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Plant Physiology and Biochemistry xxx (2015) 1e6
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Q3 Q2
A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase Nicolas Navrot a, Nicklas Skjoldager a, Jakob Bunkenborg b, c, Birte Svensson a, €gglund a, * Per Ha a
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Building 224, DK-2800 Kgs. Lyngby, Denmark Center of Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark c Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, DK-2650 Hvidovre, Denmark b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2015 Accepted 7 March 2015 Available online xxx
Monomeric and dimeric forms of recombinant barley (Hordeum vulgare subsp. vulgare) glutathione peroxidase 2 (HvGpx2) are demonstrated to display distinctly different functional properties in vitro. Monomeric HvGpx2 thus has five fold higher catalytic efficiency than the dimer towards tert-butyl hydroperoxide, but is more sensitive to inactivation by hydrogen peroxide. Treatment of the monomer with hydrogen peroxide results in dimer formation. This observed new behavior of a plant glutathione peroxidase suggests a mechanism involving a switch from a highly catalytically competent monomer to a less active, but more oxidation-resistant dimer. © 2015 Published by Elsevier Masson SAS.
Keywords: Glutathione peroxidase Thioredoxin Antioxidant Hydrogen peroxide Alkyl peroxide Oligomerization
1. Introduction Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), the superoxide (O2) and hydroxyl (OH ) radicals cause oxidative stress and macromolecular damage. ROS can also react with nitric oxide (NO ) leading to formation of toxic reactive nitrogen species (RNS) such as peroxynitrite (ONOO). Higher plants are sessile organisms and thus particularly vulnerable to the considerable amounts of ROS produced as side-products in photosynthesis and respiration. In addition, H2O2 and NO exert important signaling functions in regulation of fundamental processes, e.g. apoptosis, seed germination and responses to biotic and abiotic stresses (Bailly, 2004; Bolwell, 1999). Plants utilize multiple non-enzymatic and enzymatic antioxidant systems to cope with
Abbreviations: COOH, cumene hydroperoxide; cv., cultivar; CysGpx, thioldependent glutathione peroxidase; DTNB, 5,50 -dithiobis-(2-nitrobenzoic acid); Gpx, glutathione peroxidase; GSH, glutathione; Prx, peroxiredoxin; ROS, reactive oxygen species; SEC, size exclusion chromatography; SecGpx, selenium-dependent glutathione peroxidase; tBOOH, tert-butyl hydroperoxide; Trx, thioredoxin. * Corresponding author. E-mail address: [email protected] (P. H€ agglund).
environmental stresses and control ROS and RNS levels (Foyer and Noctor, 2005; Møller, 2001; Noctor and Foyer, 1998). Among these, the thiol-dependent non-heme peroxiredoxins (Prx) and glutathione peroxidases (Gpx) catalyze reduction of peroxynitrite and different peroxides to nitrites and alcohols, respectively (Flohe et al., 2003). Gpx is also involved in signal transduction pathways through interactions with specific regulators, e.g. the protein phosphatase ABI2 in Arabidopsis thaliana (Miao et al., 2006) or transcription factors such as YAP1 in yeast (Delaunay et al., 2002). Signaling with plant Gpx is associated with apoptosis, cell cycle regulation and plant cell differentiation (Chen et al., 2004; Faltin et al., 2010; Kadota et al., 2005). All Prx share an active site peroxidatic cysteine that gets oxidized to a sulfenic acid upon substrate turnover, but they differ in the mechanism of the reductive part of the catalytic cycle (Wood et al., 2003). Plant Prx are classified based on sequence similarity into four groups which are reduced either directly by redoxins or low-molecular weight antioxidants (1-cys Prx and Prx II) or indirectly via formation of either an inter- (2-cys Prx) - or an intramolecular (Prx Q) disulfide bond involving a so-called resolving cysteine in Prx (Rouhier and Jacquot, 2005).
http://dx.doi.org/10.1016/j.plaphy.2015.03.003 0981-9428/© 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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Gpx are divided into selenium-dependent (SecGpx) and selenium-independent (CysGpx) enzymes according to their catalytic mechanism (Flohe et al., 2011). SecGpx utilize a catalytic selenocysteine that undergoes oxidation to selenenic acid by reaction with peroxide (Fig. 1A). Reduced selenocysteine is regenerated at the expense of two molecules of reduced glutathione (GSH). The high reactivity of the active site selenocysteine makes SecGpx and extremely efficient as peroxide elimination enzymes (Flohe , 2001). The selenium-independent CysGpx by Brigelius-Flohe contrast function via dithiol-disulfide chemistry similarly to Prx (Herbette et al., 2007) involving formation of an intramolecular disulfide in turn reduced by thioredoxin (Trx) regenerating the active enzyme (Fig. 1B). Animal cells and some green algae, e.g. Chlamydomonas reinhardtii express both SecGpx and CysGpx, whereas fungi and land plants appear to only contain CysGpx (Margis et al., 2008). CysGpx are found in different plant organs and suggested to be localized in the cytosol, mitochondria and chloroplasts (Navrot et al., 2006). Plant CysGpx reduce peroxides with a similar catalytic efficiency as other Trx-dependent Prx, which is several orders of magnitude below that of SecGpx (Iqbal et al., 2006; Navrot et al., 2006; Selles et al., 2012). The rate of oxidation of the catalytic cysteine to sulfenic acid is however almost as high as for SecGpx (Fourquet et al., 2008). Dimeric poplar PtGpx5, which currently represents the only available plant CysGpx structure, undergoes major conformational changes to switch between reduced and oxidized forms presumably limiting the catalytic rate (Koh et al., 2007). Noticeably, an a-helix unfolds to bring the so-
