Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation

Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol. 11:1241–1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity.     

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 methionine sulfoxide reductases: Catalysis and substrate specificities

Oxidation of Met residues in proteins leads to the formation of methionine sulfoxides (MetSO). Methionine sulfoxide reductases (Msr) are ubiquitous enzymes, which catalyze the reduction of the sulfoxide function of the oxidized methionine residues. In vivo, the role of Msrs is described as essential in protecting cells against oxidative damages and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB, with opposite stereoselectivity towards the S and R isomers of the sulfoxide function, respectively. Both Msrs present a similar three-step catalytic mechanism. The first step, called the reductase step, leads to the formation of a sulfenic acid on the catalytic Cys with the concomitant release of Met. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the catalysis, in particular of the reductase step, and in structural specificities.     

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.     

Recharging oxidative protein repair: Catalysis by methionine sulfoxide reductases towards their amino acid, protein, and model substrates

The sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins.     

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.     

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

Oxidation of methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible posttranslational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been established as a key mechanism for direct regulation of a wide range of protein functions and cellular processes. Furthermore, recent reports suggest an interaction between Met oxidation and O-phosphorylation. Such interactions are a potentially direct interface between redox sensing and signaling, and cellular protein kinase/phosphatase-based signaling. Herein, we describe the current state of Met oxidation research, provide some mechanistic insight into crosstalk between these two major posttranslational modifications, and consider the evolutionary significance and regulatory potential of this crosstalk.     

The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin

We report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin-binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.     

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.     

Thioredoxin-dependent redox regulation of cellular signaling and stress response through reversible oxidation of methionines

The sensitive oxidations of sulfur containing amino acids (i.e., cysteines and methionines) commonly control protein function, and act as important signaling mechanisms to modify metabolic responses to environmental stressors. Mechanisms associated with cysteine oxidation to form sulfenic acid and disulfides (i.e., cystine and glutathione adducts), and their reversibility through thioredoxin-dependent mechanisms, are broadly appreciated as important regulatory mechanisms that control the function of a range of different proteins. Less commonly understood are the cellular consequences of methionine oxidation to form methionine sulfoxide, as the structural requirements for their thioredoxin-dependent reduction by methionine sulfoxide reductases limit the reversibility of methionine oxidation to sequences within surface exposed and conformationally disordered regions of proteins. Surface exposed methionines are commonly involved in molecular recognition between transient protein signaling complexes, where their oxidation disrupts productive protein-protein interactions linked to a range of cellular responses. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress responses in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the physical basis for differences in the sensitivity of individual methionines within plant and animal calmodulin to reactive oxygen species (ROS), the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes. It is suggested that, in combination with high-throughput proteomic methods and current generation informatics tools, these mechanistic insights permit useful predictions of oxidatively sensitive signaling proteins that act as redox and stress sensors in response to methionine oxidation.     

Methionine oxidation and aging

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

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     

Methionine sulfoxide reductases and methionine sulfoxide in the subterranean mole rat (Spalax): characterization of expression under various oxygen conditions

The blind subterranean mole rat (Spalax ehrenbergi) exhibits a relatively long life span, which is attributed to an efficient antioxidant defense affording protection against accumulation of oxidative modifications of proteins. Methionine residues can be oxidized to methionine sulfoxide (MetO) and then enzymatically reduced by the methionine sulfoxide reductase (Msr) system. In the current study we have isolated the cDNA sequences of the Spalax Msr genes as well as 23 additional selenoproteins and monitored the activities of Msr enzymes in liver and brain of rat (Rattus norvegicus), Spalax galili, and Spalax judaei under normoxia, hypoxia, and hyperoxia. Under normoxia, the Msr activity was lower in S. galili in comparison to S. judaei and R. norvegicus especially in the brain. The pattern of Msr activity of the three species was similar throughout the tested conditions. However, exposure of the animals to hypoxia caused a significant enhancement of Msr activity, especially in S. galili. Hyperoxic exposure showed a highly significant induction of Msr activity compared with normoxic conditions for R. norvegicus and S. galili brain. It was concluded that among all species examined, S. galili appears to be more responsive to oxygen tension changes and that the Msr system is upregulated mainly by severe hypoxia.     

