GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic reticulum

Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H₂O₂) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H₂O₂ is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H₂O₂ to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H₂O₂-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H₂O₂ production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α–GPx8 complex prevents diffusion of Ero1α-derived H₂O₂ within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.     

Macrokinetics and operational stability of immobilized glucose oxidase and catalase

Glucose oxidation by immobilized glucose oxidase (GlO) and catalase (Cat) has been investigated in batch and continuous reactions for operational studies. The macrokinetics of the process depend on coupled reaction steps and diffusion rates. The problem may be approximated by a simple pseudohomogeneous model taking into account both substrates of glucose oxidase and the intermediate reaction product H₂O₂. The effectiveness of both enzymes is enhanced in the coupled reaction path, the overall effectiveness nevertheless is very low. H₂O₂ causes the inactivation of both GlO and Cat. The rates of deactivation depend on the oxidation rates of glucose that give different quasistationary levels of H₂O₂ concentration. As a first approximation, the deactivation rates may be described by first-order reactions with respect to H₂O₂.     

Mechanistic characterization of the thioredoxin system in the removal of hydrogen peroxide

The thioredoxin system, which consists of a family of proteins, including thioredoxin (Trx), peroxiredoxin (Prx), and thioredoxin reductase (TrxR), plays a critical role in the defense against oxidative stress by removing harmful hydrogen peroxide (H₂O₂). Specifically, Trx donates electrons to Prx to remove H₂O₂ and then TrxR maintains the reduced Trx concentration with NADPH as the cofactor. Despite a great deal of kinetic information gathered on the removal of H₂O₂ by the Trx system from various sources/species, a mechanistic understanding of the associated enzymes is still not available. We address this issue by developing a thermodynamically consistent mathematical model of the Trx system which entails mechanistic details and provides quantitative insights into the kinetics of the TrxR and Prx enzymes. Consistent with experimental studies, the model analyses of the available data show that both enzymes operate by a ping-pong mechanism. The proposed mechanism for TrxR, which incorporates substrate inhibition by NADPH and intermediate protonation states, well describes the available data and accurately predicts the bell-shaped behavior of the effect of pH on the TrxR activity. Most importantly, the model also predicts the inhibitory effects of the reaction products (NADP⁺ and Trx(SH)₂) on the TrxR activity for which suitable experimental data are not available. The model analyses of the available data on the kinetics of Prx from mammalian sources reveal that Prx operates at very low H₂O₂ concentrations compared to their human parasite counterparts. Furthermore, the model is able to predict the dynamic overoxidation of Prx at high H₂O₂ concentrations, consistent with the available data. The integrated Prx–TrxR model simulations well describe the NADPH and H₂O₂ degradation dynamics and also show that the coupling of TrxR- and Prx-dependent reduction of H₂O₂ allowed ultrasensitive changes in the Trx concentration in response to changes in the TrxR concentration at high Prx concentrations. Thus, the model of this sort is very useful for integration into computational H₂O₂ degradation models to identify its role in physiological and pathophysiological functions.     

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes.

Conditions for the recovery of H₂O₂ from microsomes and for determination of the rate and extent of H₂O₂ formation during oxidation of NADPH by liver microsomes have been investigated. H₂O₂ was determined by two methods that are applicable to conditions existing during microsomal mixed function oxidation reactions, provided that contaminating catalase activity is inhibited by azide and that interference by other mixed function oxidation reactions can be excluded. To estimate the formation of H₂O₂ in absence of azide, H₂O₂ was determined indirectly by the production of HCHO during oxidation of cold and ¹⁴C-labeled methanol and an excess of exogenous catalase. As additional catalase-independent decomposition of H₂O₂ also occurs during oxidation of NADPH, the kinetics of H₂O₂ formation in microsomes is influenced by two independent processes. H₂O₂ will be produced under optimal conditions i.e., at V when O₂ and NADPH are in excess. Addition or formation of increasing amounts of H₂O₂ raises the substrate (H₂O₂) concentration and will enhance the rate of breakdown of H₂O₂.     

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H₂O₂) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H₂O₂ sensor that allows H₂O₂ monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H₂O₂ sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC_R to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H₂O₂ produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H₂O₂ levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H₂O₂ levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H₂O₂ biology in plant cells.

The establishment of a hypersensitive H₂O₂ sensor in six major compartments of the Chlamydomonas cell reveals steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments.     

