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.     

Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.     

Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases

Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.     

Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases

Methionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner.     

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.     

Reaction mechanism,evolutionary analysis,and role of zinc in Drosophila methionine-R-sulfoxide reductase

Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.     

MsrB1 (methionine-R-sulfoxide reductase 1) knock-out mice: roles of MsrB1 in redox regulation and identification of a novel selenoprotein form

Protein oxidation has been linked to accelerated aging and is a contributing factor to many diseases. Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically removed. Further studies revealed that the 5-kDa protein occurred in both mouse tissues and human HEK 293 cells; was down-regulated by MsrB1 small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations; and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with (75)Se and mass spectrometry analyses revealed that the 5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed reduced MsrB activity, with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knock-out mice also showed increased levels of malondialdehyde, protein carbonyls, protein methionine sulfoxide, and oxidized glutathione as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox control in mouse liver and kidney and identified a novel form of this protein.     

Protein maintenance in aging and replicative senescence: a role for the peptide methionine sulfoxide reductases

Cellular aging is characterized by the build-up of oxidatively modified protein that results, at least in part, from impaired redox homeostasis associated with the aging process. Protein degradation and repair are critical for eliminating oxidized proteins from the cell. Oxidized protein degradation is mainly achieved by the proteasomal system and it is now well established that proteasomal function is generally impaired with age. Specific enzymatic systems have been identified which catalyze the regeneration of cysteine and methionine following oxidation within proteins. Protein-bound methionine sulfoxide diastereoisomers S and R are repaired by the combined action of the enzymes MsrA and MsrB that are subsequently regenerated by thioredoxin/thioredoxin reductase. Importantly, the peptide methionine sulfoxide reductase system has been implicated in increased longevity and resistance to oxidative stress in different cell types and model organisms. In a previous study, we reported that peptide methionine sulfoxide reductase activity as well as gene and protein expression of MsrA are decreased in various organs as a function of age. More recently, we have shown that gene expression of both MsrA and MsrB2 (Cbs-1) is decreased during replicative senescence of WI-38 fibroblasts, and this decline is associated with an alteration in catalytic activity and the accumulation of oxidized protein. In this review, we will address the importance of protein maintenance in the aging process as well as in replicative senescence, with a special focus on regulation of the peptide methionine sulfoxide reductase systems.     

Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.     

Methionine sulfoxide reductases in prokaryotes

In living organisms, most methionine residues exposed to reactive oxygen species (ROS) are converted to methionine sulfoxides. This reaction can lead to structural modifications and/or inactivation of proteins. Recent years have brought a wealth of new information on methionine sulfoxide reductase A (MsrA) and B (MsrB) which makes methionine oxidation a reversible process. Homologs of msrA and msrB genes have been identified in most living organisms and their evolution throughout different species led to different genetic organization and different copy number per organism. While MsrA and MsrB had been the focus of multiple biochemical investigations, our understanding of their physiological role in vivo remains scarce. Yet, the recent identification of a direct link between protein targeting and MsrA/MsrB repair offers a best illustration of the physiological importance of this pathway. Repeatedly identified as a potential "virulence factor", contribution of msrA to pathogenicity is also discussed. It remains, however, unclear whether reduced virulence results from overall viability loss or relates to specific oxidized virulence factors left unrepaired. We speculate that a major issue towards assessing the in vivo role of the MsrA/MsrB repair pathway in the next future will be to decipher the interrelations, if any, between MsrA/MsrB-mediated repair and chaperone-assisted folding and/or protease-assisted degradation.     

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.     

Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli. BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.     

Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals

Msrs (methionine sulfoxide reductases), MsrA and MsrB, are repair enzymes that reduce methionine sulfoxide residues in oxidatively damaged proteins to methionine residues in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. In recent years, significant efforts have been made to explore the catalytic properties and physiological functions of these enzymes. In the current review, we present recent progress in this area, with the focus on mammalian MsrA and MsrBs including their roles in disease, evolution and function of selenoprotein forms of MsrA and MsrB, and the biochemistry of these enzymes.     

Molecular Evolution of Peptide Methionine Sulfoxide Reductases (MsrA and MsrB): On the Early Development of a Mechanism That Protects Against Oxidative Damage

Methionine sulfoxide reductases, enzymes that reverse the oxidation of methionine residues, have been described in a wide range of species. The reduction of the diastereoisomers of oxidized methionine is catalyzed by two different monomeric methionine sulfoxide reductases (MsrA and MsrB) and is best understood as an evolutionary response to high levels of oxygen either in the Earth’s atmosphere or possibly in more localized environments. Phylogenetic analyses of these proteins suggest that their distribution is the outcome of a complex history including many paralogy and lateral gene transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Methionine sulfoxide reductase A: Structure, function and role in ocular pathology

Methionine is a highly susceptible amino acid that can be oxidized to S and R diastereomeric forms of methionine sulfoxide by many of the reactive oxygen species generated in biological systems. Methionine sulfoxide reductases (Msrs) are thioredoxin-linked enzymes involved in the enzymatic conversion of methionine sulfoxide to methionine. Although MsrA and MsrB have the same function of methionine reduction, they differ in substrate specifi city, active site composition, subcellular localization, and evolution. MsrA has been localized in different ocular regions and is abundantly expressed in the retina and in retinal pigment epithelial (RPE) cells. MsrA protects cells from oxidative stress. Overexpression of MsrA increases resistance to cell death, while silencing or knocking down MsrA decreases cell survival; events that are mediated by mitochondria. MsrA participates in protein-protein interaction with several other cellular proteins. The interaction of MsrAwith α-crystallins is of utmost importance given the known functions of the latter in protein folding, neuroprotection, and cell survival. Oxidation of methionine residues in α-crystallins results in loss of chaperone function and possibly its antiapoptotic properties. Recent work from our laboratory has shown that MsrA is co-localized with αA and αB crystallins in the retinal samples of patients with age-related macular degen- eration. We have also found that chemically induced hypoxia regulates the expression of MsrA and MsrB2 in human RPE cells. Thus, MsrA is a critical enzyme that participates in cell and tissue protection, and its interaction with other proteins/growth factors may provide a target for therapeutic strategies to prevent degenerative diseases.     

The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS-1, was analyzed during replicative senescence of WI-38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS-1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age-related accumulation of oxidative damage.     

Purification and characterization of methionine sulfoxide reductases from mouse and Staphylococcus aureus and their substrate stereospecificity.

Many organisms have been shown to possess a methionine sulfoxide reductase (MsrA), exhibiting high specificity for reduction the S form of free and protein-bound methionine sulfoxide to methionine. Recently, a different form of the reductase (referred to as MsrB) has been detected in several organisms. We show here that MsrB is a selenoprotein that exhibits high specificity for reduction of the R forms of free and protein-bound methionine sulfoxide. The enzyme was partially purified from mouse liver and a derivative of the mouse MsrB gene, in which the codon specifying selenocystein incorporation was replaced by the cystein codon, was prepared, cloned, and overexpressed in Escherichia coli. The properties of the modified MsrB protein were compared directly with those of MsrA. Also, we have shown that in Staphylococcus aureus there are two MsrA and one nonselenoprotein MsrB, which demonstrates the same substrate stereospecificity as the mouse MsrB.     

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.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

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.