Acid-derived Hydroperoxides Research Articles

Side-by-side comparison of recombinant human glutathione peroxidases identifies overlapping substrate specificities for soluble hydroperoxides

Five out of eight human glutathione peroxidases (GPXs) are selenoproteins, representing proteins that contain selenium as part of the amino acid selenocysteine. The GPXs are important for reducing hydroperoxides in a glutathione-consuming manner and thus regulate cellular redox homeostasis. GPX1, GPX2, and GPX4 represent the three main cytosolic GPXs, but they differ in their expression patterns with GPX1 and GPX4 being expressed ubiquitously, whereas GPX2 is mainly expressed in epithelial cells. GPX1 and GPX2 have been described to reduce soluble hydroperoxides, while GPX4 reduces complex lipid hydroperoxides, thus protecting cells from lipid peroxidation and ferroptosis. But most of these data are derived from cells that are devoid of one of the isoforms and thus, compensation or other cellular effects might affect the conclusions. So far, the use of isolated recombinant human selenoprotein glutathione peroxidases in pure enzyme assays has not been employed to study their substrate specificities side by side. Using recombinant GPX1, GPX2, and GPX4 produced in E. coli we here assessed their GPX activities by a NADPH-consuming glutathione reductase-coupled assay with 17 different peroxides (all at 50 μM) as substrates. GPX4 was clearly the only isoform able to reduce phosphatidylcholine hydroperoxide. In contrast, small soluble hydroperoxides such as H2O2, cumene hydroperoxide, and tert-butyl hydroperoxide were reduced by all three isoforms, but with approximately 10-fold higher efficiency for GPX1 in comparison to GPX2 and GPX4. Also, several fatty acid-derived hydroperoxides were reduced by all three isoforms and again GPX1 had the highest activity. Interestingly, the stereoisomerism of the fatty acid-derived hydroperoxides clearly affected the activity of the GPX enzymes. Overall, distinct substrate specificity is obvious for GPX4, but not so when comparing GPX1 and GPX2. Clearly GPX1 was the most potent isoform of the three GPXs in terms of turnover in reduction of soluble and fatty-acid derived hydroperoxides.

Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H2O2) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H2O2, have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H2O2, cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H2O2 via its enzymatic conversion to an amino acid-derived hydroperoxide that ‘escapes’ effective reductive inactivation to engage in selective oxidative activation of key target proteins.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an aminoacid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

41 - Lipid Hydroperoxides as Mediators of Ozone-Induced Oxidative Changes in Human Airway Epithelial Cells

Human exposure to tropospheric ozone (O3) is a significant public health concern. O3 inhalation induces airway inflammation and pulmonary function decrements, and may also lead to cardiovascular morbidity. The carbon-carbon double bonds in polyunsaturated fatty acids are primary targets for oxidation by O3, leading to formation of free radicals, hydrogen peroxide (H2O2), and lipid hydroperoxides. However, the role of these intermediates in mediating downstream cellular responses to O3 inhalation is not well understood. Previous studies have observed an O3-induced increase in the glutathione redox potential (EGSH) in human airway epithelial cells (HAEC). Impairment of mitochondrial respiration has also been previously observed in mice and rats exposed to O3. We hypothesized that lipid hydroperoxides contribute to these O3-induced oxidative changes in HAEC. In the present study we monitored the EGSH using the fluorogenic sensor roGFP and mitochondrial respiration with the Seahorse Extracellular Flux Analyzer in BEAS-2B cells exposed to lipid hydroperoxides. Overexpression of catalase was used to evaluate the role of H2O2 in the observed hydroperoxide-induced changes. We found that exposure to the linoleic acid-derived hydroperoxide 9-HpODE induces dose-dependent increases in EGSH that are independent of H2O2 production. Exposure to 9-HpODE also caused a decrease in mitochondrial basal oxygen consumption, maximal respiration, and spare respiratory capacity. These results suggest that lipid hydroperoxides are primary effectors of oxidative changes induced by O3 inhalation. This abstract of a proposed presentation does not necessarily reflect EPA policy.

Lipoxygenase 2 from Cyanothece sp. controls dioxygen insertion by steric shielding and substrate fixation

The biological function of lipoxygenases depends on the regio and stereo specific formation of fatty acid-derived hydroperoxides and different concepts exist to explain the mechanism that directs dioxygen to a specific carbon atom within the substrate. Here, we report the 1.8 Å resolution crystal structure of a cyanobacterial lipoxygenase that produces bis-allylic hydroperoxides (CspLOX2). Site directed mutagenesis experiments combined with computational approaches reveal that residues around the active site direct dioxygen to a preferred carbon atom and stereo configuration in the substrate fatty acid. Modulating the cavity volume around the pentadiene system of linoleic acid shifted the product formation towards 9S-, 9R-, 13S- or 13R-hydroperoxides in correlation with the site of mutation, thus decreasing the amount of the bis-allylic 11R-hydroperoxide. Decreasing the channel size of a 9R-lipoxygenase (CspLOX1) on the other hand could in turn induce formation of the bis-allylic 11R-hydroperoxide. Together this study suggests that an active site clamp fixing the pentadiene system of the substrate together with steric shielding controls the stereo and regio specific positioning of dioxygen at all positions of the reacting pentadiene system of substrate fatty acids.