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.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Oxidation of hemoglobin by arenediazonium salts. The influence of dioxygen

The reactions of human hemoglobin with p-nitro- and p-chlorobenzenediazonium tetrafluoroborates in the presence and absence of molecular oxygen have been investigated in kinetic detail. The oxidation of iron(II) occurs with first order rate dependence on both the hemoglobin and diazonium salt concentrations, but inverse first order dependence on the concentration of molecular oxygen characterizes reactions performed in the presence of O₂. In the absence of O₂, nitrobenzene is the only product observed from hemoglobin oxidation by p-NO₂C₆H₄N₂⁺BF₄^?, and a 1:1 stoichiometry exists between nitrobenzene produced and Fe(II) oxidized. In the presence of O₂, p-nitrophenol is the dominant product, but product yield is dependent on the ratio of reactants. Electron transfer to the diazonium salt rather than its corresponding diazohydroxide or diazoate is inferred from the relative absence of pH dependence on the rate of oxidation. The composite results are consistent with a mechanism for hemoglobin oxidation that requires molecular oxygen dissociation from oxyhemoglobin prior to oxidation by the diazonium salt. Implications of this investigation for the mechanism of arylhydrazine reactions with hemoglobin are discussed.     

Hydrogen-peroxide-producing system ofPleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase

The effect of benzyl alcohol, benzaldehyde and benzoic acid on the production of extracellular hydrogen peroxide (H₂O₂) by the ligninolytic fungusPleurotus eryngii was investigated. It was found that an equilibrium between oxidative and reductive reactions of these compounds is established, leading to the continuous production of H₂O₂. A multienzymatic cyclic system is proposed in which H₂O₂ is produced extracellularly by the action of aryl-alcohol oxidase on benzyl alcohol, the most abundant compound after redox reactions, and to a lower extent on benzaldehyde. The oxidation products of these reactions, benzaldehyde and benzoic acid, are reduced by intracellular dehydrogenases.     

Hydrogen Peroxide-mediated Oxidation of Indole-3-acetic Acid by Tomato Peroxidase and Molecular Oxygen

The oxidation of indole-3-acetic acid by anionic tomato peroxidase was found to be negligible unless reaction mixtures were supplemented with H₂O₂. The addition of H₂O₂ to reaction mixtures initiated a period of rapid indole-3-acetic acid oxidation and O₂ uptake; this phase ended and O₂ uptake fell to a low level when the H₂O₂ was exhausted. The stoichiometry of the reaction, which is highly dependent on enzyme concentration and pH, suggests that H₂O₂ initiates a sequence of reactions in which indole-3-acetic acid is oxidized.     

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.     

Cinnamtannin B-1, a natural antioxidant that reduces the effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on CCK-8-evoked responses in mouse pancreatic acinar cells

This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H₂O₂) in mouse pancreatic acinar cells. We have studied Ca²⁺ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H₂O₂. We found that H₂O₂ (0.1–100 μM) increased CM-H₂DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H₂O₂-induced oxidation of CM-H₂DCFDA. CCK-8 induced oxidation of CM-H₂DCFDA in a similar way to low micromolar concentrations of H₂O₂, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H₂O₂ induced a slow and progressive increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Cinnamtannin B-1 reduced the effect of H₂O₂ on [Ca²⁺]_c, but only at the lower concentrations of the oxidant. H₂O₂ inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H₂O₂ on enzyme secretion. Finally, H₂O₂ reduced cell viability, and the antioxidant protected acinar cells against H₂O₂. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca²⁺ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.     

Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: Physiological significance of the oxidation reactions

Phenolic components and peroxidases are localized in vacuoles. Vacuolar peroxidase can oxidize phenolics when H₂O₂ is formed in vacuoles or tonoplasts, or when H₂O₂ formed outside of vacuoles is diffused into the organelles. In a mixture of phenolics containing a good and a poor substrate for peroxidase, a radical transfer reaction is possible from the radicals of the good substrate to the poor substrate, resulting in the enhancement of oxidation of the poor substrate. Phenoxyl radicals formed by peroxidase-dependent reactions are reduced by ascorbate in vacuoles. So, as long as ascorbate is present in vacuoles, the accumulation of oxidation products of phenolics is not significant. This suggests that ascorbate/phenolics/peroxidase systems in the vacuoles can scavenge H₂O₂. During aging, some phenolics are accumulated in vacuoles and the apoplast, and the accumulated phenolics are oxidized to brown components by peroxidase-dependent reactions. The brown components can produced O₂ ^? and H₂O₂ by autooxidation. The significance and the mechanisms of browning are discussed in tobacco leaves and onion scales.