called resolving cysteine close to the oxidized catalytic cysteine resulting in formation of an intramolecular disulfide bond and Trxmediated regeneration of the reduced enzyme (Fig. 1B). In the present study biochemical and structural features of the major barley seed CysGpx, HvGpx2, were investigated in relation to the redox state of the enzyme milieu. Remarkably HvGpx2 switches between a monomeric and a dimeric form that differ in catalytic efficiency and susceptibility to substrate-mediated inactivation in vitro. While this type of structural rearrangement has been described previously for Prx (Barranco-Medina et al., 2009), it is novel among CysGpx. 2. Materials and METHODS 2.1. Chemicals and proteins Recombinant barley (Hordeum vulgare subsp. vulgare) NADPHdependent thioredoxin reductase (HvNTR2), thioredoxins (HvTrxh1, HvTrxh2, and Escherichia coli Trx1) were obtained as €rnberg et al., 2012; Maeda et al., 2003; Shahpiri et al., described (Bjo 2008). All chemicals were from SigmaeAldrich (St. Louis, MO), unless stated otherwise. SDS-PAGE was performed using NuPAGE Novex 12e15% Bis-Tris minigels and electrophoresis system (Thermo Fisher Scientific, Waltham, MA). 2.2. Cloning of HvGpx2 Barley (cultivar (cv.) Barke) seeds were germinated for 3 days on wet filter paper at RT in the dark, the embryos were excised with a scalpel and RNA was extracted with Plant Mini Kit for RNA extraction (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. Specific primers HvGpx2for (50 -ATGGCCTCC TCCGCCTCCTCCGTC-30 ) and HvGpx2rev, (50 -GGATCCTTAAGAACTC GCGAGCAA-30 ) including NdeI and BamHI restriction sites, respectively, were used to amplify HvGpx2 cDNA without the predicted N-terminal signal peptide using 1-step RT-PCR kit (Qiagen) according to the manufacturer's instructions. In order to optimize the temperature for PCR the sequence encoding for three N-terminal alanines was omitted. The amplified fragment was cloned into pET15b for expression of HvGpx2 with an N-terminal 6xHis tag (the sequence of the recombinant protein is displayed in Fig. S1 in Supplementary material). Plasmid DNA sequences were confirmed by bidirectional sequencing using T7 and T7 terminator primers. 2.3. Expression and purification of HvGpx2
Fig. 1. Catalytic cycle of (A) selenium-dependent Gpx (SecGpx) and (B) seleniumindependent Gpx (CysGpx). A. Selenenic acid (Gpx-SeOH) is formed upon reaction of the catalytic selenocysteine (Gpx-SeH) in SecGpx with a hydroperoxide (ReOOH). Reduced glutathione (GSH) reacts with SeOH forming a glutathione adduct (Gpx-SeSG) that subsequently reacts with GSH, yielding Gpx-SeH and oxidized glutathione (GSSG). B. The catalytic (peroxidatic) cysteine thiol group in CysGpx (Gpx-SH) is oxidized to the sulfenic acid (Gpx-SOH) by reaction with ReOOH. A second (resolving) cysteine reacts with Gpx-SOH forming an intramolecular disulfide bond (Gpx-S-S) that reacts with reduced Trx, yielding Gpx-SH and oxidized Trx.
LB medium (1 L in a 3 L shake flask) was inoculated to OD600nm ¼ 0.1 with an overnight culture of E. coli Rosetta(DE3)™ (Merck Millipore, Darmstadt, Germany) containing pET15bHvGpx2. Cells were propagated at 37 C and induced by 0.1 mM IPTG added when the culture reached OD600nm ¼ 0.6. Cells were pelleted (4000 g, 15 min) after 16 h induction at 30 C and resuspended in 10 mL Bugbuster reagent added 1 mL benzonase (Merck Millipore). After centrifugation HvGpx2 was purified from the supernatant by Ni-ion affinity chromatography (5 mL HisTrap HP, GE Healthcare, Little Chalfont, UK) and eluted by a linear 30e200 mM imidazole gradient of loading buffer (50 mM TriseHCl pH 8, 400 mM NaCl, 10 mM imidazole) and elution buffer (loading buffer with 400 mM imidazole). Fractions containing HvGpx2 (determined by SDS-PAGE) were pooled, dialyzed (50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA), concentrated (10 kDa cutoff; Amicon Ultra 15 mL centrifugal filter unit) and subjected to size exclusion chromatography (SEC) on a Superdex 75 26/60 column (GE Healthcare) in the dialysis buffer. Fractions containing monomeric
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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and dimeric HvGpx2 were pooled separately, dialyzed against 50 mM TriseHCl pH 8, 1 mM EDTA and concentrated (as above). 2.4. Thiol titration and protein concentration determination Protein samples (2030 mM) in 100 mM TriseHCl pH 8, 8 M urea (270 mL) were mixed with 30 mL 1 mM 5,50 -dithiobis-(2nitrobenzoic acid (DTNB) and absorbance at 412 nm was measured (BioTek microplate reader, Winooski, VT) using N-acetyl cysteine (0e50 mM) as standard. The stoichiometry per protein molecule was calculated from spectrophotometric measurements using 3280 ¼ 18235 M1 cm1 and 21743 M1 cm1 determined by aid of amino acid analysis (Barkholt and Jensen, 1989), for HvGpx2 monomer and HvGpx2 dimer, respectively. 2.5. HvGpx2 activity assays Unless otherwise stated, HvTrxh1 or HvTrxh2 (final 6 mM) was mixed with H2O2, tert-butyl hydroperoxide (tBOOH) or cumene hydroperoxide (COOH) (final 1 mM) in 50 mM Tris HCl pH 8, 0.1% BSA, 0.25 mM NADPH, 0.2 mM HvNTR2 (total volume 100 mL). To determine kinetic parameters using peroxide (0.05e5 mM) and Trx (0.5e25 mM), NADPH oxidation was monitored as decrease in absorbance at 340 nm (3340 ¼ 6220 M1 cm1) for 60 s following the addition of 0.2 mM HvGpx2 and the slope was calculated by linear fitting. Background reaction without Gpx was subtracted for each measurement. The activity was expressed as the rate (mmol/s) and plotted versus the concentration of either Trx or peroxide. KM and kcat values were obtained from non-linear curve fits (Kaleidagraph, Synergy software, Reading, PA). 2.6. HvGpx2 inactivation and analytical SEC HvGpx2 monomer or dimer (20 mM) was mixed with H2O2 (0e10 mM) in 50 mM Tris HCl pH 8 (total volume 20 mL), incubated 2 h at RT, added 50 mM Tris HCl pH 8, 0.1% BSA (180 mL) and kept on ice. Activity was assayed in 100 mL 0.25 mM NADPH, 5 mM HvTrxh1, 1 mM tBOOH, 50 mM Tris HCl pH 8, 0.1% BSA containing 10 mL of H2O2-treated HvGpx2 (final 0.2 mM) and a saturating concentration (0.6 mM) of HvNTR2. Aliquots (10 mL) removed at appropriate time intervals from 100 mL reaction mixtures of HvGpx2 monomer (42 mM) and H2O2 (0, 0.5, 1, 5, 10 mM) in 50 mM Tris pH 8, 1 mM EDTA, were immediately diluted 20 fold in 50 mM Tris HCl pH 8, 1 mM EDTA, 0.1% BSA and assayed (final HvGpx2 0.1 mM) essentially as above to determine the inactivation kinetics. For analytical SEC, HvGpx2 monomer or dimer (175 mM) was treated with H2O2 (0.1, 0.5, 2, 6 and 20 mM) in 50 mM Tris HCl pH 8, 1 mM EDTA (500 mL) for 2 h at RT followed by separation (Superdex 75 16/60 column; GE Healthcare) in 50 mM Tris HCl pH 8, 200 mM NaCl, 1 mM EDTA. Monomer/dimer ratios were calculated from the maximum A280 values of the eluted monomer and dimer in the chromatograms. 