CyaC,a redox‐regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme‐B membrane anchor domain

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane‐integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra‐histidine signature that is universally present in the membrane anchors of bacterial diheme‐B succinate‐quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme‐B protein and carried a binuclear iron‐sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme‐B in the membrane and loss of AC activity. Heme‐B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q₀ or Q₀H₂). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q₀ and O₂, or NADH and Q₀H₂ respectively. We conclude that CyaC‐like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

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.     

Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

α‐Dioxygenases (α‐DOX) are heme‐containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α‐DOX oxygenate a variety of 14–20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro‐R hydrogen from the α‐carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H₂O₂). We determined the X‐ray crystal structures of wild type α‐DOX from Oryza sativa, the wild type enzyme in complex with H₂O₂, and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α‐DOX such that the carboxylate forms ionic interactions with His‐311 and Arg‐559. Thr‐316 aids in the positioning of carbon‐2 for hydrogen abstraction. Twenty‐five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H₂O₂ structures provides insight into enzyme activation. The binding of H₂O₂ at the distal face of the heme displaces residues His‐157, Asp‐158, and Trp‐159 ～2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp‐158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp‐159 and Trp‐213 reorient, and the guanidinium group of Arg‐559 is repositioned near Tyr‐379, poised to interact with the carboxylate group of the substrate.     

Comparison of aerobic and anaerobic growth of Lactobacillus plantarum in a glucose medium

The growth rate of Lactobacillus plantarum in a complex medium with 55.6 mM glucose decreased during aerobic incubation (relative to anaerobic incubation). The decrease occurred much earlier than an increase in the rate of oxygen utilization by the culture which led to H₂O₂ accumulation. The concentration of H₂O₂ accumulated in the medium was easily tolerated by the culture and elimination of the H₂O₂ did not prevent the decrease in growth rate. Increased O₂ utilization was accompanied by a switch in metabolism which resulted in acetate rather than lactate accumulation in aerobic cultures.Abbreviation MRSG Man, Rogosa and Sharpe (1960). Medium modified as in Materials and methods with glucose as fermentation substrate     

Biochemical basis of sulphenomics: how protein sulphenic acids may be stabilized by the protein microenvironment

Among protein residues, cysteines are one of the prominent candidates to ROS‐mediated and RNS‐mediated post‐translational modifications, and hydrogen peroxide (H₂O₂) is the main ROS candidate for inducing cysteine oxidation. The reaction with H₂O₂ is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H₂O₂. Indeed, only deprotonated Cys (i.e. thiolate form, ? S^?) can react with H₂O₂ leading to sulphenic acid formation (? SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable because they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, ? SO₂H and ? SO₃H, respectively), or alternatively, they can proceed towards further modifications including disulphide bond formation (? SS? ), S‐glutathionylation (? SSG) and sulphenamide formation (? SN?). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments.     

Protective Effects of Anthocyanins from Coreopsis tinctoria against Oxidative Stress Induced by Hydrogen Peroxide in MIN6 Cells

Anthocyanins (AC) from Coreopsis tinctoria possesses strong antioxidant properties, while the effects of AC on cells damage induced by reactive oxygen species (ROS) in diabetes mellitus diseases progression have not been reported. The present study was carried out to evaluate the protective property of AC against cellular oxidative stress with an experimental model, H₂O₂‐exposed MIN6 cells. AC could reverse the decrease of cell viability induced by H₂O₂ and efficiently suppressed cellular ROS production and cell apoptosis. In addition, Real‐time PCR and Western blot analyses indicated that AC could protect MIN6 cells against oxidative injury through increasing the translocation of Nrf2 into nuclear, decreasing the phosphorylation level of p38 and up‐regulating the protein expression of antioxidant enzyme (SOD1, SOD2 and CAT). Thus, this study provides evidence to support the beneficial effect of AC in inhibiting MIN6 cells from H₂O₂‐induced oxidative injury.     

An anaerobic bacterium,Bacteroides thetaiotaomicron,uses a consortium of enzymes to scavenge hydrogen peroxide

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H₂O₂ that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H₂O₂ in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H₂O₂, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H₂O₂ formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H₂O₂ was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.     