Molecular Basis of Hydroperoxide Specificity in Peroxiredoxins: The Case of AhpE from Mycobacterium tuberculosis.

Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzyme's dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.

The cytosolic or the mitochondrial glutathione peroxidase-type tryparedoxin peroxidase is sufficient to protect procyclic Trypanosoma brucei from iron-mediated mitochondrial damage and lysis.

African trypanosomes express three virtually identical glutathione peroxidase (Px)-type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid-derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild-type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I-III-deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch-deferoxamine is inefficient. In glucose-rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px-type peroxidases protect procyclic cells from an iron-mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.

Efficient and Specific Conversion of 9‐Lipoxygenase Hydroperoxides in the Beetroot. Formation of Pinellic Acid

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~103 times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pKa reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~108 and 105M−1s−1, respectively, at pH 7.4 and 25°C) were much faster than expected according to the Brønsted relationship with leaving-group pKa. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.

Natural mechanisms for cereal resistance to the accumulation of Fusarium trichothecenes

This review describes the naturally occurring mechanisms in cereals that lead to a reduction of Fusarium trichothecene mycotoxin accumulation in grains. A reduction in mycotoxin contamination in grains could also limit fungal infection, as trichothecenes have been reported to act as virulence factors. The mechanisms explaining the low toxin accumulation trait, generally referred to as type V resistance to Fusarium, can be subdivided into two classes. Class 1 includes mechanisms by which the plants chemically transform the trichothecenes, leading to their degradation or detoxification. Among the detoxification strategies, glycosylation of trichothecenes is a natural process already reported in wheat. According to the structure and the toxicity of trichothecenes, two other detoxification processes, acetylation and de-epoxidation, can be expressed, at least in transgenic plants. Class 2 comprises mechanisms that lead to reduced mycotoxin accumulation by inhibition of their biosynthesis through the action of plant endogenous compounds. These include both grain constitutive compounds and compounds induced in response to pathogen infection. There are already many compounds with antioxidant properties, like phenolic compounds, peptides or carotenoids, and with prooxidant properties, like hydrogen peroxide or linoleic acid-derived hydroperoxides, that have been described as ‘modulators’ of mycotoxin biosynthesis. This review addresses for the first time different studies reporting specific in vitro effects of such compounds on the biosynthesis of Fusarium mycotoxins. A better understanding of the natural processes limiting accumulation of trichothecenes in the plant will open the way to the development of novel breeding varieties with reduced ‘mycotoxin risk’.

Hepoxilin A 3 (HXA 3) synthase deficiency is causative of a novel ichthyosis form

Non-bullous congenital ichthyosis erythroderma (NCIE) and lamellar ichthyosis (LI) are characterized by mutations in 12 R-lipoxygenase (12 R-LOX) and/or epidermal lipoxygenase 3 (eLOX3) enzymes. The eLOX3 lacks oxygenase activity, but is capable of forming hepoxilin-type products from arachidonic acid-derived hydroperoxide from 12 R-LOX, termed 12 R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12 R-HpETE). Mutations in either of two enzymes lead to NCIE or LI. Moreover, 12 R-LOX-deficient mice exhibit severe phenotypic water barrier dysfunctions. Here, we demonstrate that 12 R-HpETE can also be transformed to 8 R-HXA 3 by hepoxilin A 3 (HXA 3) synthase (12-lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed hepoxilin A 3 synthase-linked ichthyosis (HXALI), whose scales expressed high levels of 12 R-LOX, but were deficient of HXA 3 synthase.

Synthesis of Six Epoxyketooctadecenoic Acid (EKODE) Isomers, Their Generation from Nonenzymatic Oxidation of Linoleic Acid, and Their Reactivity with Imidazole Nucleophiles

As a class of linoleic acid oxidation products, epoxyketooctadecenoic acids (EKODEs), are formed in vivo and in vitro by a free radical mechanism initiated by either enzymatic or nonenzymatic pathways. They have so far been made available in small-scale quantities, often as isomeric mixtures, from reductive decomposition of linoleic acid-derived hydroperoxides. There is major interest in these compounds owing to their highly potent biological activities and their ability to covalently modify proteins. The synthesis of six EKODE regio- and stereoisomers, two trans alpha',beta'-epoxy-alpha,beta-enones, and two trans and the two cis gamma,delta,-epoxy-alpha,beta-enones was accomplished, with the key steps being Wittig-type reactions and aldol condensations. All six EKODE isomers were confirmed by HPLC to be generated in the autoxidation of linoleic acid promoted by Fe(II)/ascorbic acid through spiking in of authentic samples. On the basis of evidence for EKODE modification of protein His residues, the reactions of Nalpha-benzoyl-L-histidine with autoxidizing linoleic acid and with the individual EKODE isomers were compared, as were the kinetics of the various EKODE reactions with imidazole nucleophiles. The structures of His-EKODE-(E)-I adducts were confirmed to reflect conjugate addition (epoxide ring remains intact) through an NMR study of the reaction of imidazole with a generic EKODE-(E)-I analog. The synthesis of the EKODE isomers makes these important molecules available for further chemical and biological evaluation.