Peroxidase Cycle Research Articles

Delineating drought-induced antioxidative traits as potential mechanisms for climate resilience in Argania spinosa

Drought stress ranks among the most critical environmental challenges facing agriculture today, causing significant impairments to plant growth and development. In Morocco, these negative impacts are projected to intensify further under the combined pressures of climate change and the worsening water shortage crisis. The imperative need for sustainable arganiculture (cultivation of Argane trees) in dry lands necessitates the expeditious identification of drought-tolerant elite Argania spinosa trees. In this context, we investigated the drought tolerance of 2-year-old A. spinosa seedlings from two contrasting provenances Lakhssas (LKS) and Aoulouz (ALZ) under severe stress by evaluating their antioxidative defense mechanisms. A total of 24 parameters related to reactive oxygen species (ROS), oxidative damage, and enzymatic and non-enzymatic antioxidant defense were measured and compared between both provenances. The results showed that the drought-stressed conditions significantly increased the activity of enzymatic antioxidants, including superoxide dismutase, catalase, peroxidase, polyphenoloxidase, glutathione peroxidase, glutathione S-transferase, and ascorbate-glutathione cycle enzymes, as well as the content of non-enzymatic antioxidants, including reduced glutathione (GSH), ascorbic acid (AsA), α-Tocopherols (α-toc), polyphenols, anthocyanins, and group thiols. The ability of A. spinosa trees to greatly enhance their antioxidant system to limit cellular damage caused by ROS production might be an important attribute linked to drought tolerance. Regarding the inter-provenance variation under drought stress conditions, LKS provenance exhibited superior antioxidative capacity through enhanced AsA-GSH cycle activity and elevated levels of AsA-GSH, α-toc, and polyphenols. However, ALZ demonstrated elevated anthocyanin levels and reduced peroxidative stress markers (hydrogen peroxide and malonyldialdehyde). Significant and positive correlations were recorded between studied ROS and antioxidants. Investigating the multifaceted antioxidative defense system underpinning drought tolerance in A. spinosa can facilitate the identification of drought-tolerant argane trees for the development of a future breeding program allowing sustainable arganiculture in dry lands.

Rational Design of Mono-Substituted Gd-DOTA as Highly Stable and Efficient MRI Contrast Agents for Hepatobiliary and Inflammation Imaging.

Hepatobiliary-specific magnetic resonance imaging contrast agents (MRI CAs) play a crucial role in the early diagnosis of hepatocellular carcinoma (HCC). However, only two acyclic CAs, Gd-BOPTA and Gd-EOB-DTPA, exhibit unfavorable kinetic inertness. Our study focused on the development of superior stable innovative macrocyclic CAs. By introducing a lipophilic benzyloxy group (OBn) into the H4DOTA ring (Gd-L1), we achieved significant enhancement in kinetic inertness. In vivo experiments in mice demonstrated that 40% of the dosage was distributed to the liver at 5 min, providing sustained hepatic enhancement for over 35 min. We also developed an MPO-responsive MRI CA (Gd-L3), which can participate in the "peroxidase cycle" as the substrate, generating oligomers with a 3.8-fold increase in relaxivity, and selectively enhance the lesion in an acute gout mouse model. Overall, our work represents a significant advancement in the field of hepatic and inflammatory MRI, offering promising avenues for early diagnosis and improved imaging outcomes.

Phytic acid-mediated enhancement of Meyerozyma caribbica biocontrol of Aspergillus carbonarius infection in grape berries through regulation of ROS metabolism

