Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Functional Characterization of Peroxiredoxins from the Human Protozoan Parasite Giardia intestinalis

The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O₂ and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O₂, NO and superoxide anion (O₂ ^−•) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H₂O₂ and peroxynitrite (ONOO⁻). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H₂O₂ and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO⁻ rapidly (k = 4×10⁵ M⁻¹ s⁻¹ and 2×10⁵ M⁻¹ s⁻¹ at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O₂ for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

UV-B induced stress responses in three rice cultivars

UV-B responses of three rice (Oryza sativa L.) cultivars (Sasanishiki, Norin 1 and Surjamkhi) with different photolyase activity were investigated. Carbon dioxide assimilation data support that Sasanishiki was less sensitive to UV-B than Norin 1 and Surjamkhi. UV-B radiation sharply decreased the content of Rubisco protein in Surjamkhi and has no effect in Sasanishiki. The photochemical activities of photosystem (PS) 1 and PS 2 was slightly affected by UV-B treatment. The content of H₂O₂ and the activities of antioxidant enzymes, catalase (CAT), peroxides (POX) and superoxide dismutase (SOD) were enhanced after UV-B treatment. The activities of CAT and POX isoenzymes in Sasanishiki were more enhanced by UV-B radiation than those in Norin 1 and Surjamkhi.     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.     

Distinct Characteristics of Two 2-Cys Peroxiredoxins of Vibrio vulnificus Suggesting Differential Roles in Detoxifying Oxidative Stress

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C_P and C_R, but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C_P-SO₃H specific immunoblotting demonstrated overoxidation of C_P to C_P-SO₂H (or C_P-SO₃H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C_P was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H₂O₂ and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H₂O₂. A mutagenesis study indicated that lack of Prx2 accumulated sufficient H₂O₂ to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H₂O₂, whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H₂O₂, and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.     

Hydrogen peroxide metabolism and sensing in human erythrocytes: A validated kinetic model and reappraisal of the role of peroxiredoxin II

Hydrogen peroxide (H₂O₂) metabolism in human erythrocytes has been thoroughly investigated, but unclear points persist. By integrating the available data into a mathematical model that accurately represents the current understanding and comparing computational predictions to observations we sought to (a) identify inconsistencies in present knowledge, (b) propose resolutions, and (c) examine their functional implications. The systematic confrontation of computational predictions with experimental observations of the responses of intact erythrocytes highlighted the following important discrepancy. The high rate constant (10⁷–10⁸ M⁻¹ s⁻¹) for H₂O₂ reduction determined for purified peroxiredoxin II (Prx2) and the high abundance of this protein indicate that under physiological conditions it consumes practically all the H₂O₂. However, this is inconsistent with extensive evidence that Prx2’s contribution to H₂O₂ elimination is comparable to that of catalase. Models modified such that Prx2’s effective peroxidase activity is just 10⁵ M⁻¹ s⁻¹ agree near quantitatively with extensive experimental observations. This low effective activity is probably due to a strong but readily reversible inhibition of Prx2’s peroxidatic activity in intact cells, implying that the main role of Prx2 in human erythrocytes is not to eliminate peroxide substrates. Simulations of the responses to physiological H₂O₂ stimuli highlight that a design combining abundant Prx2 with a low effective peroxidase activity spares NADPH while improving potential signaling properties of the Prx2/thioredoxin/thioredoxin reductase system.     

Antioxidant enzyme activities and isozyme pattern in hairy roots and regenerated tobacco plants

Hairy root disease is caused by infection of wounded higher plants with Agrobacterium rhizogenes. Transformation of tissues or plants with A. rhizogenes, as well as transformation with rol genes, in addition to hairy roots, may produce alterations in the plant secondary metabolism. H₂O₂ and other ROS are involved as signals in secondary metabolite production pathways and play a key role in plant defense reactions. In this work the effects of A. rhizogenes rol genes on nicotine content, antioxidant enzymes activity, H₂O₂ production, the pattern of peroxidase (POX) and superoxide dismutase (SOD) isozymes in hairy roots and regenerated Nicotiana tabacum plants were studied. The rise in SOD and POX activities in the transformed lines TRa and TRb and the resulting regenerated plants and a decreased level of H₂O₂ in them as compared with the untransformed lines indicates that rol gene expression decreases H₂O₂ level probably by increasing production of antioxidant enzymes. A decreased H₂O₂ content in TRc line, in spite of similarity of antioxidant enzyme activity as compared to normal roots, indicates that rol genes activate other mechanisms except SOD and POX enzymes for reducing H₂O₂.     