2.7. LC-MS/MS mapping of oxidation sites HvGpx2 in 50 mM Tris HCl pH 8 was treated with different concentrations of H2O2 (0, 1, and 10 mM) in a total volume of 50 mL. From each reaction 19 mL was removed and mixed with 12 mL 8 M urea. Then 5 mL 100 mM iodoacetamide was added and incubated for 30 min at RT in the dark. Following addition of 22.5 mL 50 mM Tris HCl pH 7.4 and 0.7 mL sequencing-grade endoproteinase Asp-N (Promega, Madison, WI), the samples were incubated overnight at RT. Reversed-phase LC-MS analysis was performed on an LTQOrbitrap Velos (Thermo Fisher Scientific) connected to an Agilent 1100 nanoflow HPLC system (Agilent, Santa Clara, CA). Database searches were performed using MaxQuant Version 1.5.2.8 (Cox and
3
Mann, 2008) against the Uniprot Hordeum vulgare database with standard orbitrap settings, allowing for proteolytic cleavage on the N-terminal side of Cys, Asp, Glu residues and the following variable modifications: mono- and dioxidation (Met, Cys, Trp, Tyr), trioxidation (Cys, Trp), and kynurenine and hydroxykynurenine formation (Trp). 3. Results and discussion 3.1. A barley glutathione peroxidase family Plants possess a family of Prx and CysGpx orthologs that have become a focal point for investigations on redox regulation and signaling in plant cells (Rouhier and Jacquot, 2005). Three barley gpx genes (encoding HvGpx1e3) were discovered in the late 90's (Churin et al., 1999). Since then, exhaustive inventories of Gpx gene families have been published for A. thaliana and Populus trichocarpa (Navrot et al., 2006; Rodriguez Milla et al., 2003). Analysis of transcripts and homologues (NCBI EST databases) of HvGpx1e3 identified two additional isoforms, here named HvGpx4 and HvGpx5. Sequence alignment (Figs. S2 and S3 in Supplementary material) reveals high identity between Gpx from barley, A. thaliana and P. trichocarpa (Tables S1 and S2 in Supplementary material). HvGpx1 and HvGpx2 are closely related to two poplar isoforms, PtGpx3.1 and PtGpx3.2, which are broadly expressed in different tissues and cellular compartments, and to A. thaliana AtGpx6 highly expressed in seeds. Analysis of gpx transcriptomic data using Genevestigator (Hruz et al., 2008) suggests that the gene coding for HvGpx2 (annotated as BarPHGPX in the database) is preferentially expressed in seeds (Fig. S4 in Supplementary material). Noticeably HvGpx2 contains an N-terminal extension potentially targeting to chloroplasts (Table S3 in Supplementary material). Furthermore, an alternative initiation site encodes a presumed cytosolic form (Fig. S3 in Supplementary material). Here, the gene corresponding to HvGpx2 was amplified without the predicted Nterminal signal peptide by RT-PCR using total barley embryo RNA extracted 3 d after start of imbibition. Bidirectional sequencing of the cloned gene revealed that Glu17 in the deposited HvGpx2 sequence from cv. Haisa (Q9SME6) is substituted by a lysine in HvGpx2 from cv. Barke (this work). BarPHGPX (AB096704) from cv. OUK305 (Sugimoto and Takeda, 2003) has identical sequence to HvGpx2 from cv. Barke. 3.2. Monomeric and dimeric HvGpx2 HvGpx2 produced recombinantly in E. coli Rosetta(DE3)™ and purified to electrophoretic homogeneity consisted of two major components (Fig. 2A) with apparent molecular mass of 26.6 and 54.9 kDa (Fig. S5 in Supplementary material), suggesting the presence of a monomeric and a dimeric form (theoretical mass 20350 and 40700 Da, respectively). This stands in contrast to the mainly dimeric poplar PtGpx5 (Koh et al., 2007; Navrot et al., 2006; Selles et al., 2012). The crystal structure of PtGpx5 reveals the dimer interface to be stabilized by several aromatic residues, of which Tyr151 appears to be of particular importance. A Tyr151Arg substitution thus shifted PtGpx5 to a mainly monomeric protein as verified by SEC (Selles et al., 2012). This finding suggests that oligomerization of plant CysGpx is influenced by subtle amino acid differences at the dimer interface. In HvGpx2 and most other CysGpx from poplar, barley and A. thaliana the Tyr151 and also Trp124 both located at the dimer interface of PtGpx5 are substituted by small aliphatic residues (Fig. S3 in Supplementary material). However, the tyrosine residue preceding Tyr151 (Tyr149 in HvGpx2) appears to be conserved in most of the plant Gpx
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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N. Navrot et al. / Plant Physiology and Biochemistry xxx (2015) 1e6 Table 1 Electron donor specificity of HvGpx2. Kinetic parameters for regeneration of monomeric and dimeric forms of HvGpx2 by HvTrxh1, HvTrxh2, and E. coli Trx1 using 1 mM tBOOH as electron acceptor.
HvTrxh1
HvTrxh2
EcTrx1
KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1)
Monomer
Dimer
4.8 ± 0.7 4.0 ± 0.2 0.83 9.2 ± 1.3 6.6 ± 0.7 0.72 60 ± 28 5.2 ± 1.5 0.09
6.6 ± 1.1 1 ± 0.05 0.15 12.3 ± 2.8 1.6 ± 0.2 0.13 n. d. n. d. n. d.
et al., 2007). SEC analysis of HvGpx2 treated with DTT revealed a minor peak with essentially the same retention time as nonreduced monomer followed by a major peak with slightly longer retention time, most likely corresponding to a reduced monomer (Fig. 2C). In addition, DTT induced a shift in the electrophoretic mobility on SDS-PAGE (Fig. S6 in Supplementary material). These results are consistent with a redox-dependent conformational change in HvGpx2. 3.3. Electron donor and acceptor specificity
Fig. 2. Size exclusion chromatography elution profiles of HvGpx2 on a Superdex 75 16/ 60 column in 30 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA. A. Separation of HvGpx2 following His-Trap purification. B. Monomeric (dashed line) or dimeric (solid line) HvGpx2 isolated from (A) and re-chromatographed under the same conditions after 48 h on ice. C. Separation of HvGpx2 following His-Trap purification and 2 h incubation at RT in 10 mM DTT, 50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA (10 mM DTT also included in elution buffer).
