystal structure of reduced
GSTF9red shows a typical GSTꢀfold. The crystal structure of GSTF9red is shown in a gray cartoon
representation with the alpha helices and beta sheets labeled. The glutathione (GSH) binding site
(Gꢀsite) and the hydrophobic substrateꢀbinding site (Hꢀsite) are indicated with arrows. A
glutathione molecule is observed in the Gꢀsite, and sulfenylated GSH (GSOH) in the Hꢀsite. The
catalytic serine (S12), the Met residues (M35, M70, M118, M123 and M184), and the Nꢀ and Cꢀ
terminus are indicated. An omit map contouring the GSH and GSOH at 3σ is shown in green.
(B) A closer view of the three methionines (M35, M118 and M123) located close to the Gꢀ and
Hꢀsite. Surface representation is shown for atoms within 4 Å of HꢀGSOH. The Hꢀbond between
the catalytic S12 and the sulfur of GSH is 3.3 Å (black dotted line).
Figure 2. GSTF9 oxidation has a different impact on the transferase and glutathione peroxidase
activity. (A) Bar graphs show the percentage of transferase activity measured in rate constants
(k) of GSTF9red and GSTF9ox (0.2 µM), where the GST catalysed conjugation of GSH (0.5 mM)
onto CDNB (3.8 mM) is followed at 340 nm. When oxidized, GSTF9ox activity significantly
(P<0.001) decreases from 100% (0.54 sꢀ1) to 70% (0.38 sꢀ1). MsrA and MsrB treatment causes
activity increase from 70% to 94% (0.51 sꢀ1), indicating that Msr enzymes are capable of
recovering GSTF9ox activity. Two control samples were used: GSTF9ox incubated with DTT and
GSTF9red incubated with MsrA, MsrB and DTT. The data from at least 3 independent
experiments are presented as a mean ± SD. (B) FOX assay was used to compare the peroxidase
activity of GSTF9red and GSTF9ox in the presence of 150 µM H2O2. Remained H2O2
concentrations were determined for samples taken after 15 and 30 min. Upon treatment of
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GSTF9 with hydrogen peroxide (GSTF9ox), glutathione peroxidase activity is not influenced. As
a control, the influence of GSH on H2O2 reduction was also recorded. The data from 3
independent experiments were normalized and presented as a mean ± SD.
Figure 3. Identification of Met35 of GSTF9 as a methionine sulfoxide. Identification of
oxidation on Met35 of GSTF9 under H2O2 exposure. The LCꢀMS spectrum shows MS2 data
obtained from a +2 parent ion with m/z 718.3. The yꢀ and bꢀ series of ions allow exact
localization of the methionine sulfoxide. A neutral loss of 64 Da is observed from the y6
daughter ion and corresponds to the release of methane sulfenic acid (CH3SOH) from the side
chain of methionine sulfoxide.
Figure 4. Oxidation leads to GSTF9 Hꢀsite disorder and oxidized glutathione (GSO3) formation
in the Gꢀsite. (A) Overlay of the 10 copies of GSTF9 in the AU of GSTF9ox (shown in gold
cartoon) and the two chains present in the AUs of both the GSTF9red (chain A and B in gray
cartoon) and GSTF9_H2O2+NaOCl structures (chain A in pink, chain B in green) shows a high
mobility for the α2ꢀβ2 and especially the α4ꢀα5 loops. Residues 120ꢀ127 in the latter loop
become entirely disordered in the GSTF9ox structure, as well as in chain A of the GSTF9_
H2O2+NaOCl structure. The side chains of Met35, Met118 and Met123 shown in sticks are highly
mobile. (BꢀC) The active site Hꢀbond network of GSFT9red (gray cartoon) has a GSH molecule
(B), which is oxidized to GSO3 (C) within the GSTF9ox Gꢀsite (gold cartoon). The residues (gold
for GSTF9ox and gray for GSTF9red) interacting with GSH and GSO3 are shown in stick
representation. Leu34 and Phe10 delineate the Gꢀsite. Hydrogen bonds are in gray or black
dashed lines. Water molecules Hꢀbonded to GSH or GSO3 are in red spheres. An omit map
contouring the ligands, GSH and GSO3, at 3σ is shown in green.
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Figure 5. Glutathione sulfonate (GSO3) inhibits GSTF9 glutathione transferase and peroxidase
activity. Increasing concentrations of GSO3 results in a decrease of the initial velocity of (A) the
GSTF9 glutathione transferase and (B) the peroxidase activity. The inhibition constant (Ki) for
the transferase activity is 34 µM. The data represent the mean ± SD of 2 independent
experiments.