     

Diffusion effects in the metabolism of hydrogen peroxide by rat liver peroxisomes.

Rat liver peroxisomes are membrane-bounded organelles containing catalase and oxidases producing H₂O₂. Diffusion effects in the metabolism of H₂O₂ and the physiological significance of the structure of peroxisomes are explored on the basis of two models. Model I considers the liver cell as consisting of two rapidly mixed compartments, the peroxisomal contents and the rest of the cell, separated by a membrane. On the basis of model I, it is concluded that in order to maintain a minimal H₂O₂ concentration in the cytoplasm, there must be an H₂O₂ destroying system in the cytoplasm, but the capacity of this system need be only a small fraction of that of the catalase in the peroxisomes. Model II takes account of the detailed morphology of peroxisomes and includes the effect of peroxisomal membrane permeability to H₂O₂ and H₂O₂ diffusion inside and outside the peroxisomes. On the basis of previously published experimental data and model II, it is concluded that the latency of catalase activity in intact peroxisomes is due chiefly to a permeability barrier to H₂O₂ at the peroxisomal membrane rather than to a restriction of H₂O₂ diffusion within the peroxisomes. Peroxisomes are calculated to be very efficient at destroying the H₂O₂ produced within them, whether the H₂O₂ is produced in the catalase-free core or in the catalase-containing matrix. Less than 2% of the H₂O₂ produced in peroxisomes leaves the particles. The efficiency of H₂O₂ trapping is the consequence of the membrane permeability barrier. A similar H₂O₂ trapping efficiency could be achieved by particles without a membrane barrier only if H₂O₂ diffusion within such particles were reduced by many orders of magnitude.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Electron acceptor function of O2 in radical N-demethylation reactions catalyzed by hemeproteins

In aerobic solutions, O₂ consumption correlated well with N-demethylation of N,N-dimethyl-$\text{p}$-toluidine catalyzed by horseradish peroxidase, in the presence or absence of H₂O₂. In the absence of added H₂O₂, superoxide dismutase stimulated, and catalase inhibited, both reactions; in the presence of H₂O₂, argon inhibition of formaldehyde production increased with increasing concentration of horseradish peroxidase. These results provide evidence for competing reactions of the enzymatically-generated substrate radical: oxidation by O₂ increases formaldehyde production, while radical dimerization decreases the yield of this product. Implications of these findings for similar reactions catalyzed by microsomal cytochrome P-450 are suggested.     

Lipoperoxide radical production during oxidation of cardiolipin in the complex with cytochrome c

The key stage of apoptosis is lipid peroxidation which causes cytochrome c efflux from mitochondria. Cardiolipin-bound cytochrome c on the surface of the inner mitochondrial membrane is supposed to be a main lipoperoxidation catalyst. In this work, lipoperoxide radical (LOO·) production in the complex of cytochrome c (Cyt C) with bovine heart cardiolipin (BCL) was investigated with the method of chemiluminescence (CL) in the presence of a physical activator, coumarin dye C-525. It was shown that a CL flash with a half quenching time of 1.12 min was observed after the addition of Cyt C to a BCL+C-525 solution in the absence of hydrogen peroxide. At H₂O₂ concentrations of 0.1–0.5 mM, quenching time reduced at constant CL flash amplitude and at H₂O₂ concentrations of 1–5 mM, the amplitude of CL increased with the growth of peroxide concentration. It testifies to different mechanisms of BCL oxidation: the lipoxygenase mechanism in the absence of H₂O₂ and at low H₂O₂ concentrations, and the peroxidase mechanism at higher H₂O₂ concentrations. When small H₂O₂ amounts were added, another CL flash was observed in the course of a lipoxygenase reaction whose light sum increased with time in parallel with the extent of the following inhibition of CL. Iron chelators EDTA and o-phenanthroline made no significant effect on the CL associated with cytochrome c lipoxygenase action, while desferal, a well-known peroxidase and lipoxygenase inhibitor, inhibited CL by half in a concentration of 18 μM. A scheme of reactions resulting in LOO· radical production on BCL oxidation by the Cyt C-cardiolipin complex in the absence and in the presence of H₂O₂ was suggested.     

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

Identification of the <Emphasis Type="Italic">Vibrio vulnificus ahpC</Emphasis>l gene and its influence on survival under oxidative stress and virulence

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H₂O₂. In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H₂O₂ and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H₂O₂ in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.