Recycling of oxidized HvGpx2 by the major barley seed Trx (HvTrxh1 and HvTrxh2) was assayed using tBOOH as electron acceptor and steady-state saturation kinetics were obtained (Table 1). Even though the overall catalytic efficiency (measured as kcat/KM) with both electron donors were similar, HvGpx2 had twofold higher KM for HvTrxh2 than HvTrxh1 (Table 1). E. coli Trx1 also recycled HvGpx2, albeit with nearly 10-fold lower efficiency than barley Trx. In terms of substrate specificity HvGpx2 reduced tBOOH and H2O2 with similar efficiency using HvTrxh1 as electron donor (Table 2). In contrast, the related poplar PtGpx5 reduced tBOOH less efficiently than COOH and H2O2 (Selles et al., 2012). Site-directed mutagenesis correlated this low efficiency of PtGpx5 with Glu79, which is one of the four amino acids forming the so-called catalytic tetrad that surrounds the catalytic cysteine at the active site (Fig. S3 in Supplementary material). The PtGpx5 Glu79Gln mutant generated to introduce the more frequently occurring glutamine at this position displayed increased activity toward tBOOH reaching the same level as activity for H2O2 (kcat/KM of 14.7 and 8.66 M1 s1, respectively), suggesting that Glu79 critically affects binding of the bulky alkyl peroxide substrate in the active site of PtGpx5 (Selles et al., 2012). 3.4. Monomeric HvGpx2 is inactivated by H2O2 Remarkably, the HvGpx2 monomer was significantly more
sequences investigated. Isolated monomer and dimer HvGpx2 forms were stable in solution for at least 48 h (on ice) as indicated by SEC rechromatography (Fig. 2B) and contained 0.96 ± 0.3 and 0.54 ± 0.2 thiol group/monomer unit, respectively. The lower thiol content of the dimer indicates that thiol groups are either partially oxidized or inaccessible to DTNB. SDS-PAGE analysis of HvGpx2 in the absence and presence of DTT suggests that the dimer is stabilized by an intermolecular disulfide bond (Fig. S6A in Supplementary material). In contrast to HvGpx2, PtGpx5 does not appear to be stabilized by an intermolecular disulfide bond since the three conserved cysteine residues are positioned distal from the dimer interface in both the reduced and oxidized forms of the crystal structure (Koh
Table 2 Electron acceptor specificity of monomeric HvGpx2. Kinetic parameters for reduction of different peroxide substrates by HvGpx2 using 5 mM HvTrxh1 as electron donor. H2O2
tBOOH
COOH
KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1)
0.8 ± 0.22 8.93 ± 0.06 11.2 0.76 ± 0.09 8.66 ± 0.09 10.8 1.59 ± 0.23 8.64 ± 0.09 5.4
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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competent in reducing tBOOH showing five-fold higher catalytic efficiency (kcat/Km) than the dimer (Table 1). Treatment of monomeric HvGpx2 with 0.1, 1 and 10 mM H2O2 for 2 h at room temperature resulted in approximately 20, 40 and 75% loss of activity, respectively, whereas the activity of the dimeric HvGpx2 was not significantly affected (Fig. 3). Kinetic analysis demonstrated that the activity of monomeric HvGpx2 dropped to a minimum activity level of around 25% after 30 min in 10 mM H2O2 (Fig. S7 in Supplementary material) or 16 h in 1 mM H2O2 (data not shown). SEC revealed dimer formation after H2O2 treatment of the monomer for 2 h (Fig. 4). At lower concentration of H2O2 (0.1e1 mM) up to about 85% occurred in the dimeric state, while less dimer was formed above 1 mM H2O2. No formation of monomeric HvGpx2 was observed when these H2O2 treatments were applied to the HvGpx2 dimer (data not shown). As demonstrated by SEC, the dimer obtained after incubation in 1 mM H2O2 partially dissociates following incubation in 10 mM DTT (Fig. S8 in Supplementary material). Similar to what was observed for non-H2O2-treated HvGpx2 (Fig. 2C), reaction with DTT results in a shift in the retention time for the monomer, supporting a redox-dependent conformational change. Furthermore, SDS-PAGE analysis suggests that the dimer formed upon H2O2 treatment was stabilized by an intermolecular disulfide bond (Fig. S6B in Supplementary material). Mass spectrometric analysis of H2O2-treated HvGpx2 revealed additional peaks of increased molecular mass (in the range of 30e70 Da) compared to untreated protein (Fig. S9 in Supplementary material). These results indicate that formation of oxidative modifications is not restricted to disulfide bonds. We hypothesized that such modifications may include over-oxidized cysteines as has been observed for other thiol-dependent peroxidases (Rabilloud et al., 2002; Selles et al., 2012). To pinpoint possible sites of modifications HvGpx2 was thus digested by endoproteinase Asp-N (EC 3.4.24.33) from Pseudomonas fragi which cleaves peptides bonds on the N-terminal side of aspartate and glutamate, and also cysteine residues oxidized to sulfinic (SO2H) or sulfonic (SO3H) acids (Drapeau, 1980). Mass spectrometric analysis of HvGpx2 treated with 1 or 10 mM H2O2 indeed identified Asp-N digested peptides
5
Fig. 4. Dimerization of HvGpx2 monomer treated with H2O2. HvGpx2 monomer (175 mM) was incubated 2 h in H2O2 (020 mM) at RT and immediately chromatographed on a Superdex 75 16/60 column, in 50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA. Monomer/dimer ratios were calculated from maximum A280 values of the respective eluting forms in the chromatograms.
with Cys72 and Cys91 oxidized to sulfonic acids in the respective Ntermini (Table S4 in Supplementary material). Cys91 corresponds to the so-called resolving cysteine involved in the catalytic mechanism of CysGpx (Fig. 1B), whereas the functional role of Cys72 has not been established (Navrot et al., 2006). Furthermore oxidization of Trp132 and Tyr149 was observed both in samples treated with H2O2 and the untreated control (Table S4 in Supplementary material). By integrating enzyme kinetics and the SEC data a bimodal mechanism can be suggested for HvGpx2, where monomeric HvGpx2 acts as an efficient peroxide reductant, but at H2O2 levels
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Plant Physiology and Biochemistry xxx (2015) 1e6
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Q3 Q2
A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase Nicolas Navrot a, Nicklas Skjoldager a, Jakob Bunkenborg b, c, Birte Svensson a, €gglund a, * Per Ha a
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Building 224, DK-2800 Kgs. Lyngby, Denmark Center of Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark c Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, DK-2650 Hvidovre, Denmark b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 February 2015 Accepted 7 March 2015 Available online xxx
Monomeric and dimeric forms of recombinant barley (Hordeum vulgare subsp. vulgare) glutathione peroxidase 2 (HvGpx2) are demonstrated to display distinctly different functional properties in vitro. Monomeric HvGpx2 thus has five fold higher catalytic efficiency than the dimer towards tert-butyl hydroperoxide, but is more sensitive to inactivation by hydrogen peroxide. Treatment of the monomer with hydrogen peroxide results in dimer formation. This observed new behavior of a plant glutathione peroxidase suggests a mechanism involving a switch from a highly catalytically competent monomer to a less active, but more oxidation-resistant dimer. © 2015 Published by Elsevier Masson SAS.