Sour rot, an infection caused by Aspergillus carbonarius is one of the detrimental diseases that damage grape vines after harvest. To investigate the biocontrol of this mycotoxigenic fungus, the current study explored the efficiency of Meyerozyma caribbica pretreated with phytic acid (PA) in reducing sour rot in table grapes through the regulation of reactive oxygen species (ROS) production. The content of hydrogen peroxide (H2O2,), superoxide (O2.−) production rate, and the degree of lipid peroxidation were measured. Furthermore, antioxidant enzymes activities, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and enzymes of the ascorbate–glutathione (AsA-GSH) and glutathione peroxidase (GPX) cycles were investigated. Enzymatic activity results of select genes were confirmed using real-time quantitative polymerase chain reaction (RT-qPCR). Meyerozyma caribbica pretreated with 10 μmol/mL PA (YE) was most efficient in reducing sour decay in grape berries as indicated by an 81.1 % reduction in disease incidence and 1.5 mm lesion diameter by the third day. Phytic acid treated M. caribbica showed a significant reduction in the contents of malondialdehyde (MDA), H2O2, and production rate of O2.− which was complemented by a marked rise in ROS scavenging enzymes’ activities. The H2O2 content in YE was the lowest (24.09 nmol/g FW) by the third day and O2.− production rate was about half that of CK; control (4.19 mmol/min.g FW) on the same day. The SOD, POD, and CAT activities were significantly higher in YE treated fruits (6.63, 18, and 6.64 U/mg FW, respectively) compared to the CK by the fifth day. Subsequently, AsA-GSH and GPX cycles also showed higher activity in grapes inoculated with YE compared to other treatments. Genes encoding SOD, POD, CAT, DHAR, GR, and APX were significantly overexpressed in grapes inoculated with YE compared to the CK, yeast alone, and PA treatments. These findings indicate that PA likely enhances the biocontrol efficacy of M. caribbica, leading to the induction of ROS scavenging mechanisms and subsequent mortality of A. carbonarius in table grapes. Consequently, M. caribbica pre-treated with PA holds promise as a potential biological control agent for A. carbonarius in future applications

The biocontrol potentiality of Bacillus amyloliquefaciens against postharvest soft rot of tomatoes and insights into the underlying mechanisms

Tomatoes can easily be affected by soft rot, one of the main postharvest infections caused by a bacterium called Pectobacterium carotovorum. This study examined the soft rot control effectiveness by Bacillus amyloliquefaciens in tomatoes, and identify the underlying action mechanisms from the perspective of the inhibition effects of antibacterial metabolites derived from B. amyloliquefaciens on pathogen and the influences of this antagonist on reactive oxygen species (ROS) scavenging capability of the fruit. The results demonstrated that B. amyloliquefaciens effectively lessened postharvest soft rot occurrence in tomatoes. The non-volatile metabolites synthesized by B. amyloliquefaciens suppressed P. carotovorum subsp. brasiliense (Pcb) growth in vitro and in vivo. The antibacterial ingredients presented in methanol-soluble sediments obtained from non-volatile metabolites. UHPLC-ESI-MS spectra revealed that the primary antibacterial ingredients were C12-C15 surfactin analogues, along with C14-C16 iturins and C16-C17 fengycins. Moreover, B. amyloliquefaciens could raise the activity of enzymatic ROS scavenging system in tomatoes, including several antioxidases and the enzymes related to glutathione peroxidase (GPX) and ascorbate-glutathione (AsA-GSH) cycles. In addition, B. amyloliquefaciens increased the levels of non-enzymatic system for ROS scavenging, including AsA and GSH. Together, O2-·production rate, H2O2 and malondialdehyde (MDA) levels were reduced. It suggested that B. amyloliquefaciens enhanced the ROS scavenging ability of tomatoes, thereby improving their resistance to the pathogen.

Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase-Insights into the Catalytic Cycle of Bacterial Peroxidases.

Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

Transcriptomics-based analysis of genes related to lead stress and their expression in the roots of Pogonatherum crinitum.

Revealing plants' tolerance and transport genes to heavy metal stress play an important role in exploring the potential of phytoremediation. Taking the heavy metal lead (Pb) hyperaccumulator plant Pogonatherum crinitum (Thunb.) Kunth as the research object, a hydroponic simulation stress experiment was set up to determine the physiological indicators such as antioxidant enzymes and non-enzymatic antioxidants in the roots of P. crinitum under different Pb concentrations (0, 300, 500, 1000, 2000 mg·L-1). RNA-Seq was performed, the Unigenes obtained by transcriptome sequencing were enriched and annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the differential expression genes (DEGs) of root were screened and verified by quantitative real-time polymerase chain reaction (qRT-PCR). The results are as follows: with the increase of Pb concentration, superoxide dismutase (SOD), catalase (CAT), and ascorbic acid (AsA) content increased. Peroxidase (POD), malondialdehyde (MDA), and ascorbic acid-glutathione (AsA-GSH) cycles showed low promotion with high inhibition. A total of 38.21 Gb of bases were obtained by transcriptome sequencing, and the base quality of each sample reached Q20 and Q30, accounting for 90%, making the sequencing results reliable. Combined with transcriptome sequencing, functional annotation, and qRT-PCR validation results, 17 root Pb-tolerant genes of P. crinitum were screened out, which were related to antioxidation, transportation, and transcription functions. Moreover, qRT-PCR verification results under different Pb stress concentrations were consistent with the transcriptome sequencing results and changes in physiological indicators. In brief, the root of P. crinitum can adapt to the Pb stress environment by up-regulating the expression of related genes to regulate the physiological characteristics.

