Formation Of Methionine Sulfoxide Research Articles

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.

Cloning, Expression, and Characterization of a Methionine Sulfoxide Reductase B Gene from Nicotiana tabacum

Reactive oxygen species (ROS) are generated during normal aerobic metabolism and in plants exposed to environmental stress. Methionine (Met) residues are particularly sensitive to ROS-mediated oxidation, leading to the formation of methionine sulfoxide (MetSO) under mild oxidative conditions. Methionine sulfoxide reductase (MSR) repairs oxidized Met and protects plants from oxidative damage. Herein, we identified a tobacco (Nicotiana tabacum) MSRB gene, referred to as NtMSRB3. Analysis of the sequence showed that the NtMSRB3 protein had four typical motifs in a SelR domain, which is known as the catalytic region of MSRBs. NtMSRB3 specifically reduced the R epimer of MetSO and converted either free MetSO or Dabsyl-MetSO in the presence of dithiothreitol. Escherichia coli cells harboring NtMSRB3 displayed relative high viability under H₂O₂ stress. Subcellular localization of NtMSRB3 revealed that it was a plastid-targeted protein. Furthermore, the semi-quantitative reverse transcription polymerase chain reaction assay showed that NtMSRB3 was upregulated apparently by abscisic acid, salt, cold, and methyl viologen stress within 24 h of treatment.

Expression and Biological Properties of a Novel Methionine Sulfoxide Reductase A in Tobacco (Nicotiana tabacum)

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21kDa, possessed the highly conserved motif, 'GCFWG' in the N-terminus and 'KGCNDPIRCY' motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42°C and pH 9.0 were optimum for NtMSRA4 activity. The K m and K cat values of NtMSRA4 were determined to be 40.04μM and 0.048S(-1) in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H2O2 toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.

Conjugation of Glutathione to Oxidized Tyrosine Residues in Peptides and Proteins

Tyrosine residues are sensitive to oxidation and can be converted to hydroperoxides either by superoxide reacting with the Tyr radical or by singlet oxygen. These hydroperoxides rearrange to bicyclic derivatives that are readily reduced to more stable hydroxides. The aromatic character of tyrosine is lost, but the product contains an α-β unsaturated carbonyl group and is, therefore, an electrophile. We have generated hydroxide derivatives of several Tyr-containing peptides and shown using liquid chromatography/mass spectrometry that they undergo Michael addition with GSH. For Tyr-Gly, rate constants of 9.2 and 11.8 m(-1)min(-1) were measured for the two chromatographically distinct isomers. Unusual for GSH addition to an electrophile, the reaction is reversible, with a half-life of many hours for the reverse reaction. These kinetics indicate that with a typical cellular concentration of 5 mm GSH, >95% Tyr-Gly hydroxide would become conjugated with a half-life of ∼15 min. Sperm whale myoglobin forms a hydroperoxide on Tyr-151 in a hydrogen peroxide/superoxide-dependent reaction. We show that its hydroxide derivative reacts with GSH to form a conjugate. Detection of the conjugate required stabilization by reduction; otherwise, the reverse reaction occurred during tryptic digestion and analysis. Our findings represent a novel mechanism for peptide or protein glutathionylation involving a carbon-sulfur cross-link between oxidized Tyr and Cys. As with other electrophiles, the oxidized Tyr should undergo a similar reaction with Cys residues in proteins to give intramolecular or intermolecular protein cross-links. This mechanism could give rise to protein cross-linking in conditions of oxidative stress.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.

Protein Oxidative Modification in the Aging Organism and the Role of the Ubiquitin Proteasomal System

Living in an oxygen containing environment is automatically connected to oxidative stress. Beside lipids and nucleic acids, especially proteins are very susceptible for oxidative modifications. These oxidative modifications comprise alterations of single amino acids, like the formation of protein carbonyls and methionine sulfoxide, or the aggregation of whole proteins. Due to the ongoing accumulation of protein aggregates during the aging process, the cellular protein quality control system becomes more and more overwhelmed. One essential element of the protein quality control machinery is the ubiquitin proteasomal system which plays therefore a crucial part in the aging process, too. Ubiquitination of proteins is a three step mechanism to tag proteins with a polyubiquitin chain for the proteasome. The proteasome is a regulated, barrel-shaped multi-enzyme complex which is responsible for the degradation of proteins. Although there is no drastic loss of all proteasomal subunits during the aging process, there is a functional decline of the proteasome activity in aging organisms. Impairment of the ubiquitin proteasome system leads to increasing protein aggregation and cellular death. A lot of age related diseases are closely connected to an inhibition of the proteasome and the formation of large protein aggregates. Especially skin aging, atherosclerosis, age-dependent macula degeneration, cataract formation and several neurodegenerative diseases are directly connected to the decline of proteasome function. This review outlines the connections between aging, oxidative stress and protein oxidation, as well as the influence on the ubiquitin proteasomal system and several associated diseases.

Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13

The leukocyte serine proteases (LSPs) elastase, proteinase 3 and cathepsin G cleave von Willebrand factor (VWF) near or at the same cleavage site (Tyr1605-Met1606) as ADAMTS-13, the metalloprotease that specifically controls the proteolytic processing of VWF. Recent studies have shown that oxidation of VWF at Met1606 with formation of methionine sulfoxide (MetSO) severely impairs its proteolysis by ADAMTS-13. This study was aimed at assessing whether or not oxidation of VWF by reactive oxygen species (ROS) can also affect its cleavage by elastase, proteinase 3, and cathepsin G. In this study, the catalytic specificity of hydrolysis by LSPs of the VWF peptide substrate VWF74 and full-length VWF, both unaltered and in the oxidized form, was measured by RP-HPLC, electrophoretic and mass spectrometry methods. LSPs cleaved both VWF multimers and VWF74 near or at the same peptide bond as is cleaved by ADAMTS-13, with k(cat)/K(m) values similar to those of the metalloprotease. However, unlike ADAMTS-13, cathepsin G cleaved VWF74 containing a MetSO residue at position 1606 with a k(cat)/K(m) value higher than that for VWF74, whereas the catalytic efficiencies of both elastase and proteinase 3 were unaffected by the replacement of Met1606 with MetSO. Likewise, oxidation of VWF multimers by hypochlorous acid and ROS, produced by activated leukocytes, improved their hydrolysis by LSPs. Oxidation by leukocyte ROS has a net positive effect on the cleavage of VWF multimers by LSPs, under conditions where high concentrations of oxidant species would severely reduce the proteolytic efficiency of ADAMTS-13.

Combined Raman and IR spectroscopic study on the radical-based modifications of methionine

Among damages reported to occur on proteins, radical-based changes of methionine (Met) residues are one of the most important convalent post-translational modifications. The combined application of Raman and infrared (IR) spectroscopies for the characterisation of the radical-induced modifications of Met is described here. Gamma-irradiation was used to simulate the endogenous formation of reactive species such as hydrogen atoms (•H), hydroxyl radicals (•OH) and hydrogen peroxide (H(2)O(2)). These spectroscopic techniques coupled to mass experiments are suitable tools in detecting almost all the main radical-induced degradation products of Met that depend on the nature of the reactive species. In particular, Raman spectroscopy is useful in revealing the radical-induced modifications in the sulphur-containing moiety, whereas the IR spectra allow decarboxylation and deamination processes to be detected, as well as the formation of other degradation products. Thus, some band patterns useful for building a library of spectra-structure correlation for radical-based degradation of Met were identified. In particular, the bands due to the formation of methionine sulfoxide, the main oxidation product of Met, have been identified. All together, these results combine to produce a set of spectroscopic markers of the main processes occurring as a consequence of radical stress exposure, which can be used in a spectroscopic protocol for providing a first assessment of Met modifications in more complex systems such as peptides and proteins, and monitoring their impact on protein structure.

Redox Proteomics of Protein-bound Methionine Oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.

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.

Characterization by mass spectrometry and IRMPD spectroscopy of the sulfoxide group in oxidized methionine and related compounds

Methionine protein residues are prone to oxidation. To unravel the controversy about the mechanism of its one-electron oxidation, we characterised the main biological product, methionine sulfoxide, using mass spectrometry and IR multiple photon dissociation spectroscopy. Gas phase IR spectra in the 800–2000 cm −1 range of protonated methionine and its sulfoxide were recorded and compared to those computed for the lowest energy structures. The signature of the S O bond was clearly identified at around 1000 cm −1. Oxidation of methionine–lysine dipeptide by OH radicals in the presence of catalase revealed the formation of methionine sulfoxide upon one-electron oxidation.

Site-Directed Mutagenesis Combined with Oxidative Methionine Labeling for Probing Structural Transitions of a Membrane Protein by Mass Spectrometry

Exposure of the membrane protein bacteriorhodopsin (BR) to SDS induces partial breakdown of the native conformation. The exact structural properties of this SDS state remain a matter of debate, despite its widespread use in BR folding experiments. The current work employs hydroxyl radical (·OH) labeling in conjunction with mass spectrometry (MS)-based peptide mapping for probing the solvent accessibility of individual BR segments in the presence of SDS. Previous work revealed methionine sulfoxide formation to be the dominant oxidative pathway. Those data suggested extensive unfolding of helices A and D in SDS. Unfortunately, the lack of Met residues in helices C and F implies that no direct information on the behavior of the latter two elements could be obtained. Here, we address this problem by employing two variants with additional Met residues, L93M (helix C) and V179M (helix F). The oxidation behavior of the resulting 11 methionines can be grouped into three categories: (1) extensively labeled both in native BR and in SDS (loop residues M32, M68, and M163); (2) protected in the native state but not in SDS (M20, M118); (3) always protected (M56, M60, M93, M145, M179, M209). These data show that a solvent-inaccessible core is retained in SDS. This core consists of partially intact helices B, C, E, F, and G. The termini of these helices are highly dynamic and/or unraveled, particularly on the cytoplasmic side. Overall, this work demonstrates how the use of engineered ·OH labeling sites can provide insights into structural properties of membrane proteins.