Keywords: Glutathione peroxidase Thioredoxin Antioxidant Hydrogen peroxide Alkyl peroxide Oligomerization
1. Introduction Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), the superoxide (O2) and hydroxyl (OH ) radicals cause oxidative stress and macromolecular damage. ROS can also react with nitric oxide (NO ) leading to formation of toxic reactive nitrogen species (RNS) such as peroxynitrite (ONOO). Higher plants are sessile organisms and thus particularly vulnerable to the considerable amounts of ROS produced as side-products in photosynthesis and respiration. In addition, H2O2 and NO exert important signaling functions in regulation of fundamental processes, e.g. apoptosis, seed germination and responses to biotic and abiotic stresses (Bailly, 2004; Bolwell, 1999). Plants utilize multiple non-enzymatic and enzymatic antioxidant systems to cope with
Abbreviations: COOH, cumene hydroperoxide; cv., cultivar; CysGpx, thioldependent glutathione peroxidase; DTNB, 5,50 -dithiobis-(2-nitrobenzoic acid); Gpx, glutathione peroxidase; GSH, glutathione; Prx, peroxiredoxin; ROS, reactive oxygen species; SEC, size exclusion chromatography; SecGpx, selenium-dependent glutathione peroxidase; tBOOH, tert-butyl hydroperoxide; Trx, thioredoxin. * Corresponding author. E-mail address: [email protected] (P. H€ agglund).
environmental stresses and control ROS and RNS levels (Foyer and Noctor, 2005; Møller, 2001; Noctor and Foyer, 1998). Among these, the thiol-dependent non-heme peroxiredoxins (Prx) and glutathione peroxidases (Gpx) catalyze reduction of peroxynitrite and different peroxides to nitrites and alcohols, respectively (Flohe et al., 2003). Gpx is also involved in signal transduction pathways through interactions with specific regulators, e.g. the protein phosphatase ABI2 in Arabidopsis thaliana (Miao et al., 2006) or transcription factors such as YAP1 in yeast (Delaunay et al., 2002). Signaling with plant Gpx is associated with apoptosis, cell cycle regulation and plant cell differentiation (Chen et al., 2004; Faltin et al., 2010; Kadota et al., 2005). All Prx share an active site peroxidatic cysteine that gets oxidized to a sulfenic acid upon substrate turnover, but they differ in the mechanism of the reductive part of the catalytic cycle (Wood et al., 2003). Plant Prx are classified based on sequence similarity into four groups which are reduced either directly by redoxins or low-molecular weight antioxidants (1-cys Prx and Prx II) or indirectly via formation of either an inter- (2-cys Prx) - or an intramolecular (Prx Q) disulfide bond involving a so-called resolving cysteine in Prx (Rouhier and Jacquot, 2005).
http://dx.doi.org/10.1016/j.plaphy.2015.03.003 0981-9428/© 2015 Published by Elsevier Masson SAS.
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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Gpx are divided into selenium-dependent (SecGpx) and selenium-independent (CysGpx) enzymes according to their catalytic mechanism (Flohe et al., 2011). SecGpx utilize a catalytic selenocysteine that undergoes oxidation to selenenic acid by reaction with peroxide (Fig. 1A). Reduced selenocysteine is regenerated at the expense of two molecules of reduced glutathione (GSH). The high reactivity of the active site selenocysteine makes SecGpx and extremely efficient as peroxide elimination enzymes (Flohe , 2001). The selenium-independent CysGpx by Brigelius-Flohe contrast function via dithiol-disulfide chemistry similarly to Prx (Herbette et al., 2007) involving formation of an intramolecular disulfide in turn reduced by thioredoxin (Trx) regenerating the active enzyme (Fig. 1B). Animal cells and some green algae, e.g. Chlamydomonas reinhardtii express both SecGpx and CysGpx, whereas fungi and land plants appear to only contain CysGpx (Margis et al., 2008). CysGpx are found in different plant organs and suggested to be localized in the cytosol, mitochondria and chloroplasts (Navrot et al., 2006). Plant CysGpx reduce peroxides with a similar catalytic efficiency as other Trx-dependent Prx, which is several orders of magnitude below that of SecGpx (Iqbal et al., 2006; Navrot et al., 2006; Selles et al., 2012). The rate of oxidation of the catalytic cysteine to sulfenic acid is however almost as high as for SecGpx (Fourquet et al., 2008). Dimeric poplar PtGpx5, which currently represents the only available plant CysGpx structure, undergoes major conformational changes to switch between reduced and oxidized forms presumably limiting the catalytic rate (Koh et al., 2007). Noticeably, an a-helix unfolds to bring the so-
called resolving cysteine close to the oxidized catalytic cysteine resulting in formation of an intramolecular disulfide bond and Trxmediated regeneration of the reduced enzyme (Fig. 1B). In the present study biochemical and structural features of the major barley seed CysGpx, HvGpx2, were investigated in relation to the redox state of the enzyme milieu. Remarkably HvGpx2 switches between a monomeric and a dimeric form that differ in catalytic efficiency and susceptibility to substrate-mediated inactivation in vitro. While this type of structural rearrangement has been described previously for Prx (Barranco-Medina et al., 2009), it is novel among CysGpx. 2. Materials and METHODS 2.1. Chemicals and proteins Recombinant barley (Hordeum vulgare subsp. vulgare) NADPHdependent thioredoxin reductase (HvNTR2), thioredoxins (HvTrxh1, HvTrxh2, and Escherichia coli Trx1) were obtained as €rnberg et al., 2012; Maeda et al., 2003; Shahpiri et al., described (Bjo 2008). All chemicals were from SigmaeAldrich (St. Louis, MO), unless stated otherwise. SDS-PAGE was performed using NuPAGE Novex 12e15% Bis-Tris minigels and electrophoresis system (Thermo Fisher Scientific, Waltham, MA). 