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.     

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.     

Kinetic characterization of the catalytic mechanism of methionine sulfoxide reductase B from Neisseria meningitidis

Methionine sulfoxide reductases catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The methionine sulfoxide reductases family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the sulfoxide function. Both enzymes are monomeric and share a similar three-step chemical mechanism. First, in the reductase step, a sulfenic acid intermediate is formed with a concomitant release of 1 mol of methionine per mol of enzyme. Then, an intradisulfide bond is formed. Finally, Msrs return back to reduced forms via reduction by thioredoxin. In the present study, it is shown for the Neisseria meningitidis MsrB that (1) the reductase step is rate-determining in the process leading to formation of the disulfide bond and (2) the thioredoxin-recycling process is rate-limiting. Moreover, the data suggest that within the thioredoxin-recycling process, the rate-limiting step takes place after the two-electron chemical exchange and thus is associated with the release of oxidized thioredoxin.     

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.     

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.     

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.     

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.     

Calorimetry and mass spectrometry study of oxidized calmodulin interaction with target and differential repair by methionine sulfoxide reductases

Calmodulin is known to be a target for oxidation, which leads to conversion of methionine residues to methionine sulfoxides. Previously, we reported that both methionine sulfoxide reductases MsrA and MsrB were able to reduce methionine sulfoxide residues in oxidized calmodulin. In the present study, we have made use of the interaction between calmodulin and RS20, a peptide model for calmodulin targets, to probe the structural consequences of oxidation and mode of repair both by MsrA and MsrB. Isothermal titration calorimetry and differential scanning calorimetry showed that oxidized calmodulin interacts with RS20 via its C-terminal domain only, resulting in a non-productive complex. As shown by spectrofluorometry, oxidized calmodulin treated with MsrA exhibited native binding affinity for RS20. In contrast, MsrB-treatment of oxidized calmodulin resulted in 10-fold reduced affinity. Mass spectrometry revealed that the sulfoxide derivative of methionine residue 124 was differentially repaired by MsrA and MsrB. This provided a basis for rationalizing the difference in binding affinities of oxidized calmodulin reported above, since Met124 residue had been shown to be critical for interaction with some targets. This study provides the first evidence that in an oxidized polypeptide chain MetSO residues might be differentially repaired by the two Msr enzymes.     

The enzymology and biochemistry of methionine sulfoxide reductases

The methionine sulfoxide reductase (Msr) family is composed of two structurally unrelated classes of monomeric enzymes named MsrA and MsrB, which display opposite stereo-selectivities towards the sulfoxide function. MsrAs and MsrBs, characterized so far, share the same chemical mechanism implying sulfenic acid chemistry. The mechanism includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). This scheme is in accordance with the kinetic mechanism of both Msrs which is of ping-pong type. For both Msrs, the reductase step is rate-determining in the process leading to the formation of the disulfide bond. The overall rate-limiting step takes place within the thioredoxin-recycling process, likely being associated with oxidized thioredoxin release. The kinetic data support structural recognition between oxidized Msr and reduced thioredoxin. The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO. About 50% of the MsrBs binds a zinc atom, the location of which is in an opposite direction from the active site. Introducing or removing the zinc binding site modulates the catalytic efficiency of MsrB.     

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.     

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.     

Catalytic advantages provided by selenocysteine in methionine-S-sulfoxide reductases

Methionine sulfoxide reductases are key enzymes that repair oxidatively damaged proteins. Two distinct stereospecific enzyme families are responsible for this function: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In the present study, we identified multiple selenoprotein MsrA sequences in organisms from bacteria to animals. We characterized the selenocysteine (Sec)-containing Chlamydomonas MsrA and found that this protein exhibited 10-50-fold higher activity than either its cysteine (Cys) mutant form or the natural mouse Cys-containing MsrA, making this selenoenzyme the most efficient MsrA known. We also generated a selenoprotein form of mouse MsrA and found that the presence of Sec increased the activity of this enzyme when a resolving Cys was mutated in the protein. These data suggest that the presence of Sec improves the reduction of methionine sulfoxide by MsrAs. However, the oxidized selenoprotein could not always be efficiently reduced to regenerate the active enzyme. Overall, this study demonstrates that sporadically evolved Sec-containing forms of methionine sulfoxide reductases reflect catalytic advantages provided by Sec in these and likely other thiol-dependent oxidoreductases.     

Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B

Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism.     

Kinetic characterization of the chemical steps involved in the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

Oxidation of methionine into methionine sulfoxide is associated with many pathologies and is described to exert regulatory effects on protein functions. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, have been described to reduce the S and the R isomers of the sulfoxide of methionine sulfoxide back to methionine, respectively. Although MsrAs and MsrBs display quite different x-ray structures, they share a similar, new catalytic mechanism that proceeds via the sulfenic acid chemistry and that includes at least three chemical steps with 1) the formation of a sulfenic acid intermediate and the concomitant release of methionine; 2) the formation of an intra-disulfide bond; and 3) the reduction of the disulfide bond by thioredoxin. In the present study, it is shown that for the Neisseria meningitidis MsrA, 1) the rate-limiting step is associated with the reduction of the Cys-51/Cys-198 disulfide MsrA bond by thioredoxin; 2) the formation of the sulfenic acid intermediate is very efficient, thus suggesting catalytic assistance via amino acids of the active site; 3) the rate-determining step in the formation of the Cys-51/Cys-198 disulfide bond is that leading to the formation of the sulfenic intermediate on Cys-51; and 4) the apparent affinity constant for methionine sulfoxide in the methionine sulfoxide reductase step is 80-fold higher than the Km value determined under steady-state conditions.     

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.     

Characterization of the methionine sulfoxide reductase activities of PILB, a probable virulence factor from Neisseria meningitidis.

PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/MsrB) of N. meningitidis were produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding protein-bound MetSO more efficiently than free MetSO; 3) the carbon Calpha is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols.     

Overexpression, purification, and preliminary X-ray crystallographic studies of methionine sulfoxide reductase B from Bacillus subtilis

The peptide methionine sulfoxide reductases Msrs) are enzymes that catalyze the reduction of methionine sulfoxide back to methionine. Because of two enantiomers of methionine sulfoxide (S and R forms), this reduction reaction is carried out by two structurally unrelated classes of enzymes, MsrA (E.C. 1.8.4.11) and MsrB (E.C. 1.8.4.12). Whereas MsrA has been well characterized structurally and functionally, little information on MsrB is available. The recombinant MsrB from Bacillus subtilis has been purified and crystallized by the hanging-drop vapor-diffusion method, and the functional and structural features of MsrB have been elucidated. The crystals belong to the trigonal space group P3, with unit-cell parameters a=b=136.096, c=61.918 , and diffracted to 2.5 resolution using a synchrotron-radiation source at Pohang Light Source. The asymmetric unit contains six subunits of MsrB with a crystal volume per protein mass (VM) of 3.37 A3 Da(-1) and a solvent content of 63.5%.     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.     

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.