The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite

Both peroxynitrous acid and peroxynitrite react with methionine, k(acid) = (1.7 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 8.6 +/- 0.2 M(-1) s(-1), respectively, and with N-acetylmethionine k(acid) = (2.8 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 10.0 +/- 0.1 M(-1) s(-1), respectively, to form sulfoxides. In contrast to the results of Pryor et al. (1994, Proc. Natl. Acad. Sci. USA 91, 11173-11177), a linear correlation between k(obs) and [met] was obtained. Surprisingly, for every two sulfoxides and nitrites formed, one peroxynitrite is converted to nitrate. Thus, methionine also catalyzes the isomerization of peroxynitrite to nitrate. Neither the pH nor the concentration of methionine affected the distribution of the yields of nitrite, nitrate, and methionine sulfoxide, which were the only products detected. No products other than nitrite, nitrate, and methioninesulfoxide could be detected. The reactions of methionine and N-acetylmethionine with peroxynitrous acid and peroxynitrite are simple bimolecular reactions that do not involve an activated form of peroxynitrous acid or of peroxynitrite. Nitrite, produced together with methionine sulfoxide, or present as a contamination in the peroxynitrite preparation, is not innocuous, but oxidizes methionine by one electron, which leads to the formation of methional and ethylene.     

Radical production by hydrogen peroxide/bicarbonate and copper uptake in mammalian cells: modulation by Cu(II) complexes

The presence of the bicarbonate/carbon dioxide pair is known to accelerate the transition metal ion-catalysed oxidation of various biotargets. It has been shown that stable Cu(II) complexes formed with imine ligands that allow redox cycling between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures. It is also reported that Cu(II)-tetraglycine can form a stable Cu(III) complex. In the present study, radical generation from H₂O₂ and H₂O₂/HCO₃⁻ in the presence of these two different classes of Cu(II) complexes was evaluated by monitoring the oxidation of dihydrorhodamine 123 and NADH and by the quantitative determination of thiobarbituric acid reactive substances (TBARs method). Cu(II)-imine complexes produced low levels of reactive species whereas Cu(II)-Gly-derived complexes, as well as the free Cu(II) ion, produced oxygen-derived radicals in significantly larger amounts. The effects of these two classes of complexes on mammalian tumour cell viability were equally distinct, in that Cu(II)-imine complexes caused apoptosis, entered in cell and remained almost unaffected in high levels whilst, at the same concentrations, Cu(II)-Gly peptide complexes and Cu(II) sulphate stimulated cell proliferation, with the cell managing copper efficiently. Taken together, these results highlight the different biological effects of Cu(II) complexes, some of which have been recently studied as anti-tumour drugs and radical system generators, and also update the effects of reactive oxygen species generation on cell cycle control.     

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Among various metal ions of physiological interest, Cu²⁺ is uniquely capable of catalyzing the oxidation of NADH by H₂O₂. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu²⁺-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H₂O₂ utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD⁺. D₂O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding¹O₂ as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H₂O₂.     

Protection of GroEL by its methionine residues against oxidation by hydrogen peroxide

GroEL undergoes an important functional and structural transition when oxidized with hydrogen peroxide (H2O2) concentrations between 15 and 20mM. When GroEL was incubated for 3h with 15 mM H2O2, it retained its quaternary structure, chaperone and ATPase activities. Under these conditions, GroEL's cysteine and tyrosine residues remained intact. However, all the methionine residues of the molecular chaperone were oxidized to the corresponding methionine-sulfoxides under these conditions. The oxidation of the methionine residues was verified by the inability of cyanogen bromide to cleave at the carboxyl side of the modified methionine residues. The role for the proportionately large number (23) of methionine residues in GroEL has not been identified. Methionine residues have been reported to have an antioxidant activity in proteins against a variety of oxidants produced in biological systems including H2O2. The carboxyl-terminal domain of GroEL is rich in methionine residues and we hypothesized that these residues are involved in the protection of GroEL's functional structure by scavenging H2O2. When GroEL was further incubated for the same time, but with increasing concentrations of H2O2 (>15 mM), the oxidation of GroEL's cysteine residues and a significant decrease of the tyrosine fluorescence due to the formation of dityrosines were observed. Also, at these higher concentrations of H2O2, the inability of GroEL to hydrolyze ATP and to assist the refolding of urea-unfolded rhodanese was observed.     

The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide

The initiation of lipid peroxidation by Fe2+ and H2O2 (Fenton's reagent) is often proposed to be mediated by the highly reactive hydroxyl radical. Using Fe2+, H2O2, and phospholipid liposomes as a model system, we have found that lipid peroxidation, as assessed by malondialdehyde formation, is not initiated by the hydroxyl radical, but rather requires Fe3+ and Fe2+. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and the bleaching of para-nitrosodimethylaniline confirmed the generation of the hydroxyl radical in this system. Accordingly, catalase and the hydroxyl radical scavengers mannitol and benzoate efficiently inhibited the generation and the detection of hydroxyl radical. However, catalase, mannitol, and benzoate could either stimulate or inhibit lipid peroxidation. These unusual effects were found to be consistent with their ability to modulate the extent of Fe2+ oxidation by H2O2 and demonstrated that lipid peroxidation depends on the Fe3+:Fe2+ ratio, maximal initial rates occurring at 1:1. These studies suggest that the initiation of liposomal peroxidation by Fe2+ and H2O2 is mediated by an oxidant which requires both Fe3+ and Fe2+ and that the rate of the reaction is determined by the absolute Fe3+:Fe2+ ratio.     