Figure 6. Oxidized GSTF9 reduces hydrophobic peroxides less efficiently. A difference plot of
the peroxide concentration (µM) (H2O2, tꢀbutyl peroxide, CuOOH) reduced after 30 min with
GSTF9ox and GSTF9red versus the hydrophobicity index (ALogP) of the peroxides is presented.
The more hydrophobic the peroxide, the larger the difference between GSTF9red and GSTF9ox
peroxide reduction (∆[peroxide]). The data represent the mean ± SD of 3 independent
experiments. The reduction scheme for peroxides (X) with the release of oxidized glutathione
(GSSG) and a water molecule is shown. The structures of the three peroxides (H2O2, tꢀbutyl
peroxide, CuOOH) are generated using ChemBioDraw 14.0.
Figure 7. Arrhenius (A) and Eyring (B) plot of GSTF9red and GSTF9ox. Temperature
dependence of lnk and ln (k/T) of the glutathione transferase catalyzed reaction by reduced and
oxidized GSTF9 at pH 6.4 in a temperature range from 17°C to 37°C is shown. The linearity of
the plots suggests that there is no change in the rateꢀlimiting step or protein inactivation within
this temperature range.
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TABLES
Table I. GSTF9 X-ray data collection and refinement statistics.
Dataset
PDB code
GSTF9red
6EZY
GSTF9H2O2+NaOCl
GSTF9ox
6F05
6F01
Data collection
Beamline
Wavelength
IO4@DLS$
0.9795
IO4@DLS$
0.9795
IO4@DLS$
0.9795
Processing
Space group
P31 2 1
P31 2 1
P1
Cell parameters (Å/°)
61.43 93.97 107.71 93.19 101.57 114.76 114.76
61.43 93.97
107.71
93.19 101.57
101.97
101.97
90.31
90 90 120
Resolution, Å (outer
shell)(a)
48.13-2.34 (2.48-2.34)
66.84-2.5 (2.57- 62.7-2.2 (2.26-2.2)
2.5)
Total reflections
Unique reflections
Completeness (%)
Multiplicity
282989 (43703)
2880 (4594)
98.7 (96.8)
9.9 (9.5)
158033(11796)
24179 (1781)
99.8 (99.9)
6.5 (6.6)
268584 (19908)
116579 (8561)
97.8 (97.1)
2.3 (2.3)
CC1/2 (%)
Rmeas (%)
<I/σ(I)>
99.9 (55.9)
11.2 (137.9)
18.3 (1.64)
99.5 (71.1)
17.7 (124.8)
11.56 (2.09)
99.7 (73.3)
7.4 (60.4)
10.67 (2.08)
Refinement
Resolution range (Å)
Percentage observed (%)
Rcryst (%)(b)
49.41-2.35
98.79
15.63
66.84-2.3
99.84
16.43
65.73-2.06
97.66
16.15
Rfree (%)(c)
19.66
20.89
20.93
RMS
Bonds (Å)
Angles (°)
0.012
1.267
0.010
1.268
0.008
1.18
Ramachandran Plot
Most favored (%)
Additionally allowed (%)
Disallowed (%)
97.83
1.69
0.48
97.31
2.2
0.49
97.76
1.68
0.56
GSH (G-site) and
GSOH (H-site)
GSO3 (G-site) and
GSOH (H-site)
GSO3 (G-site)
Bound Ligand
(a) Values between brackets are for the highest resolution shell; (b) Rcryst = Σ||Fobs|–|Fcalc||/Σ|Fobs|, Fobs and Fcalc are
observed and calculated structure factor amplitudes; (c) Rfree is the same as Rcryst, but using a random subset of 5%
of the data excluded from the refinement; $Diamond Light Source (DLS)
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Table II. Steady-state kinetics parameters of GSTF9 under reducing and oxidizing
conditions. Kinetic parameters were determined using varying concentrations of GSH or
CDNB and the values were plotted using the Michaelis-Menten equation.
[GSH]
[CDNB]
F9red
F9ox
F9red
F9ox
131 ± 9 µM 71 ± 7 µM
1.3 ± 0.1 mM 1.8 ± 0.2 mM
KM
k
cat (s-1)
0.85 ± 0.02 0.50 ± 0.01
0.98 ± 0.02
7.5 x 102
0.72 ± 0.04
4.0 x 102
6.5 x 103
7.0 x 103
kcat/KM (M-1s-1)
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