Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases

Oxidative damage to proteins is considered to be one of the major causes of aging and age-related diseases, and thus mechanisms have evolved to prevent or reverse these modifications. Methionine is one of the major targets of reactive oxygen species (ROS), where it is oxidized to methionine sulfoxide (MetO). Recently, evidence has accumulated suggesting that methionine (Met) oxidation may play an important role in the development and progression of neurodegenerative diseases like Alzheimer's and Parkinson's diseases. Oxidative alteration of Met to Met(O) is reversed by the methionine sulfoxide reductases (consisting of MsrA enzymes that reduce S-MetO and MsrB enzymes that reduce R-MetO, respectively). A major biological role of the Msr system is suggested by the fact that the MsrA null mouse (MT) exhibits a neurological disorder in the form of ataxia ("tip toe walking"), is more sensitive to oxidative stress, and has a shorter life span (by approximately 40%) than wild-type (WT) mice. By their action, the Msr enzymes can regulate protein function, be involved in signal-transduction pathways, and prevent cellular accumulation of faulty proteins. Malfunction of the Msr system can lead to cellular changes resulting in compromised antioxidant defense, enhanced age-associated diseases involving neurodegeneration, and shorter life span. In this review, the function and possible roles of the Msr system in prokaryotes and eukaryotes, in general, and in neurodegenerative diseases, in particular, will be discussed.     

Functional characterization of the Aspergillus nidulans methionine sulfoxide reductases (msrA and msrB)

Proteins are subject to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological function, leading to an alteration in cellular homeostasis. Sulfur-containing amino acids as methionine are the most vulnerable to oxidation by ROS, resulting in the formation of methionine sulfoxide [Met(O)] residues. This modification can be repaired by methionine sulfoxide reductases (Msr). Two distinct classes of these enzymes, MsrA and MsrB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Here, we describe the homologs of methionine sulfoxide reductases, msrA and msrB, in the filamentous fungus Aspergillus nidulans. Both single and double inactivation mutants were viable, but more sensitive to oxidative stress agents as hydrogen peroxide, paraquat, and ultraviolet light. These strains also accumulated more carbonylated proteins when exposed to hydrogen peroxide indicating that MsrA and MsrB are active players in the protection of the cellular proteins from oxidative stress damage.     

Anoxia,acidosis, and intergenic interactions selectively regulate methionine sulfoxide reductase transcriptions in mouse embryonic stem cells

Methionine sulfoxide reductases (Msr) belong to a gene family that contains one MsrA and three MsrBs (MsrB1, MsrB2, and MsrB3). We have identified all four of the genes that are expressed in mouse embryonic stem cell cultures. The vital cellular functions of the Msr family of genes are to protect cells from oxidative damage by enzymatically reducing the oxidized sulfide groups of methionine residues in proteins from the sulfoxide form (? SO) back to sulfide thus restoring normal protein functions as well as reducing intracellular reactive oxygen species (ROS). We have performed studies on the Msr family genes to examine the regulation of gene expression. Our studies using real‐time RT‐PCR and Western blotting have shown that expression levels of the four Msr family genes are under differential regulation by anoxia/reoxygenation treatment, acidic culture conditions and interactions between MsrA and MsrB. Results from these in vitro experiments suggest that although these genes function as a whole in oxidative stress protection, each one of the Msr genes could be responsive to environmental stimulants differently at the tissue level. J. Cell. Biochem. 112: 98–106, 2011. © 2010 Wiley‐Liss, Inc.     

Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite

Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-( S )-sulphoxide, and MsrB, active against methionine-( R )-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects Δ msrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N -acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.     

The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.     

Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pylori

The ability of Helicobacter pylori to colonize the stomach requires that it combat oxidative stress responses imposed by the host. The role of methionine sulfoxide reductase (Msr), a methionine repair enzyme, in H. pylori stress resistance was evaluated by a mutant analysis approach. An msr mutant strain lacked immunologically detectable sulphoxide reductase protein and also showed no enzyme activity when provided with oxidized methionines as substrates. The mutant strain showed diminished growth compared to the parent strain in the presence of chemical oxidants, and showed rapid viability loss when exposed to oxidizing conditions. The stress resistance and enzyme activity could be recovered by complementing the mutant with a functional copy of the msr gene. Upon fractionation of parent strain and the complemented mutant cells into membranes and cytoplasmic proteins, most of the immunologically detectable Msr was localized to the membrane, and this fraction contained all of the Msr activity. Qualitative detection of the whole cell protein pattern using 2,4-dinitro phenyl hydrazine (DNPH) showed a far greater number of oxidized protein species in the mutant than in the parent strain when the cells were subjected to oxygen, peroxide or s-nitrosoglutathione (GSNO) induced stress. Importantly, no oxidized proteins were discerned in either strain upon incubation in anaerobic conditions. A mutant strain that synthesized a truncated Msr (corresponding to the MsrA domain) was slightly more resistant to oxidative stress than the msr strain. Mouse colonization studies showed Msr is an important colonization factor, especially for effective longer-term (14 and 21 days) colonization. Complementation of the mutant msr strain by chromosomal insertion of a functional gene restored mouse colonization ability.     