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.     

Metal-catalyzed oxidation of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide

alpha-Synuclein is a component of abnormal protein depositions of Lewy bodies and senile plaques found in Parkinson's and Alzheimer's diseases, respectively. By using chemical coupling reagents such as dicyclohexylcarbodiimide or N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, the protein was shown to experience self-oligomerization in the presence of either copper(II) or Abeta25-35. The oligomers which appeared as a ladder on a 10-20% Tricine/SDS-PAGE have been suggested to participate in the formation of protein aggregations by possibly providing a nucleation center. Since oxidatively modified protein could increase its own tendency toward protein aggregation, metal-catalyzed oxidation of alpha-synuclein has been examined with copper(II) and hydrogen peroxide in the absence of the coupling reagent. Intriguingly, the protein was also self-oligomerized into an SDS-resistant ladder on the gel. This biochemically specific copper-mediated oxidative oligomerization was shown to be dependent upon the acidic C-terminus of alpha-synuclein because the C-terminally truncated proteins such as alpha-syn114 and alpha-syn97 were not affected by the metal and hydrogen peroxide. More importantly, the oxidative oligomerization was synergistically enhanced by the presence of Abeta25-35, indicating that the peptide interaction with alpha-synuclein facilitated the copper(II) binding to the acidic C-terminus and subsequent oxidative crosslinking. It has been, therefore, suggested that abnormalities in copper and H(2)O(2) homeostasis and certain pathological factors functionally similar to the Abeta25-35 could play critical roles in the metal-catalyzed oxidative oligomerization of alpha-synuclein, which may lead to possible protein aggregation and neurodegenerations.     

Potential role of methionine sulfoxide in the inactivation of the chaperone GroEL by hypochlorous acid (HOCl) and peroxynitrite (ONOO-)

GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (.NO) or hydrogen peroxide (H(2)O(2)), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl). For the exposure of 17.5 microm GroEL to 100-250 microm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met(111) and Met(114) to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO(-) suggests that this protein may be a target for bacterial killing by phagocytes.     

NAD(P)H oxidation by hydrogen peroxide in Escherichia coli

A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity. This activity is compared to and found to be distinct from well-known E. Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.     

Interaction of peroxynitrite and hydrogen peroxide with dinitrosyl iron complexes containing thiol ligands in vitro

The interaction of peroxynitrite with thiolate dinitrosyl iron complexes (DNIC) has been examined and compared with the interaction with H2O2. Peroxynitrite oxidized DNIC containing various thiolate ligands--cysteine, glutathione, and bovine serum albumin. Analysis of the oxidation suggested a two-electron reaction and gave third-order rate constants of (9.3 +/- 0.5).109 M-2.sec-1 for DNIC with BSA, (4.0 +/- 0.3).108 M-2.sec-1 for DNIC with cysteine, and (1. 8 +/- 0.3).107 M-2.sec-1 for DNIC with glutathione at 20 degrees C and pH 7.6. Peroxynitrite was more reactive towards DNIC than towards sulfhydryls. Addition of sodium dithionite after the reaction led to significant restoration of the EPR signal of DNIC with cysteine. The reaction of glutathione DNIC with H2O2 was about 600 times slower than with ONOO- and not reversed by sodium dithionite. Thus peroxynitrite, in contrast to hydrogen peroxide, changes the pool of nitrosocompounds which can be responsible for interconversion, storage, and transportation of nitric oxide in vivo.     

Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/micrograms in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence. Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3'-diamino-4,4'-dimethylazobenzene and 3,3'-diamino-4,4'-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 microliters S9 per plate, respectively.     

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II.

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/μg in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence.Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3′-diamino-4,4′-dimethylazobenzene and 3,3′ diamino-4,4′-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 μl S9 per plate, respectively.     

Interaction between iron(II) and hydroxamic acids: oxidation of iron(II) to iron(III) by desferrioxamine B under anaerobic conditions

Interaction between iron(II) and acetohydroxamic acid (Aha), alpha-alaninehydroxamic acid (alpha-Alaha), beta-alaninehydroxamic acid (beta-Alaha), hexanedioic acid bis(3-hydroxycarbamoyl-methyl)amide (Dha) or desferrioxamine B (DFB) under anaerobic conditions was studied by pH-metric and UV-Visible spectrophotometric methods. The stability constants of complexes formed with Aha, alpha-Alaha, beta-Alaha and Dha were calculated and turned out to be much lower than those of the corresponding iron(II) complexes. Stability constants of the iron(II)-hydroxamate complexes are compared with those of other divalent 3d-block metal ions and the Irving-Williams series of stabilities was found to be observed. Above pH 4, in the reactions between iron(II) and desferrioxamine B, the oxidation of the metal ion to iron(III) by the ligand was found. The overall reaction that resulted in the formation of the tris-hydroxamato complex [Fe(HDFB)]+ and monoamide derivative of DFB at pH 6 is: 2Fe2+ + 3H4DFB+ = 2[Fe(HDFB)]+ + H3DFB-monoamide+ + H2O + 4H+. Based on these results, the conclusion is that desferrioxamine B can uptake iron in iron(III) form under anaerobic conditions.     

Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.