Silicon nanoparticles in higher plants: Uptake, action, stress tolerance, and crosstalk with phytohormones, antioxidants, and other signalling molecules

Silicon is absorbed as uncharged mono-silicic acid by plant roots through passive absorption of Lsi1, an influx transporter belonging to the aquaporin protein family. Lsi2 then actively effluxes silicon from root cells towards the xylem from where it is exported by Lsi6 for silicon distribution and accumulation to other parts. Recently, it was proposed that silicon nanoparticles (SiNPs) might share a similar route for their uptake and transport. SiNPs then initiate a cascade of morphophysiological adjustments that improve the plant physiology through regulating the expression of many photosynthetic genes and proteins along with photosystem I (PSI) and PSII assemblies. Subsequent improvement in photosynthetic performance and stomatal behaviour correspond to higher growth, development, and productivity. On many occasions, SiNPs have demonstrated a protective role during stressful environments by improving plant-water status, source-sink potential, reactive oxygen species (ROS) metabolism, and enzymatic profile. The present review comprehensively discusses the crop improvement potential of SiNPs stretching their role during optimal and abiotic stress conditions including salinity, drought, temperature, heavy metals, and ultraviolet (UV) radiation. Moreover, in the later section of this review, we offered the understanding that most of these upgrades can be explained by SiNPs intricate correspondence with phytohormones, antioxidants, and signalling molecules. SiNPs can modulate the endogenous phytohormones level such as abscisic acid (ABA), auxins (IAAs), cytokinins (CKs), ethylene (ET), gibberellins (GAs), and jasmonic acid (JA). Altered phytohormones level affects plant growth, development, and productivity at various organ and tissue levels. Similarly, SiNPs regulate the activities of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and ascorbate-glutathione (AsA-GSH) cycle leading to an upgraded defence system. At the cellular and subcellular levels, SiNPs crosstalk with various signalling molecules such as Ca2+, K+, Na+, nitric oxide (NO), ROS, soluble sugars, and transcription factors (TFs) was also explained.

Controlling black spot of postharvest broccoli by Meyerozyma guilliermondii and its regulation on ROS metabolism of broccoli

Black spot caused by Alternaria brassicicola is one of the most common fungal diseases of postharvest broccoli. The present study investigated the biocontrol efficacy of Meyerozyma guilliermondii against black spot of postharvest broccoli and the mechanisms involved in the enhanced disease resistance of broccoli based on its effects on reactive oxygen species (ROS) metabolism. Our investigation proved that M. guilliermondii could decrease the disease index of postharvest broccoli caused by A. brassicicola. This antagonistic yeast could potentially increase peroxidase, catalase and superoxide dismutase activities, and the activities of enzymes involved in ascorbate–glutathione cycle and glutathione peroxidase cycle, such as ascorbate peroxidase, glutathione peroxidase and glutathione reductase. Furthermore, the contents of non-enzymatic antioxidants, including ascorbic acid and glutathione, were enhanced by this yeast. Accordingly, the accumulation of ROS, including hydrogen peroxide (H2O2) and superoxide anion (O2–), was reduced by M. guilliermondii, as well as malondialdehyde amount which was one of the membrane lipid peroxides. Our results suggested that M. guilliermondii could improve the activities of ROS scavenging enzymatic system and the levels of antioxidant substances to scavenge excessive ROS, and then protect cells from oxidative damage, which enhanced the disease resistance of broccoli to pathogens. Overall, our study provides a new disease control strategy for postharvest broccoli by improving the ability of ROS scavenging to resist pathogens.