Functions and evolution of selenoprotein methionine sulfoxide reductases

Methionine sulfoxide reductases (Msrs) are thiol-dependent enzymes which catalyze conversion of methionine sulfoxide to methionine. Three Msr families, MsrA, MsrB, and fRMsr, are known. MsrA and MsrB are responsible for the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide residues in proteins, respectively, whereas fRMsr reduces free methionine-R-sulfoxide. Besides acting on proteins, MsrA can additionally reduce free methionine-S-sulfoxide. Some MsrAs and MsrBs evolved to utilize catalytic selenocysteine. This includes MsrB1, which is a major MsrB in cytosol and nucleus in mammalian cells. Specialized machinery is used for insertion of selenocysteine into MsrB1 and other selenoproteins at in-frame UGA codons. Selenocysteine offers catalytic advantage to the protein repair function of Msrs, but also makes these proteins dependent on the supply of selenium and requires adjustments in their strategies for regeneration of active enzymes. Msrs have roles in protecting cellular proteins from oxidative stress and through this function they may regulate lifespan in several model organisms.     

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol‐located and mitochondria‐enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications. J. Cell. Biochem. 111: 94–103, 2010. © 2010 Wiley‐Liss, Inc.     

Specific activity of methionine sulfoxide reductase in CD-1 mice is significantly affected by dietary selenium but not zinc

Reactive oxygen species-mediated oxidation of methionine residues in protein results in a racemic mixture of R and S forms of methionine sulfoxide (MetO). MetO is reduced back to methionine by the methionine sulfoxide reductases MsrA and MsrB. MsrA is specific toward the S form and MsrB is specific toward the R form of MetO. MsrB is a selenoprotein reported to contain zinc (Zn). To determine the effects of dietary selenium (Se) and Zn on Msr activity, CD-1 mice (N=16/group) were fed, in a 2×2 design, diets containing 0 or 0.2 μg Se/g and 3 or 15 ∥ Zn/g. As an oxidative stress, half of the mice received L-buthionine sulfoximine (BSO; ip; 2 mmol/kg, three times per week for the last 3 wk); the others received saline. After 9.5 wk, Msr (the combined specific activities of MsrA and MsrB) was measured in the brain, kidney, and liver. Se deficiency decreased (p<0.0001) Msr in all three tissues, but Zn had no direct effect. BSO treatment was expected to result in increased Msr activity; this was not seen. Additionally, we found that the ratio of MetO to methionine in liver protein was increased (indicative of oxidative damage) by Se deficiency. The results show that Se deficiency increases oxidation of methionyl residues in protein, that Se status affects Msr (most likely through effects on the selenoprotein MsrB), and that marginal Zn deficiency has little effect on Msr in liver and kidney. Finally, the results show that the oxidative effects of limited BSO treatment did not upregulate Msr activity.     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met(O)) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.Key words: immunoglobulin gamma antibody, methionine sulfoxide, oxidation, photo-oxidation, methionine sulfoxide reductase     

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.     

Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism

Almost all forms of reactive oxygen species (ROS) oxidize methionine residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide. Because organisms contain methionine sulfoxide reductases (Msr's) that can catalyze the thioredoxin-dependent reduction of the sulfoxides back to methionine, it was proposed that the cyclic oxidation/reduction of methionine residues might serve as antioxidants to scavenge ROS, and also to facilitate the regulation of critical enzyme activities. We summarize here results of studies showing that organisms possess two different forms of Msr – namely, MsrA that catalyzes reduction of the S-isomer and MsrB that catalyzes the reduction of the R-isomer. Deletion of the msrA gene in mice leads to increased sensitivity to oxidative stress and to a decrease (40%) in the maximum lifespan. This suggests that elimination of both Msr's would have more serious consequences.     

Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase.

Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turn-over rates of 0.6 min(-1) and 20 min(-1), respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.     