Methionine Sulfoxide Reductase B1 (MsrB1) Recovers TRPM6 Channel Activity during Oxidative Stress

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.

Protein Carbonyl and the Methionine Sulfoxide Reductase System

The formation and accumulation of protein-carbonyl by reactive oxygen species may serve as a marker of oxidative stress, aging, and age-related diseases. Enzymatic reversal of the protein-carbonyl modification has not yet been detected. However, an enzymatic reversal of protein-methionine sulfoxide modification exists and is mediated by the methionine sulfoxide reductase (Msr) system. Methionine sulfoxide modifications to proteins may precede the formation of protein-carbonyl adducts because of consequent structural changes that increase the vulnerability of amino acid residues to carbonylation. Supportive evidence for this possibility arises from the elevated protein-carbonyl accumulations observed in organisms, such as yeast and mice, lacking the methionine sulfoxide reductase A (MsrA) enzyme. In addition, advanced age or enhanced oxidative-stress conditions foster the accumulations of protein-carbonyls. This review discusses the possible involvement of methionine sulfoxide formation in the occurrence of protein-carbonyl adducts and their relevance to the aging process and neurodegenerative diseases.

Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (T(m)) from 58.7 degrees C for native apoA-I to 48.2 degrees C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 A and 9.92 A, indicating a cross-beta amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C-terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) and hydroxyl radicals (OH(*)) was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H(2)O(2) system differed from that of CCK8 in the Fenton system, in which OH(*) are produced. In the H(2)O(2) system, (28)Met and (31)Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, (28)Met and (31)Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that (28)Met and (31)Met residues of CCK8 are susceptible to oxidation by ROS.

Superoxide-mediated Formation of Tyrosine Hydroperoxides and Methionine Sulfoxide in Peptides through Radical Addition and Intramolecular Oxygen Transfer

The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/O(2)(.) system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from O(2)(.) to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of O(2)(.) to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of O(2)(.) and have major implications.

Characterization of peptide Methionine oxidation and Methionine sulfoxide reduction using Methionine‐rich proteins

Methionine (Met) is one of the amino acids particularly susceptible to oxidation. Methionine oxidation mediated by reactive oxygen species (ROS) may result in formation of methionine sulfoxide (MetO), alter protein structure/function, and accelerate age‐related diseases. However, MetO can be reduced back to Met by methionine sulfoxide reductases MsrA and MsrB. Therefore, MetO is a candidate for biomarker of oxidative stress and Msr deficiency. The conventional methods to monitor MetO formation and reduction rely on HPLC or mass spectrometry procedures. Here, we computationally identified six proteins rich in methionine residues (>20%), termed them Met‐rich proteins (MRPs), and used them to examine Met oxidation and repair by in‐gel and immunoblot assays. Met oxidation and MetO reduction could be easily monitored by the mobility shift of these proteins on SDS‐PAGE gels, and by immunoblot assays with antibodies specific for the oxidized forms of MRPs. The oxidation of MRPs with hydrogen peroxide was more selective to Met than that with hypochlorite or other oxidants. All MetO residues in MRPs could by reduced by MsrA and MsrB either in DTT or Trx dependent assays. Our research suggests that MRPs may be a useful and convenient tool to characterize Met oxidation, MetO reduction and Msr activity.

Role of the Sulfur Atom on the Reactivity of Methionine toward OH Radicals: Comparison with Norleucine

Density functional theory has been used to model the OH reaction with Gly-Met-Gly and Gly-Nle-Gly tripeptides. The first one is predicted to be about 100 times faster than the second one. Therefore, if a methionine fragment is replaced by norleucine, the overall reactivity of the peptide toward free radicals is expected to be significantly reduced, which is in agreement with previous experimental findings. Since the most reactive sites were found to be located in the central backbone for Nle and in the terminal fragment of the side chain for Met, this decrease is expected to be even more critical for large-sized free radicals. The S atom seems to activate not only those alkyl sites next to it but also those located an odd number of bonds apart. In addition the viability of different paths explaining the formation of methionine sulfoxide has been tested, and it is proposed that this process involves the formation of R-SO radical and formaldehyde. The results from the present work offer an explanation to the role of sulfur atom on the reactivity of methionine toward free radicals. They also support the preponderant role of Met35 on the development of the Alzheimer disease.