Pichia caribbica improves disease resistance of cherry tomatoes by regulating ROS metabolism

Black spot caused by Alternaria alternata is one of major diseases of postharvest cherry tomatoes. This study intends to investigate biocontrol efficacy of Pichia caribbica against black spot and explore the mechanisms involved in improved disease resistance of cherry tomatoes through analyzing reactive oxygen species (ROS) metabolism. The results demonstrated that P. caribbica significantly decreased the occurrence of black spot in cherry tomatoes. P. caribbica improved the activities of enzymatic system for ROS scavenging in cherry tomatoes, including some antioxidant enzymes, the enzymes in ascorbic acid-glutathione (AsA-GSH) and glutathione peroxidase (GPX) cycles, and glutathione S-transferase (GST). Furthermore, P. caribbica maintained the high level of the key antioxidant substances and then decreased the production rate of O2¯·, the contents of H2O2 and malondialdehyde (MDA). These results revealed that P. caribbica induced the enzymatic and non-enzymatic systems to remove ROS, and then improved the disease resistance of cherry tomatoes against pathogen. This study will provide new strategy for controlling postharvest disease of fruits and vegetables by improving the ability of ROS scavenging to defense against pathogens.

Propofol inhibits the myeloperoxidase activity by acting as substrate through a redox process

BackgroundPropofol (2,6-diisopropylphenol) is frequently used as intravenous anesthetic agent, especially in its injectable form (Diprivan), to initiate and maintain sedative state during surgery or in intensive care units. Numerous studies have reported the antioxidant and anti-inflammatory effect of propofol. The oxidant enzyme myeloperoxidase (MPO), released from activated neutrophils, plays a key role in host defense. An increase of the circulating MPO concentration has been observed in patients admitted in intensive care unit and presenting a systemic inflammatory response related to septic shock or trauma. MethodsThis study investigates the immunomodulatory action of propofol and Diprivan as inhibitor of the oxidant activity of MPO. The understanding of the redox action mechanism of propofol and Diprivan on the myeloperoxidase chlorination and peroxidase activities has been refined using the combination of fluorescence and absorption spectroscopies with docking and cyclic voltammetry. ResultsPropofol acts as a reversible MPO inhibitor. The molecule interacts as a reducing substrate in the peroxidase cycle and promotes the accumulation of compound II. At acidic pH (5.5), propofol and Diprivan do not inhibit the chlorination activity, but their action increases at physiological pH (7.4). The main inhibitory action of Diprivan could be attributed to its HOCl scavenging property. General significancePropofol can act as a reversible MPO inhibitor at clinical concentrations. This property could, in addition to other previously proven anti-inflammatory actions, induce an immunomodulatory action, beneficial during clinical use, particularly in the treatment of systemic inflammation response syndrome.

Hemoglobins from Scapharca subcrenata (Bivalvia: Arcidae) likely play an bactericidal role through their peroxidase activity.

Hemoglobin (Hb) is an iron-containing respiratory protein present in all vertebrates and some invertebrates. The blood clam Scapharca subcrenata is one of the few invertebrates that have Hb-containing red hemocytes. In this study, we purified Hb (Ss-Hb), including Ss-HbI and Ss-HbII, from S. subcrenata hemocytes using gel chromatography with a recovery rate of 70.71%, and then characterized their peroxidase activities. Both Ss-Hbs possessed peroxidase activity with high affinity to the substrates guaiacol and H2O2. Moreover, both Ss-Hbs had structural similarities, such as type b heme, proximal histidine (His), distal His, and heme pocket arginine (Arg), with other peroxidases. The optimal peroxidase activity of both Ss-Hbs was at pH5 and 35°C, but this was inhibited in the presence of Cu2+ and Fe2+. Ss-Hbs produced [Formula: see text] in the presence of H2O2. β-phenylethylamine, a substrate of peroxidase, increased the [Formula: see text] generation, while Cu2+, an inhibitor of peroxidase, inhibited this reaction. These results indicated that the peroxidase cycle of Ss-Hb was involved in the production of [Formula: see text] . A large amount of [Formula: see text] may be generated by the peroxidase cycle if the substrate is sufficient. During the incubation of Ss-Hbs with Bacillus subtilis, it was speculated that trace H2O2, probably from autoxidation of Ss-Hbs or generated by B. subtilis, started the peroxidase cycle of Ss-Hb. and produced a large amount of [Formula: see text] in the presence of sufficient substrate in the culture medium. It is therefore reasonable to assume that Ss-Hbs played an antibacterial role owing to their peroxidase activity, which produced [Formula: see text] .

Radiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605

BackgroundRadiation exposure of tissues is associated with inflammatory cell influx. Myeloperoxidase (MPO) is an enzyme expressed in granulocytes, such as neutrophils (PMN) and macrophages, responsible for active chlorine species (ACS) generation. The present study aimed to: 1) determine whether exposure to γ-irradiation induces MPO-dependent ACS generation in murine PMN; 2) elucidate the mechanism of radiation-induced ACS generation; and 3) evaluate the effect of the synthetic lignan LGM2605, known for ACS scavenging properties. MethodsMPO-dependent ACS generation was determined by using hypochlorite-specific 3′-(p-aminophenyl) fluorescein (APF) and a highly potent MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and confirmed in PMN derived from MPO−/− mice. Radiation-induced MPO activation was determined by EPR spectroscopy and computational analysis identified tyrosine, serine, and threonine residues near MPO's active site. Resultsγ-radiation increased MPO-dependent ACS generation dose-dependently in human MPO and in wild-type murine PMN, but not in PMN from MPO−/− mice. LGM2605 decreased radiation-induced, MPO-dependent ACS. Protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSTP) inhibitors decreased the radiation-induced increase in ACS. Peroxidase cycle results demonstrate that tyrosine phosphorylation blocks MPO Compound I formation by preventing catalysis on H2O2 in the active site of MPO. EPR data demonstrate that γ-radiation increased tyrosyl radical species formation in a dose-dependent manner. ConclusionsWe demonstrate that γ-radiation induces MPO-dependent generation of ACS, which is dependent, at least in part, by protein tyrosine and Ser/Thr dephosphorylation and is reduced by LGM2605. This study identified for the first time a novel protein dephosphorylation-dependent mechanism of radiation-induced MPO activation.

Dynamics of a Key Conformational Transition in the Mechanism of Peroxiredoxin Sulfinylation.

Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein–thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation.

3-Hydroxy-3-Methylglutaric Acid Impairs Redox and Energy Homeostasis, Mitochondrial Dynamics, and Endoplasmic Reticulum-Mitochondria Crosstalk in Rat Brain.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a neurometabolic disorder characterized by predominant accumulation of 3-hydroxy-3-methylglutaric acid (HMG) in tissues and biological fluids. Patients often present in the first year of life with metabolic acidosis, non-ketotic hypoglycemia, hypotonia, lethargy, and coma. Since neurological symptoms may be triggered or worsened during episodes of metabolic decompensation, which are characterized by high urinary excretion of organic acids, this study investigated the effects of HMG intracerebroventricular administration on redox homeostasis, citric acid cycle enzyme activities, dynamics (mitochondrial fusion and fission), and endoplasmic reticulum (ER)-mitochondria crosstalk in the brain of neonatal rats euthanized 1 (short term) or 20 days (long term) after injection. HMG induced lipid peroxidation and decreased the activities of glutathione peroxidase (GPx) and citric acid cycle enzymes, suggesting bioenergetic and redox disruption, 1 day after administration. Levels of VDAC1, Grp75, and mitofusin-1, proteins involved in ER-mitochondria crosstalk and mitochondrial fusion, were increased by HMG. Furthermore, HMG diminished synaptophysin levels and tau phosphorylation, and increased active caspase-3 content, indicative of cell damage. Finally, HMG decreased GPx activity and synaptophysin levels, and changed MAPK phosphorylation 20 days after injection, suggesting that long-term toxicity is further induced by this organic acid. Taken together, these data show that HMG induces oxidative stress and disrupts bioenergetics, dynamics, ER-mitochondria communication, and signaling pathways in the brain of rats soon after birth. It may be presumed that these mechanisms underlie the onset and progression of symptoms during decompensation occurring in HL-deficient patients during the neonatal period.