2.2. Cloning of HvGpx2 Barley (cultivar (cv.) Barke) seeds were germinated for 3 days on wet filter paper at RT in the dark, the embryos were excised with a scalpel and RNA was extracted with Plant Mini Kit for RNA extraction (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. Specific primers HvGpx2for (50 -ATGGCCTCC TCCGCCTCCTCCGTC-30 ) and HvGpx2rev, (50 -GGATCCTTAAGAACTC GCGAGCAA-30 ) including NdeI and BamHI restriction sites, respectively, were used to amplify HvGpx2 cDNA without the predicted N-terminal signal peptide using 1-step RT-PCR kit (Qiagen) according to the manufacturer's instructions. In order to optimize the temperature for PCR the sequence encoding for three N-terminal alanines was omitted. The amplified fragment was cloned into pET15b for expression of HvGpx2 with an N-terminal 6xHis tag (the sequence of the recombinant protein is displayed in Fig. S1 in Supplementary material). Plasmid DNA sequences were confirmed by bidirectional sequencing using T7 and T7 terminator primers. 2.3. Expression and purification of HvGpx2
Fig. 1. Catalytic cycle of (A) selenium-dependent Gpx (SecGpx) and (B) seleniumindependent Gpx (CysGpx). A. Selenenic acid (Gpx-SeOH) is formed upon reaction of the catalytic selenocysteine (Gpx-SeH) in SecGpx with a hydroperoxide (ReOOH). Reduced glutathione (GSH) reacts with SeOH forming a glutathione adduct (Gpx-SeSG) that subsequently reacts with GSH, yielding Gpx-SeH and oxidized glutathione (GSSG). B. The catalytic (peroxidatic) cysteine thiol group in CysGpx (Gpx-SH) is oxidized to the sulfenic acid (Gpx-SOH) by reaction with ReOOH. A second (resolving) cysteine reacts with Gpx-SOH forming an intramolecular disulfide bond (Gpx-S-S) that reacts with reduced Trx, yielding Gpx-SH and oxidized Trx.
LB medium (1 L in a 3 L shake flask) was inoculated to OD600nm ¼ 0.1 with an overnight culture of E. coli Rosetta(DE3)™ (Merck Millipore, Darmstadt, Germany) containing pET15bHvGpx2. Cells were propagated at 37 C and induced by 0.1 mM IPTG added when the culture reached OD600nm ¼ 0.6. Cells were pelleted (4000 g, 15 min) after 16 h induction at 30 C and resuspended in 10 mL Bugbuster reagent added 1 mL benzonase (Merck Millipore). After centrifugation HvGpx2 was purified from the supernatant by Ni-ion affinity chromatography (5 mL HisTrap HP, GE Healthcare, Little Chalfont, UK) and eluted by a linear 30e200 mM imidazole gradient of loading buffer (50 mM TriseHCl pH 8, 400 mM NaCl, 10 mM imidazole) and elution buffer (loading buffer with 400 mM imidazole). Fractions containing HvGpx2 (determined by SDS-PAGE) were pooled, dialyzed (50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA), concentrated (10 kDa cutoff; Amicon Ultra 15 mL centrifugal filter unit) and subjected to size exclusion chromatography (SEC) on a Superdex 75 26/60 column (GE Healthcare) in the dialysis buffer. Fractions containing monomeric
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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and dimeric HvGpx2 were pooled separately, dialyzed against 50 mM TriseHCl pH 8, 1 mM EDTA and concentrated (as above). 2.4. Thiol titration and protein concentration determination Protein samples (2030 mM) in 100 mM TriseHCl pH 8, 8 M urea (270 mL) were mixed with 30 mL 1 mM 5,50 -dithiobis-(2nitrobenzoic acid (DTNB) and absorbance at 412 nm was measured (BioTek microplate reader, Winooski, VT) using N-acetyl cysteine (0e50 mM) as standard. The stoichiometry per protein molecule was calculated from spectrophotometric measurements using 3280 ¼ 18235 M1 cm1 and 21743 M1 cm1 determined by aid of amino acid analysis (Barkholt and Jensen, 1989), for HvGpx2 monomer and HvGpx2 dimer, respectively. 2.5. HvGpx2 activity assays Unless otherwise stated, HvTrxh1 or HvTrxh2 (final 6 mM) was mixed with H2O2, tert-butyl hydroperoxide (tBOOH) or cumene hydroperoxide (COOH) (final 1 mM) in 50 mM Tris HCl pH 8, 0.1% BSA, 0.25 mM NADPH, 0.2 mM HvNTR2 (total volume 100 mL). To determine kinetic parameters using peroxide (0.05e5 mM) and Trx (0.5e25 mM), NADPH oxidation was monitored as decrease in absorbance at 340 nm (3340 ¼ 6220 M1 cm1) for 60 s following the addition of 0.2 mM HvGpx2 and the slope was calculated by linear fitting. Background reaction without Gpx was subtracted for each measurement. The activity was expressed as the rate (mmol/s) and plotted versus the concentration of either Trx or peroxide. KM and kcat values were obtained from non-linear curve fits (Kaleidagraph, Synergy software, Reading, PA). 2.6. HvGpx2 inactivation and analytical SEC HvGpx2 monomer or dimer (20 mM) was mixed with H2O2 (0e10 mM) in 50 mM Tris HCl pH 8 (total volume 20 mL), incubated 2 h at RT, added 50 mM Tris HCl pH 8, 0.1% BSA (180 mL) and kept on ice. Activity was assayed in 100 mL 0.25 mM NADPH, 5 mM HvTrxh1, 1 mM tBOOH, 50 mM Tris HCl pH 8, 0.1% BSA containing 10 mL of H2O2-treated HvGpx2 (final 0.2 mM) and a saturating concentration (0.6 mM) of HvNTR2. Aliquots (10 mL) removed at appropriate time intervals from 100 mL reaction mixtures of HvGpx2 monomer (42 mM) and H2O2 (0, 0.5, 1, 5, 10 mM) in 50 mM Tris pH 8, 1 mM EDTA, were immediately diluted 20 fold in 50 mM Tris HCl pH 8, 1 mM EDTA, 0.1% BSA and assayed (final HvGpx2 0.1 mM) essentially as above to determine the inactivation kinetics. For analytical SEC, HvGpx2 monomer or dimer (175 mM) was treated with H2O2 (0.1, 0.5, 2, 6 and 20 mM) in 50 mM Tris HCl pH 8, 1 mM EDTA (500 mL) for 2 h at RT followed by separation (Superdex 75 16/60 column; GE Healthcare) in 50 mM Tris HCl pH 8, 200 mM NaCl, 1 mM EDTA. Monomer/dimer ratios were calculated from the maximum A280 values of the eluted monomer and dimer in the chromatograms. 2.7. LC-MS/MS mapping of oxidation sites HvGpx2 in 50 mM Tris HCl pH 8 was treated with different concentrations of H2O2 (0, 1, and 10 mM) in a total volume of 50 mL. From each reaction 19 mL was removed and mixed with 12 mL 8 M urea. Then 5 mL 100 mM iodoacetamide was added and incubated for 30 min at RT in the dark. Following addition of 22.5 mL 50 mM Tris HCl pH 7.4 and 0.7 mL sequencing-grade endoproteinase Asp-N (Promega, Madison, WI), the samples were incubated overnight at RT. Reversed-phase LC-MS analysis was performed on an LTQOrbitrap Velos (Thermo Fisher Scientific) connected to an Agilent 1100 nanoflow HPLC system (Agilent, Santa Clara, CA). Database searches were performed using MaxQuant Version 1.5.2.8 (Cox and