Reactive oxygen species production and antioxidative defense in pea (Pisum sativum L.) root nodules after short-term aluminum treatment

Pea plants (Pisum sativum L.) were treated with 50???M aluminum chloride at pH 4.5 for 2 or 24?h at room temperature. Following treatment, root nodule Al uptake, the generation of reactive oxygen species (ROS, O ₂ ^?· and H₂O₂), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) were investigated. Aluminum accumulation was found chiefly in the apoplast of the nodule cortex, endodermis and meristem, while the formation of peroxide was detected in the nodule cortex, infection threads and bacteroidal tissue. Further, there were increased levels of superoxide in the meristem and bacteroidal tissue. The activity of SOD (EC 1.15.1.1) and POX (EC 1.11.1.7) increased in the Al-treated nodules and the roots of pea plants, whereas CAT (EC 1.11.1.6) activity decreased. The Al absorbed by the nodules induced ROS production. The POX and SOD are important ROS-scavengers in Al-stressed nodules.     

Antioxidant Enzyme Activities during in vitro Morphogenesis of Gladiolus and the Effect of Application of Antioxidants on Plant Regeneration

Activity of antioxidant enzymes was evaluated during somatic embryogenesis and shoot organogenesis from cultured leaf segments of Gladiolus hybridus Hort. The effect of exogenous antioxidants on somatic embryogenesis and shoot organogenesis has also been monitored. Activity of superoxide dismutase (SOD) gradually increased during somatic embryogenesis. while activities of catalase (CAT) and peroxidase (POX) decreased. In contrast, increase in CAT and POX activity and a concomitant decrease in SOD activity were noted during shoot organogenesis. Exogenous application of antioxidants such as glutathione (GSH), α-tocopherol and ascorbate (AA) inhibited somatic embryogenesis but stimulated shoot organogenesis. The frequency of somatic embryogenesis increased with the addition of H₂O₂. However, H₂O₂ inhibited shoot organogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salicylic acid increases tolerance to oxidative stress induced by hydrogen peroxide accumulation in leaves of cadmium-exposed flax (Linum usitatissimum L.)

The aim of this study is to investigate the impacts of exogenous salicylic acid (SA) pretreatments on hydrogen peroxide (H₂O₂) accumulation, protein oxidation, and H₂O₂-scavenging enzymes in leaves of Cd-treated flax seedlings. Cd-enhanced H₂O₂ levels were related to increased activities of guaiacol peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), and were independent of changes in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activities. In control flax seedlings, exogenous SA pretreatments inhibited the activity of CAT, resulted in an enhanced production of H₂O₂ suggesting that SA requires H₂O₂ to initiate an oxidative stress. However, although leaves of Cd-free flax seedlings pretreated with SA accumulated in vivo H₂O₂ by 1.2-fold compared with leaves of Cd-only exposed ones; the damage to growth and proteins after the exposure to Cd was significantly less, indicating that SA can regulate the Cd-induced oxidative stress. Moreover, the Cd-treated seedlings primed with SA exhibited a higher level of total antioxidant capacities and increased activities of H₂O₂-detoxifying enzymes.     