A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.     

Free-energy simulations of the oxidation of c-terminal methionines in calmodulin

In the course of aging or under conditions of oxidative stress, methionine residues of calmodulin undergo oxidation, leading to loss of biological activity of the protein. We have performed free-energy simulations of the effects of C-terminal methionine side-chain oxidation on the properties of calmodulin. The simulation results indicate that oxidation should have a destabilizing effect on all three protein functional states: calcium free, calcium loaded, and with both calcium and target peptide bound. Because the different states are destabilized by different amounts, this leads to a more complex pattern in the observable effects on protein thermal stability, calcium affinity, and binding of a target peptide. The influence of oxidation on the free energy of CaM unfolding is estimated by comparing the free-energy cost of oxidizing a Met residue in a Gly-Met-Gly peptide and in the protein. The protein thermal stability of the oxidized forms is lowered by a moderate amount 1-3 kcal/mol, in qualitative agreement with experimental results of 0.3 kcal/mol. The calculated changes in affinity for calcium and for the target peptide show opposing trends. Oxidation at position 144 is predicted to enhance peptide binding and weaken calcium binding, whereas oxidation at 145 weakens peptide binding and enhances affinity for calcium. The lower affinity of Met 145-oxidized calmodulin toward the target peptide correlates with experimentally observed lowering of calmodulin-activated Ca-ATPase activity when oxidized calmodulin from aged rat brains is used. Thus, our simulations suggest that Met 145 is the oxidation site in the C-terminal fragment of calmodulin. The microscopic mechanism behind the calculated free energy changes appears to be a greater affinity for water of the oxidized Met side-chain relative to normal Met. Structures with Met exposed to solvent had consistently lower free energies than those with buried Met sidechains.     

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]     

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.     

Diastereoselective protein methionine oxidation by reactive oxygen species and diastereoselective repair by methionine sulfoxide reductase

Recent studies have shown that the "calcium-sensor" protein calmodulin (CaM) suffers an age-dependent oxidation of methionine (Met) to methionine sulfoxide (MetSO) in vivo. However, MetSO did not accumulate on the Met residues that show the highest solvent-exposure. Hence, the pattern of Met oxidation in vivo may give hints as to which reactive oxygen species and oxidation mechanisms participate in the oxidation of this important protein. Here, we have exposed CaM under a series of different reaction conditions (pH, [Ca(2+)], [KCl]) to various biologically relevant reactive oxygen species and oxidizing systems (peroxides, HOCl, peroxynitrite, singlet oxygen, metal-catalyzed oxidation, and peroxidase-catalyzed oxidation) to investigate whether one of these systems would lead to an oxidation pattern of CaM similar to that observed in vivo. However, generally, these oxidizing conditions led to a preferred or exclusive oxidation of the C-terminal Met residues, in contrast to the oxidation pattern of CaM observed in vivo. Hence, none of the employed oxidizing conditions was able to mimic the age-dependent oxidation of CaM in vivo, indicating that other, yet unidentified oxidation mechanisms may be important in vivo. Some oxidizing species showed a quite-remarkable diastereoselectivity for the formation of either L-Met-D-SO or L-Met-L-SO. Diastereoselectivity was dependent on the nature of the oxidizing species but was less a function of the location of the target Met residue in the protein. In contrast, diastereoselective reduction of L-Met-D-SO by protein methionine sulfoxide reductase (pMSR) was efficient regardless of the position of the L-Met-D-SO residue in the protein and the presence or absence of calcium. With only the L-Met-D-SO diastereomer being a substrate for pMSR, any preferred formation of L-Met-L-SO in vivo may cause the accumulation of MetSO unless the oxidized protein is substrate for (accelerated) protein turnover.     

Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met₁₀₁ and Met₁₃₃, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met₁₀₁ and Met₁₃₃ residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.