The soluble curcumin derivative NDS27 inhibits superoxide anion production by neutrophils and acts as substrate and reversible inhibitor of myeloperoxidase

A water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-β-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼107 M−1 s−1) but not for Compound II (∼103 M−1 s−1) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-β-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-β-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO.

Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control.

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.

Determination of kinetic parameters of the enzymatic reaction between soybean peroxidase and natural antioxidants using chemometric tools

The oxidation of eugenol, isoeugenol and vanillin natural antioxidants catalyzed by the soybean peroxidase enzyme was studied using uv–vis spectroscopy. An experimental design was used to optimize the different variables. The multivariate curve resolution method was used to obtain the profiles of antioxidant absorbance’s as a function of time due to uv–vis absorption bands of both antioxidants and the enzymatic reaction product/s show a strong overlap. From these results, apparent Michaelis-Menten constants as well as the kinetic parameters k1 and k3 involved in the catalytic cycle of peroxidases were calculated. The antioxidant apparent acidity constants were also determined at different pH’s from uv–vis spectrophotometric measurements. Values of k1 were (0.6 ± 0.1) × 105 M−1 s−1, (2.0 ± 0.2) × 105 M−1 s−1 and (7.0 ± 0.5) × 106 M−1 s−1 and k3 (4.0 ± 0.2) × 105 M−1 s−1, (6.0 ± 0.6) × 105 M−1 s−1 and (6.0 ± 0.9) × 106 M−1 s−1 for eugenol, isoeugenol and vanillin, respectively.

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

Morphine, a potential inhibitor of myeloperoxidase activity

Morphine is an opioid alkaloid commonly used in clinical practice for its analgesic properties. The phenolic hydroxyl group of that phenanthrene derivative is pivotal for binding to opioid receptors but it may also be responsible for the antioxidant behavior of morphine reported in several in vitro experiments. In this study, we assessed the effect of morphine on myeloperoxidase (MPO), a hemic enzyme from azurophilic granules of polymorphonuclear neutrophils involved in the production of cytotoxic and microbicidal reactive oxidants during inflammatory response. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling (docking) were performed to study the potential anti-catalytic action of morphine on MPO in comparison with the inhibitory effects of reference antioxidant molecules quercetin, gallic acid and ascorbic acid. The reducing action of morphine on the MPO peroxidase cycle has been investigated using electron paramagnetic resonance (EPR) and UV-visible absorption spectroscopy. Morphine acted as a reducing substrate in the peroxidase cycle of MPO and therefore protected the enzyme against the suicide action of its natural substrate, hydrogen peroxide. The SIEFED experiments associated with the docking study, further demonstrated a lack of strong and sustained anti-catalytic activity of morphine. In summary, from the results of this study, it appears that morphine acts as a weak and reversible inhibitor of MPO that may nonetheless contribute to immunomodulatory and antioxidant effects of this opioid analgesic in vivo.

P-104 - Cytochrome c - cardiolipin interaction leads to the cytochrome c modification and degradation via formation of cardiolipin hydroperoxides

Recently, the complex of cytochrome c (cyt c), a small heme mitochondrial protein, with mitochondria specific lipid cardiolipin (CL) was linked to the control of apoptotic cell death. Under apoptotic conditions cyt c - CL complex exhibits peroxidase activity, leading to the formation of CL- hydroperoxides. CL-hydroperoxides promote cyt c release to the cytoplasm, where cyt c participates in apoptosis pathway. However, not much known about the modifications of cyt c as a part of cyt c - CL complex. In the present work, the systematic study on the peroxidase activity of CL-cyt c complex in CL-containing liposomes was performed. CL-hydroperoxides stimulate cyt c peroxidase activity in the absence of hydrogen peroxide. Furthermore, in the presence of oxygen, CL-hydroperoxides are generated as the products of the CL - cyt c peroxidase reaction. During peroxidase cycle cyt c amino acid residues are modified by reactive unsaturated aldehydes including 4-hydroxy- and 4-oxo-nonenal, derived from CL- peroxidation. This leads to the consecutive cyt c inactivation and heme degradation accompanied by the release of free iron. The current study on CL-driven cyt c degradation and iron release probably plays an important role in the mitochondrial pathway of apoptosis induction.