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Mann, 2008) against the Uniprot Hordeum vulgare database with standard orbitrap settings, allowing for proteolytic cleavage on the N-terminal side of Cys, Asp, Glu residues and the following variable modifications: mono- and dioxidation (Met, Cys, Trp, Tyr), trioxidation (Cys, Trp), and kynurenine and hydroxykynurenine formation (Trp). 3. Results and discussion 3.1. A barley glutathione peroxidase family Plants possess a family of Prx and CysGpx orthologs that have become a focal point for investigations on redox regulation and signaling in plant cells (Rouhier and Jacquot, 2005). Three barley gpx genes (encoding HvGpx1e3) were discovered in the late 90's (Churin et al., 1999). Since then, exhaustive inventories of Gpx gene families have been published for A. thaliana and Populus trichocarpa (Navrot et al., 2006; Rodriguez Milla et al., 2003). Analysis of transcripts and homologues (NCBI EST databases) of HvGpx1e3 identified two additional isoforms, here named HvGpx4 and HvGpx5. Sequence alignment (Figs. S2 and S3 in Supplementary material) reveals high identity between Gpx from barley, A. thaliana and P. trichocarpa (Tables S1 and S2 in Supplementary material). HvGpx1 and HvGpx2 are closely related to two poplar isoforms, PtGpx3.1 and PtGpx3.2, which are broadly expressed in different tissues and cellular compartments, and to A. thaliana AtGpx6 highly expressed in seeds. Analysis of gpx transcriptomic data using Genevestigator (Hruz et al., 2008) suggests that the gene coding for HvGpx2 (annotated as BarPHGPX in the database) is preferentially expressed in seeds (Fig. S4 in Supplementary material). Noticeably HvGpx2 contains an N-terminal extension potentially targeting to chloroplasts (Table S3 in Supplementary material). Furthermore, an alternative initiation site encodes a presumed cytosolic form (Fig. S3 in Supplementary material). Here, the gene corresponding to HvGpx2 was amplified without the predicted Nterminal signal peptide by RT-PCR using total barley embryo RNA extracted 3 d after start of imbibition. Bidirectional sequencing of the cloned gene revealed that Glu17 in the deposited HvGpx2 sequence from cv. Haisa (Q9SME6) is substituted by a lysine in HvGpx2 from cv. Barke (this work). BarPHGPX (AB096704) from cv. OUK305 (Sugimoto and Takeda, 2003) has identical sequence to HvGpx2 from cv. Barke. 3.2. Monomeric and dimeric HvGpx2 HvGpx2 produced recombinantly in E. coli Rosetta(DE3)™ and purified to electrophoretic homogeneity consisted of two major components (Fig. 2A) with apparent molecular mass of 26.6 and 54.9 kDa (Fig. S5 in Supplementary material), suggesting the presence of a monomeric and a dimeric form (theoretical mass 20350 and 40700 Da, respectively). This stands in contrast to the mainly dimeric poplar PtGpx5 (Koh et al., 2007; Navrot et al., 2006; Selles et al., 2012). The crystal structure of PtGpx5 reveals the dimer interface to be stabilized by several aromatic residues, of which Tyr151 appears to be of particular importance. A Tyr151Arg substitution thus shifted PtGpx5 to a mainly monomeric protein as verified by SEC (Selles et al., 2012). This finding suggests that oligomerization of plant CysGpx is influenced by subtle amino acid differences at the dimer interface. In HvGpx2 and most other CysGpx from poplar, barley and A. thaliana the Tyr151 and also Trp124 both located at the dimer interface of PtGpx5 are substituted by small aliphatic residues (Fig. S3 in Supplementary material). However, the tyrosine residue preceding Tyr151 (Tyr149 in HvGpx2) appears to be conserved in most of the plant Gpx
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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N. Navrot et al. / Plant Physiology and Biochemistry xxx (2015) 1e6 Table 1 Electron donor specificity of HvGpx2. Kinetic parameters for regeneration of monomeric and dimeric forms of HvGpx2 by HvTrxh1, HvTrxh2, and E. coli Trx1 using 1 mM tBOOH as electron acceptor.
HvTrxh1
HvTrxh2
EcTrx1
KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1)
Monomer
Dimer
4.8 ± 0.7 4.0 ± 0.2 0.83 9.2 ± 1.3 6.6 ± 0.7 0.72 60 ± 28 5.2 ± 1.5 0.09
6.6 ± 1.1 1 ± 0.05 0.15 12.3 ± 2.8 1.6 ± 0.2 0.13 n. d. n. d. n. d.
et al., 2007). SEC analysis of HvGpx2 treated with DTT revealed a minor peak with essentially the same retention time as nonreduced monomer followed by a major peak with slightly longer retention time, most likely corresponding to a reduced monomer (Fig. 2C). In addition, DTT induced a shift in the electrophoretic mobility on SDS-PAGE (Fig. S6 in Supplementary material). These results are consistent with a redox-dependent conformational change in HvGpx2. 3.3. Electron donor and acceptor specificity
Fig. 2. Size exclusion chromatography elution profiles of HvGpx2 on a Superdex 75 16/ 60 column in 30 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA. A. Separation of HvGpx2 following His-Trap purification. B. Monomeric (dashed line) or dimeric (solid line) HvGpx2 isolated from (A) and re-chromatographed under the same conditions after 48 h on ice. C. Separation of HvGpx2 following His-Trap purification and 2 h incubation at RT in 10 mM DTT, 50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA (10 mM DTT also included in elution buffer).
