Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria.

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H(2)O(2) in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H(2)O(2). In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.     

Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: the roles of glutathione and glutathione peroxidase

Three different types of red blood cells (RBC) were used: (i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH and (iii) RBC from high-GSH sheep treated with CDNB to deplete GSH. Incubation of these RBC with t-butyl hydroperoxide (tBHP, 3 mM) for 10 min caused the formation of TBARS, oxidation of haemoglobin and degradation and aggregation of membrane proteins in RBC from low-GSH sheep and GSH-depleted RBC. By contrast, RBC from high-GSH sheep (normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreitol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TBARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted RBC or normal RBCs in the presence of 1.5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degradation and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymatic system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known antioxidants, Butylated hydroxytoluene, ascorbate, alpha-tocopherol and desferrioxamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insult.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.     

NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase

The chloroplast-localized NADPH-dependent thioredoxin reductase (NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding protochlorophyllide formation. A specific involvement of NTRC during biosynthesis of protochlorophyllide is indicated from in vitro aerobic cyclase assays in which the conversion of Mg-protoporhyrin monomethyl ester into protochlorophyllide is stimulated by addition of the NTRC/2-Cys peroxiredoxin system. These findings support the hypothesis that this NADPH-dependent hydrogen peroxide scavenging system is particularly important during periods with limited reducing power from photosynthesis, e.g. under chloroplast biogenesis.     

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.     

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.     

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.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

The oxidation of oxytocin and vasopressin by peroxidase/H2O2 system

Summary Oxytocin and vasopressin are oxidized by horseradish peroxidase and by lactoperoxidase, in the presence of hydrogen peroxide. Spectrophotometric measurements are indicative of the formation of dityrosine. Kinetic parameters indicate that the affinity of horseradish peroxidase is slightly higher for oxytocin with respect to vasopressin and that the two hormones are better substrates for both peroxidases than free tyrosine.     

Signaling the signal, cyclic AMP-dependent protein kinase inhibition by insulin-formed H2O2 and reactivation by thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.     

Metabolism of hydrogen peroxide in isolated hepatocytes: Relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H2O2

The compartmentation of hydrogen peroxide catabolism was studied in isolated hepatocytes. Hydrogen peroxide generation in the peroxisomal compartment was stimulated by addition of glycolate and in the endoplasmic reticular compartment (cytosolic compartment) by ethylmorphine. The rate of catabolism by catalase was estimated from the concentration of methanol required to decrease the steady-state concentration of catalase Compound I to the half-maximal value. The rate of catabolism by glutathione peroxidase was assessed in a semiquantitative manner by the rate of GSSG efflux. The relationship of GSSG efflux to catalase-dependent metabolism of H₂O₂ in the presence of increasing concentrations of glycolate was sigmoidal. This indicates that the function of glutathione peroxidase is small relative to that of catalase at low rates of H₂O₂ production in the peroxisomal fraction, but that the contribution of the former system increases as the peroxisomal H₂O₂ production rate is enhanced, and suggests that the accumulation of a steady-state concentration of H₂O₂ in the nanomolar range in the peroxisomes is sufficient to allow diffusion of H₂O₂ into the cytosol. Following pretreatment of animals with aminotriazole to inhibit catalase, glycolate caused GSSG release at rates nearly double those in control cells. This indicates that even incomplete inhibition of catalase in cells can result in enhanced release of H₂O₂ into the cytosol and demonstrates the relationship of GSSG release to H₂O₂ production under these conditions. An estimate of the rate of H₂O₂ diffusion to catalase during ethylmorphine metabolism was made from the steady-state level of Compound I and measured formate concentrations. This rate increased threefold as the rate of GSH loss increased from 1 to 2 nmol/10⁶ cells per min, indicating that as the rate of H₂O₂ production in the endoplasmic reticulum becomes maximally stimulated in the presence of ethylmorphine, the rate of H₂O₂ metabolism by catalase becomes larger. A comparison of ethylmorphine-stimulated rates of GSSG efflux from cells of control and aminotriazole-treated rats shows that, unlike experiments with glycolate, no difference in the rate of efflux is observed. These results support the conclusion that in hepatocytes catalase has a relatively minor role in catabolism of H₂O₂ at low rates of H₂O₂ generation in the endoplasmic reticulum, but that the catalase function increases as the rate of H₂O₂ production is enhanced.     

Spectral studies on the oxidation of organic sulfides (thioanisoles) by horseradish peroxidase compounds I and II

Using both rapid-scan and conventional spectrophotometry, oxygenation of p-substituted thioanisoles by horseradish peroxidase compounds I and II was investigated at pH 5, 7 and 9. The pH-jump technique was applied to the compound II reactions at acidic and neutral pH. The rate of oxidation of the sulfides is dependent on pH, concentration of substrate and on the different substituents in the para position of the benzene ring. Our results, based on transient state observations of the enzyme intermediates, are in agreement with the results of Kobayashi, S., Minoru, N., Kimura, T. and Schaap, A.P. (Biochemistry (1987) 26, 5019-5022), obtained using 18O-labelling and studies of product formation, in which formation of a sulfur cation radical from compound I is proposed. We consider two reaction mechanisms for the compound II reaction: one a one-electron oxidation of the thioanisole, analogous to the compound I reaction, and the other, the attack of the hydroxyl radical originating from compound II on the sulfur-cation radical.     

A novel and efficient polymerization of lignosulfonates by horseradish peroxidase/H2O2 incubation

Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H₂O₂. Gel permeation chromatography showed a significant increase in weight-average molecular weight (M _w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry (¹H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M _w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4’ type, as the ¹H-NMR spectra indicated. Moreover, the HRP/H₂O₂ incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H₂O₂ incubation is a promising approach for industrial applications of LSs.     

The histones H2A/H2B and H3/H4 are imported into the yeast nucleus by different mechanisms

Proteins are imported from the cytoplasm into the nucleus by importin beta-related transport receptors. The yeast Saccharomyces cerevisiae contains ten of these importins, but only two of them are essential. After transfer through the nuclear pore, importins release their cargo upon binding to the Ran GTPase, the key regulator of nuclear transport. We investigated the import of the core histones in yeast and found that four importins are involved. The essential Pse1p and the nonessential importins Kap114p, Kap104p, and Yrb4p/Kap123p specifically bind to histones H2A and H2B. Release of H2 histones from importins requires Ran-GTP and DNA simultaneously suggesting a function of the importins in intranuclear targeting. H3 and H4 associate mainly with Pse1p and the dissociation requires Ran but not DNA, which points to a different import mechanism. Import of green fluorescent protein fusions to H2A and H2B requires primarily Pse1p and Kap114p, whereas Yrb4p plays an auxiliary role. Pse1p is predominantly necessary for nuclear uptake of H3 and H4, while Kap104p and Yrb4p also support import. We conclude from our in vivo and in vitro experiments that import of the essential histones is mediated mainly by the essential importin Pse1p, while the non-essential Kap114p functions in a parallel import pathway for H2A and H2B.