Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily

Thiols represent preferential targets of peroxynitrite in biological systems. In this work, we investigated the mechanisms and kinetics of the reaction of peroxynitrite with the dithiol dihydrolipoic acid (DHLA) and its oxidized form, lipoic acid (LA). Peroxynitrite reacted with DHLA being oxidation yields higher at alkaline pH. The stoichiometry for the reaction was two thiols oxidized per peroxynitrite. LA formation accounted for approximately 50% DHLA consumption at pH 7.4, probably reflecting secondary reactions between LA and peroxynitrite. Indeed, peroxynitrous acid reacted with LA with an apparent second-order rate constant (k(2app)) of 1400 M(-1) s(-1) at pH 7.4 and 37 degrees C. Nitrite and LA-thiosufinate were formed as reaction products. Surprisingly, the k(2app) for peroxynitrite-dependent DHLA oxidation was only 250 M(-1) s(-1) per thiol, at pH 7.4 and 37 degrees C. Testing various low-molecular-weight thiols, we found that an increase in the thiol pK (pK(SH)) value correlated with a decrease of k(2app) for the reaction with peroxynitrite at pH 7.4. The pK(SH) for DHLA is 10.7, in agreement with its modest reactivity with peroxynitrite.     

Biochemical insight into physiological effects of H₂S: reaction with peroxynitrite and formation of a new nitric oxide donor, sulfinyl nitrite

The reaction of hydrogen sulfide (H2S) with peroxynitrite (a key mediator in numerous pathological states) was studied in vitro and in different cellular models. The results show that H2S can scavenge peroxynitrite with a corresponding second order rate constant of 3.3 ± 0.4 × 103 M?1·s?1 at 23°C (8 ± 2 × 103 M?1·s?1 at 37°C). Activation parameters for the reaction (ΔH?, ΔS? and ΔV?) revealed that the mechanism is rather associative than multi-step free-radical as expected for other thiols. This is in agreement with a primary formation of a new reaction product characterized by spectral and computational studies as HSNO? (thionitrate), predominantly present as sulfinyl nitrite, HS(O)NO. This is the first time a thionitrate has been shown to be generated under biologically relevant conditions. The potential of HS(O)NO to serve as a NO donor in a pH-dependent manner and its ability to release NO inside the cells has been demonstrated. Thus sulfide modulates the chemistry and biological effects of peroxynitrite by its scavenging and formation of a new chemical entity (HSNO?) with the potential to release NO, suppressing the pro-apoptotic, oxidative and nitrative properties of peroxynitrite. Physiological concentrations of H?S abrogated peroxynitrite-induced cell damage as demonstrated by the: (i) inhibition of apoptosis and necrosis caused by peroxynitrite; (ii) prevention of protein nitration; and (iii) inhibition of PARP-1 [poly(ADP-ribose) polymerase 1] activation in cellular models, implying that a major part of the cytoprotective effects of hydrogen sulfide may be mediated by modulation of peroxynitrite chemistry, in particular under inflammatory conditions.     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Trypanosoma brucei and Trypanosoma cruzi tryparedoxin peroxidases catalytically detoxify peroxynitrite via oxidation of fast reacting thiols

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m(-1) s(-1), respectively, at pH 7.4 and 37 degrees C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 x 10(5) and 7.2 x 10(5) m(-1) s(-1) at pH 7.4 and 37 degrees C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn(3+)-porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo.     

Peroxynitrite efficiently mediates the interconversion of redox intermediates of myeloperoxidase

Nitric oxide-derived oxidants (e.g., peroxynitrite) are believed to participate in antimicrobial activities as part of normal host defenses but also in oxidative tissue injury in inflammatory disorders. A similar role is ascribed to the heme enzyme myeloperoxidase (MPO), the most abundant protein of polymorphonuclear leukocytes, which are the terminal phagocytosing effector cells of the innate immune system. Concomitant production of peroxynitrite and release of millimolar MPO are characteristic events during phagocytosis. In order to understand the mode of interaction between MPO and peroxynitrite, we have performed a comprehensive stopped-flow investigation of the reaction between all physiological relevant redox intermediates of MPO and peroxynitrite. Both iron(III) MPO and iron(II) MPO are rapidly converted to compound II by peroxynitrite in monophasic reactions with calculated rate constants of (6.8+/-0.1) x 10(6) M(-1)s(-1) and (1.3+/-0.2) x 10(6) M(-1)s(-1), respectively (pH 7.0 and 25 degrees C). Besides these one- and two-electron reduction reactions of peroxynitrite, which produce nitrogen dioxide and nitrite, a one-electron oxidation to the oxoperoxonitrogen radical must occur in the fast monophasic transition of compound I to compound II mediated by peroxynitrite at pH 7.0 [(7.6+/-0.1) x 10(6) M(-1)s(-1)]. In addition, peroxynitrite induced a steady-state transition from compound III to compound II with a rate of (1.0+/-0.3) x 10(4) M(-1)s(-1). Thus, the interconversion among the various oxidation states of MPO that is prompted by peroxynitrite is remarkable. Reaction mechanisms are proposed and the physiological relevance is discussed.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid

Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k = 155 M(-1) s(-1) (T = 37 degrees C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k(U(rate))(/plasma) = 0.05 s(-1) (T = 37 degrees C, pH 7.4; assuming [uric acid](plasma) = 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO(2) is one of the fastest with a pseudo-first-order rate constant k(CO(2))(/plasma) = 46 s(-1) (T = 37 degrees C, pH 7.4; assuming [CO(2)](plasma) = 1 mM). Thus peroxynitrite reacts with CO(2) in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO(2). The therapeutic effects of uric acid may be related to the scavenging of the radicals CO(*-)(3) and NO(*)(2) that are formed from the reaction of peroxynitrite with CO(2). We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO(2) may represent a new and viable approach for ameliorating the adverse effects associated with peroxynitrite in many diseases.     