Recycling of oxidized HvGpx2 by the major barley seed Trx (HvTrxh1 and HvTrxh2) was assayed using tBOOH as electron acceptor and steady-state saturation kinetics were obtained (Table 1). Even though the overall catalytic efficiency (measured as kcat/KM) with both electron donors were similar, HvGpx2 had twofold higher KM for HvTrxh2 than HvTrxh1 (Table 1). E. coli Trx1 also recycled HvGpx2, albeit with nearly 10-fold lower efficiency than barley Trx. In terms of substrate specificity HvGpx2 reduced tBOOH and H2O2 with similar efficiency using HvTrxh1 as electron donor (Table 2). In contrast, the related poplar PtGpx5 reduced tBOOH less efficiently than COOH and H2O2 (Selles et al., 2012). Site-directed mutagenesis correlated this low efficiency of PtGpx5 with Glu79, which is one of the four amino acids forming the so-called catalytic tetrad that surrounds the catalytic cysteine at the active site (Fig. S3 in Supplementary material). The PtGpx5 Glu79Gln mutant generated to introduce the more frequently occurring glutamine at this position displayed increased activity toward tBOOH reaching the same level as activity for H2O2 (kcat/KM of 14.7 and 8.66 M1 s1, respectively), suggesting that Glu79 critically affects binding of the bulky alkyl peroxide substrate in the active site of PtGpx5 (Selles et al., 2012). 3.4. Monomeric HvGpx2 is inactivated by H2O2 Remarkably, the HvGpx2 monomer was significantly more
sequences investigated. Isolated monomer and dimer HvGpx2 forms were stable in solution for at least 48 h (on ice) as indicated by SEC rechromatography (Fig. 2B) and contained 0.96 ± 0.3 and 0.54 ± 0.2 thiol group/monomer unit, respectively. The lower thiol content of the dimer indicates that thiol groups are either partially oxidized or inaccessible to DTNB. SDS-PAGE analysis of HvGpx2 in the absence and presence of DTT suggests that the dimer is stabilized by an intermolecular disulfide bond (Fig. S6A in Supplementary material). In contrast to HvGpx2, PtGpx5 does not appear to be stabilized by an intermolecular disulfide bond since the three conserved cysteine residues are positioned distal from the dimer interface in both the reduced and oxidized forms of the crystal structure (Koh
Table 2 Electron acceptor specificity of monomeric HvGpx2. Kinetic parameters for reduction of different peroxide substrates by HvGpx2 using 5 mM HvTrxh1 as electron donor. H2O2
tBOOH
COOH
KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1) KM (mM) kcat (s1) kcat/KM (mM1 s1)
0.8 ± 0.22 8.93 ± 0.06 11.2 0.76 ± 0.09 8.66 ± 0.09 10.8 1.59 ± 0.23 8.64 ± 0.09 5.4
Please cite this article in press as: Navrot, N., et al., A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase, Plant Physiology and Biochemistry (2015), http://dx.doi.org/10.1016/j.plaphy.2015.03.003
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competent in reducing tBOOH showing five-fold higher catalytic efficiency (kcat/Km) than the dimer (Table 1). Treatment of monomeric HvGpx2 with 0.1, 1 and 10 mM H2O2 for 2 h at room temperature resulted in approximately 20, 40 and 75% loss of activity, respectively, whereas the activity of the dimeric HvGpx2 was not significantly affected (Fig. 3). Kinetic analysis demonstrated that the activity of monomeric HvGpx2 dropped to a minimum activity level of around 25% after 30 min in 10 mM H2O2 (Fig. S7 in Supplementary material) or 16 h in 1 mM H2O2 (data not shown). SEC revealed dimer formation after H2O2 treatment of the monomer for 2 h (Fig. 4). At lower concentration of H2O2 (0.1e1 mM) up to about 85% occurred in the dimeric state, while less dimer was formed above 1 mM H2O2. No formation of monomeric HvGpx2 was observed when these H2O2 treatments were applied to the HvGpx2 dimer (data not shown). As demonstrated by SEC, the dimer obtained after incubation in 1 mM H2O2 partially dissociates following incubation in 10 mM DTT (Fig. S8 in Supplementary material). Similar to what was observed for non-H2O2-treated HvGpx2 (Fig. 2C), reaction with DTT results in a shift in the retention time for the monomer, supporting a redox-dependent conformational change. Furthermore, SDS-PAGE analysis suggests that the dimer formed upon H2O2 treatment was stabilized by an intermolecular disulfide bond (Fig. S6B in Supplementary material). Mass spectrometric analysis of H2O2-treated HvGpx2 revealed additional peaks of increased molecular mass (in the range of 30e70 Da) compared to untreated protein (Fig. S9 in Supplementary material). These results indicate that formation of oxidative modifications is not restricted to disulfide bonds. We hypothesized that such modifications may include over-oxidized cysteines as has been observed for other thiol-dependent peroxidases (Rabilloud et al., 2002; Selles et al., 2012). To pinpoint possible sites of modifications HvGpx2 was thus digested by endoproteinase Asp-N (EC 3.4.24.33) from Pseudomonas fragi which cleaves peptides bonds on the N-terminal side of aspartate and glutamate, and also cysteine residues oxidized to sulfinic (SO2H) or sulfonic (SO3H) acids (Drapeau, 1980). Mass spectrometric analysis of HvGpx2 treated with 1 or 10 mM H2O2 indeed identified Asp-N digested peptides
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Fig. 4. Dimerization of HvGpx2 monomer treated with H2O2. HvGpx2 monomer (175 mM) was incubated 2 h in H2O2 (020 mM) at RT and immediately chromatographed on a Superdex 75 16/60 column, in 50 mM TriseHCl pH 8, 200 mM NaCl, 1 mM EDTA. Monomer/dimer ratios were calculated from maximum A280 values of the respective eluting forms in the chromatograms.
with Cys72 and Cys91 oxidized to sulfonic acids in the respective Ntermini (Table S4 in Supplementary material). Cys91 corresponds to the so-called resolving cysteine involved in the catalytic mechanism of CysGpx (Fig. 1B), whereas the functional role of Cys72 has not been established (Navrot et al., 2006). Furthermore oxidization of Trp132 and Tyr149 was observed both in samples treated with H2O2 and the untreated control (Table S4 in Supplementary material). By integrating enzyme kinetics and the SEC data a bimodal mechanism can be suggested for HvGpx2, where monomeric HvGpx2 acts as an efficient peroxide reductant, but at H2O2 levels