Mechanistic characterization of the thioredoxin system in the removal of hydrogen peroxide

The thioredoxin system, which consists of a family of proteins, including thioredoxin (Trx), peroxiredoxin (Prx), and thioredoxin reductase (TrxR), plays a critical role in the defense against oxidative stress by removing harmful hydrogen peroxide (H₂O₂). Specifically, Trx donates electrons to Prx to remove H₂O₂ and then TrxR maintains the reduced Trx concentration with NADPH as the cofactor. Despite a great deal of kinetic information gathered on the removal of H₂O₂ by the Trx system from various sources/species, a mechanistic understanding of the associated enzymes is still not available. We address this issue by developing a thermodynamically consistent mathematical model of the Trx system which entails mechanistic details and provides quantitative insights into the kinetics of the TrxR and Prx enzymes. Consistent with experimental studies, the model analyses of the available data show that both enzymes operate by a ping-pong mechanism. The proposed mechanism for TrxR, which incorporates substrate inhibition by NADPH and intermediate protonation states, well describes the available data and accurately predicts the bell-shaped behavior of the effect of pH on the TrxR activity. Most importantly, the model also predicts the inhibitory effects of the reaction products (NADP⁺ and Trx(SH)₂) on the TrxR activity for which suitable experimental data are not available. The model analyses of the available data on the kinetics of Prx from mammalian sources reveal that Prx operates at very low H₂O₂ concentrations compared to their human parasite counterparts. Furthermore, the model is able to predict the dynamic overoxidation of Prx at high H₂O₂ concentrations, consistent with the available data. The integrated Prx–TrxR model simulations well describe the NADPH and H₂O₂ degradation dynamics and also show that the coupling of TrxR- and Prx-dependent reduction of H₂O₂ allowed ultrasensitive changes in the Trx concentration in response to changes in the TrxR concentration at high Prx concentrations. Thus, the model of this sort is very useful for integration into computational H₂O₂ degradation models to identify its role in physiological and pathophysiological functions.     

Sphaerophysa kotschyana, an endemic species from Central Anatolia: antioxidant system responses under salt stress

Sphaerophysa kotschyana is a Turkish endemic and endangered plant that grows near Salt Lake, in Konya, Turkey. However, little is known about the ability of this plant to generate/remove reactive oxygen species (ROS) or its adaptive biochemical responses to saline environments. After exposure of S. kotschyana to 0, 150, and 300 mM NaCl for 7 and 14 days, we investigated (1) the activities and isozyme compositions of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), and glutathione reductase (GR); (2) the oxidative stress parameters NADPH oxidase (NOX) activity, lipid peroxidation (MDA), total ascorbate (tAsA) content, and total glutathione content (tGlut); and (3) ROS levels for superoxide anion radical (O ₂ ^·? ), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH·), and histochemical staining of O ₂ ^·? and H₂O₂. H₂O₂ content increased after 14 days of salt stress, which was consistent with the results from histochemical staining and NOX activity measurements. In contrast, oxidative stress induced by 150 mM NaCl was more efficiently prevented, as indicated by low malondialdehyde (MDA) levels and especially at 7 days, by increased levels of SOD, POX, APX, and GR. However, at 300 mM NaCl, decreased levels of protective enzymes such as SOD, CAT, POX, and GR, particularly with long-term stress (14 days), resulted in limited ROS scavenging activity and increased MDA levels. Moreover, at 300 mM NaCl, the high H₂O₂ content caused oxidative damage rather than inducing protective responses against H₂O₂. These results suggest that S. kotschyana is potentially tolerant to salt-induced damage only at low salt concentrations.     

Changes in antioxidant enzymes and some key metabolites in some genetically diverse cultivars of radish (Raphanus sativus L.)