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

The oxidized protein repair methionine sulfoxide reductase (Msr) system has been implicated in aging, in longevity, and in the protection against oxidative stress. This system is made of two different enzymes (MsrA and MsrB) that catalyze the reduction of the two diastereoisomers S- and R-methionine sulfoxide back to methionine within proteins, respectively. Due to its role in cellular protection against oxidative stress that is believed to originate from its reactive oxygen species scavenging ability in combination with exposed methionine at the surface of proteins, the susceptibility of MsrA to hydrogen-peroxide-mediated oxidative inactivation has been analyzed. This study is particularly relevant to the oxidized protein repair function of MsrA in both fighting against oxidized protein formation and being exposed to oxidative stress situations. The enzymatic properties of MsrA indeed rely on the activation of the catalytic cysteine to the thiolate anion form that is potentially susceptible to oxidation by hydrogen peroxide. The residual activity and the redox status of the catalytic cysteine were monitored before and after treatment. These experiments showed that the enzyme is only inactivated by high doses of hydrogen peroxide. Although no significant structural modification was detected by near- and far-UV circular dichroism, the conformational stability of oxidized MsrA was decreased as compared to that of native MsrA, making it more prone to degradation by the 20S proteasome. Decreased conformational stability of oxidized MsrA may therefore be considered as a key factor for determining its increased susceptibility to degradation by the proteasome, hence avoiding its intracellular accumulation upon oxidative stress.     

Obesity reduces methionine sulphoxide reductase activity in visceral adipose tissue

Visceral obesity is linked to insulin resistance and cardiovascular disease. A recent genetic study indicated that the gene locus for the anti-oxidant defense enzyme methionine sulphoxide reductase A (MsrA) is positively associated with the development of visceral adiposity. This work tested the hypothesis that Msr activity is diminished in visceral fat as a result of obesity. It used two animal models of obesity, wild-type rats fed a high-fat (45% of calories from fat) diet and Zucker rats fed a 10% fat calorie diet. The data indicate that MsrA activity was selectively reduced by ～ 25% in the visceral adipose, but not subcutaneous adipose or liver, of both rat models as compared to control, wild type rats receiving a 10% fat calorie diet. MsrB activity was similarly reduced only in visceral fat. The data indicate that Msr activity is reduced by obesity and may alter oxidative stress signalling of obesity.     

Structural and kinetic analysis of an MsrA–MsrB fusion protein from Streptococcus pneumoniae

Methionine sulphoxide reductases (Msr) catalyse the reduction of oxidized methionine to methionine. These enzymes are divided into two classes, MsrA and MsrB, according to substrate specificity. Although most MsrA and MsrB exist as separate enzymes, in some bacteria these two enzymes are fused to form a single polypeptide (MsrAB). Here, we report the first crystal structure of MsrAB from Streptococcus pneumoniae ( Sp MsrAB) at 2.4 Å resolution. Sp MsrAB consists of an N-terminal MsrA domain, a C-terminal MsrB domain and a linker. The linker is composed of 13 residues and contains one 3₁₀-helix and several hydrogen bonds interacting with both MsrA and MsrB domains. Interestingly, our structure includes the MsrB domain complexed with an SHMAEI hexa-peptide that is the N-terminal region of neighbouring MsrA domain. A kinetic analysis showed that the apparent K _m of Sp MsrAB for the R -form-substrate was 20-fold lower than that for the S -form substrate, indicating that the MsrB domain had a much higher affinity for the substrate than the MsrA domain. Our study reveals the first structure of the MsrAB by providing insights into the formation of a disulphide bridge in the MsrB, the structure of the linker region, and the distinct structural nature of active site of each MsrA and MsrB domain.     

Ecdysone induction of MsrA protects against oxidative stress in Drosophila

The methionine sulfoxide reductases MsrA and MsrB reduce Met(O) to Met in epimer-specific fashion. In Drosophila, the major ecdysone induced protein is MsrA, which is regulated by the EcR-USP complex. We tested Kc cells for induction of MsrA, MsrB, EcR, and CAT by ecdysone and found that MsrA and the EcR were induced by ecdysone, but MsrB and CAT were not. When we tested for resistance to 20mM H2O2 toxicity, viability of Kc cells was reduced 3-fold. Pretreatment with 0.2 microM ecdysone for 48 h prior to exposure to H2O2, increased viability to 77% of controls. The EcR-deficient L57-3-11 knockout line was not responsive to ecdysone, and H2O2 resistance of both control and ecdysone-treated L57-3-11 cells was similar to that of the ecdysone-untreated Kc cells. These results show that hormonal regulation of MsrA is implicated in conferring protection against oxidative stress in the Drosophila model.     

Methionine sulphoxide reductases revisited: free methionine as a primary target of H2O2 stress in auxotrophic fission yeast

Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over‐oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H₂O₂ sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H₂O₂ stress decreases by 100‐fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H₂O₂ stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.