Permeation of human plasma lipoproteins in human carotid endarterectomy tissues: measurement by optical coherence tomography

Atherosclerosis is an inflammatory process occurring in arterial tissue, involving the subintimal accumulation of LDL. Measurement of the rate at which LDL and other lipoproteins, such as HDL and VLDL, enter and exit the tissue can provide insight into the mechanisms involved in the development of atherosclerotic lesions. Permeation of VLDL, LDL, HDL, and glucose was measured for both normal and atherosclerotic human carotid endarterectomy tissues (CEA) at 20°C and 37°C using optical coherence tomography (OCT). The rates for LDL permeation through normal CEA tissue were (3.16 ± 0.37) × 10(-5) cm/s at 20°C and (4.77 ± 0.48) × 10(-5) cm/s at 37°C, significantly greater (P < 0.05) than the rates for atherosclerotic CEA tissue at these temperatures [(1.97 ± 0.34) × 10(-5) cm/s at 20°C and (2.01 ± 0.23) × 10(-5) cm/s at 37°C]. This study effectively used OCT to measure the rates at which naturally occurring lipoproteins enter both normal and diseased carotid intimal tissue.     

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.     

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.     

Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid

Alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) may have a role as antioxidants against nitric oxide-derived oxidants. We previously reported that peroxynitrite reacts with LA and DHLA with second-order rate constants of 1400 and 500 M(-1) s(-1), respectively, but indicated that these direct reactions are not fast enough to protect against peroxynitrite-mediated damage in vivo. Moreover, the mechanism of the reaction of peroxynitrite with LA has been recently challenged (J. Biol. Chem.279:9693-9697; 2004). Pulse radiolysis studies indicate that LA and DHLA react with peroxynitrite-derived nitrogen dioxide (*NO2) (k2 = 1.3 x 10(6) and 2.9 x 10(7) M(-1) s(-1), respectively) and carbonate radicals (CO(3-)) (k2 = 1.6 x 10(9) and 1.7 x 10(8) M(-1) s(-1), respectively). Carbonate radical-mediated oxidation of LA led to the formation of the potent one-electron oxidant LA radical cation. LA inhibited peroxynitrite-mediated nitration of tyrosine and of a hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE), incorporated into liposomes but enhanced tyrosine dimerization. Moreover, while LA competitively inhibited the direct oxidation of glutathione by peroxynitrite, it was poorly effective against the radical-mediated thiol oxidation. The mechanisms of reaction defined herein allow to rationalize the biochemistry of peroxynitrite based on direct and free radical-mediated processes and contribute to the understanding of the antioxidant actions of LA and DHLA.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Diffusion of peroxynitrite in the presence of carbon dioxide.

Peroxynitrite, the reactive species formed in vivo by the reaction of nitric oxide with superoxide anion, is capable of diffusing across erythrocyte membranes via anion channels and passive diffusion (A. Denicola, J. M. Souza, and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566-3571, 1998). However, peroxynitrite diffusion could be limited by extracellular targets, with the reaction with CO(2) (k(2) = 4.6 x 10(4) at 37 degrees C and pH 7.4) the most relevant. Herein, we studied the influence of physiological concentrations of CO(2) on peroxynitrite diffusion across intact red blood cells. The presence of CO(2) inhibited the oxidation of intracellular oxyhemoglobin by externally added peroxynitrite. However, the inhibition by CO(2) decreased at increasing red blood cell densities. At 45% hematocrit, 1.3 mM CO(2) (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25 degrees C) only inhibited 30% of intracellular oxyhemoglobin oxidation. This partial inhibition was also observed in red blood cells pretreated with the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ruling out a competition between peroxynitrite and bicarbonate for the transport through the anion channel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracellular oxyhemoglobin, at different red blood cell densities, and in the presence or absence of CO(2). The theoretical model correlated well with the experimental data. Our results indicate that, even in the presence of CO(2), peroxynitrite is able to diffuse and reach the inside of the erythrocyte.     

Carbon dioxide stimulates the production of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite

Reaction of peroxynitrite with the biological ubiquitous CO(2) produces about 35% yields of two relatively strong one-electron oxidants, CO(3) and ( small middle dot)NO(2), but the remaining of peroxynitrite is isomerized to the innocuous nitrate. Partial oxidant deactivation may confound interpretation of the effects of HCO3-/CO(2) on the oxidation of targets that react with peroxynitrite by both one- and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that HCO3-/CO(2) partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the effects of HCO3-/CO(2) on two- and one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that HCO3-/CO(2) diverts thiol oxidation by peroxynitrite from two- to one-electron mechanisms particularly at neutral pH. At acid pH values, thiol oxidation to free radicals predominates even in the absence of HCO3-/CO(2). In addition to the previously characterized thiyl radicals (RS.), we also characterized radicals derived from them such as the corresponding sulfinyl (RSO.) and disulfide anion radical (RSSR.-) of both GSH and cysteine. Thiyl, RSO. and RSSR.- are reactive radicals that may contribute to the biodamaging and bioregulatory actions of peroxynitrite.     

Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.     

Reaction of phenylaminoethyl selenides with peroxynitrite and hydrogen peroxide

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.     

Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and formation of histidinyl radical

Human recombinant copper-zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC(50)) was approximately 100 microM at 5 microM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 +/- 1.0) x 10(3) M(-1) s(-1) per monomer at pH 7.5 and 37 degrees C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

Oxidation of low-density lipoprotein by iron at lysosomal pH: implications for atherosclerosis

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO(4) (5-50 μM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidation of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.