Salt-induced changes in the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and lipid peroxidation in terms of malondialdehyde (MDA), level of H₂O₂, and some key metabolites such as soluble proteins, free proline and phenolics in the leaves of six radish cultivars (Radish Red Neck, Radish Lal Pari, Radish Mino Japani, Radish 40 Days, Mannu Early and Desi) were investigated. Varying levels of NaCl (0, 80 and 160 mM) applied for 40 days adversely affected the shoot fresh weight, chlorophyll contents and soluble proteins, while increased the levels of proline, and the activities of SOD, POD and CAT. However, leaf H₂O₂ and total phenolic contents were not affected by salt stress. Cultivars Mannu Early, Radish 40 Days and Desi were relatively higher in shoot fresh weight (percent of control) while cvs. Radish Mino Japani and Mannu Early in proline, and cvs. Radish 40 Days and Desi in total soluble proteins at 160 mM of NaCl. However, levels of H₂O₂ and phenolics were higher in cvs. Desi, Radish Lal Pari and Mannu Early and SOD, POD and CAT activities only in Radish Lal Pari and Mannu Early than the other cultivars under saline conditions. Overall, the differential salt tolerance of radish cultivars observed in the present study was not found to be associated with higher antioxidant enzyme activities and other key metabolites analyzed, so these attributes cannot be considered as selection criteria for salt tolerance in radish.     

Effect of Coronatine on Antioxidant Enzyme Response of Chickpea Roots to Combination of PEG-Induced Osmotic Stress and Heat Stress

Abiotic stresses, such as high temperature and drought, are major limiting factors of crop production and growth. Coronatine (COR), a structural and functional analog of jasmonates, is suggested to have a role in abiotic stress tolerance. The aim of our study was to examine whether pretreatment with COR enhances the tolerance of chickpea (Cicer arietinum L. cv ICC 4958) roots to PEG-induced osmotic stress, heat stress, and their combination. Therefore, seedlings raised hydroponically in a growth chamber for 15 days were pretreated with or without COR at 0.01 μM for 24 h and then exposed to 6 % PEG 6000-induced osmotic stress or heat (starting at 35 °C and then gradually increased 1 °C every 15 min and kept at 44 °C for 1 h) stress for 3 days. After different treatment periods, the changes in relative growth rate (RGR); malondialdehyde (MDA), proline (Pro), and hydrogen peroxide (H₂O₂) contents; and the activities of antioxidant enzymes/isoenzymes in roots of chickpea seedlings with or without 0.01 μM COR application were studied. RGR in roots was increased by COR application. Under all stress conditions, H₂O₂, MDA, and Pro levels increased sharply, but pretreatment with COR significantly reduced them. Moreover, COR increased the activities of H₂O₂ scavenger enzymes such as catalase (CAT) under heat stress, ascorbate peroxidase (POX) under PEG stress, and CAT and POX under combined stresses. Therefore, COR might alleviate adverse effects of PEG stress and heat stress and combined stresses on roots of chickpea by reduction of H₂O₂ production, enhancing or keeping the existent activity of antioxidant enzymes, thereby preventing membrane peroxidation.     

Mitochondrial 1-Cys-peroxiredoxin/thioredoxin system protects manganese-containing superoxide dismutase (Mn-SOD) against inactivation by peroxynitrite in Saccharomyces cerevisiae

Peroxynitrite is a reactive nitrogen species that can mediate protein tyrosine nitration, inactivating many proteins. We show that yeast mitochondrial peroxiredoxin (Prx1p), which belongs to the group 1-Cys-Prx, has thioredoxin-dependent peroxynitrite reductase activity. This activity was characterised in vitro with the recombinant mitochondrial Prx1p, the thioredoxin reductase Trr2p and the thioredoxin Trx3p, using a generator of peroxynitrite (SIN-1). Purified mitochondria from wild-type and null Prx1p or Trx3p yeast strains, exposed to SIN-1, showed a differential inactivation of manganese-containing superoxide dismutase activity. The above yeast strains were exposed to SIN-1 and examined under confocal microscopy. Prx1p or Trx3p-null cells showed a greater accumulation of peroxynitrite than wild-type ones. Our results indicate that this 1-Cys-Prx is a peroxynitrite reductase activity that uses reducing equivalents from NADPH through the mitochondrial thioredoxin system. Therefore, mitochondrial 1-Cys-peroxiredoxin/thioredoxin system constitutes an essential antioxidant defence against oxidative and nitrosative stress in yeast mitochondria.     

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H₂O₂-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H₂O₂. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.