Preparation of co‐Co3O4/carbon nanotube/carbon foam for glucose sensor

Co‐Co₃O₄/carbon nanotube/carbon foam (Co‐Co₃O₄/CNT/CF) nanocomposites were prepared by soaking melamine foam into a solution of Co(NO₃)₂·6H₂O, followed by calcination in N₂ and air in sequence. The obtained Co‐Co₃O₄/CNT/CF nanocomposites were characterized with scanning electron microscopy and cyclic voltammetry. It was found that Co₃O₄ nanoparticles were grown on the external of CF successfully, while CNTs were grown on the surfaces of CF in a large amount, which further improved the electrical conductivity of the. The prepared Co‐Co₃O₄/CNT/CF nanocomposites were then used to construct nonenzymatic sensor to detect glucose in alkaline solution. The sensor showed detection range from 1.2 μM to 2.29 mM with a detection limit of 0.4 μM (S/N =3) and a high sensitivity of 637.5 μA^?1 cm^?2. The developed sensor also showed an instant response, favorable reproducibility, and high selectivity. The results attest that Co‐Co₃O₄/CNT/CF composites have great potential in the development of nonenzymatic sensors for glucose.     

Controlled feeding of hydrogen peroxide as oxygen source improves production of 5-ketofructose From L-sorbose using engineered pyranose 2-oxidase from Peniophora gigantea

5‐Keto‐D ‐fructose is a useful starting material for the synthesis of pyrrolidine iminosugars. It can be prepared by regioselective oxidation of L ‐sorbose using pyranose 2‐oxidase (P2Ox) and O₂ as a cosubstrate. As the solubility of O₂ in aqueous solution is low and the affinity of P2Ox for O₂ is poor, we developed a new and efficient process for the production of 5‐keto‐D ‐fructose based on engineered P2Ox from Peniophora gigantea and in situ generation of O₂ from H₂O₂ with catalase. This kind of oxygen supply required efficient mixing of the bioreactor which was achieved by controlled feeding of H₂O₂ close to the impeller tip where energy dissipation rate is highest. Thus bubbling, known to affect enzyme stability, was largely avoided, and the process could be run up to 145% oxygen super‐saturation which speeds‐up P2Ox activity. Under these conditions quantitative oxidation of 180 g L^?1 L ‐sorbose to 5‐keto‐D ‐fructose could be achieved within 4 h, resulting in a threefold higher overall productivity of the process compared to a process using gaseous oxygen supply. In addition, in situ generation of O₂ from H₂O₂ lowered the oxygen demand of the process by a factor of 100 compared to gaseous oxygen supply. Biotechnol. Bioeng. 2012; 109: 2941–2945. © 2012 Wiley Periodicals, Inc.     

Extraction and characterisation of hemicelluloses from maize stem

Introduction – Extraction and characterisation of hemicelluloses are very important for converting them into functional materials and chemicals. Objective – To develop a method for isolation of hemicelluloses from all cell walls. Methodology – Sequential steps using 90% dioxane, 80% acidic dioxane, 100% dimethyl sulphoxide and 8% NaOH were used for extraction of the hemicellulosic preparations (H₁, H₂, H₃ and H₄) from maize stem. Advanced NMR techniques were used for the analysis of native hemicelluloses. Results – Hemicelluloses with high yieldd were isolated from all cell walls, and contained arabinoxylan as the major polysaccharide. H₃ was substituted by α‐l ‐arabinofuranose, α‐d ‐xylopyranose, and acetyl groups (degree of saturation = 0.12/0.09) at O‐3/O‐2 of xylan. H₄ had a long continuous side chain of arabinose residues, and associated closely with non‐cellulosic glucose. The hemicelluloses formed more linkages with guaiacyl lignins, and some p‐coumaric acids built a bridge between hemicelluloses and lignin in maize stem. Conclusion – This modified method is successful for the isolation of hemicelluloses with high yields from all cell walls of maize stem. Copyright © 2010 John Wiley & Sons, Ltd.     

Growth inhibition by glucose oxidase system of enterotoxic Escherichia coli and Salmonella derby: in vitro studies

The antibacterial effect of different glucose oxidase (GOX)/glucose combinations was studied on two food-poisoning organisms, enterotoxic Escherichia coli PM 015 and Salmonella derby BP 177. Growth of E. coli in nutrient broth (NB) was clearly inhibited by 4.0 mg/ml glucose after 24 h when combined with 2.0 U/ml GOX and after 48 h when combined with 0.5 or 1.0 U/ml GOX. Salmonella derby was more resistant than E. coli, but showed clear growth inhibition only after 48 h when inoculated in tubes where 2 mg glucose/ml and 2 U GOX/ml (or 4 mg glucose/ml and 1 U GOX/ml) were combined. In order to understand if the enzyme effect on microbial growth can be attributed to hydrogen peroxide or to pH decrease as a result of the production of gluconic acid, catalase (CAT) was added to GOX/glucose system. Since CAT decomposes H₂O₂ to H₂O and O₂, the antibacterial effect was ascribed to a pH decrease as a result of gluconic acid in the system.     

H2O2‐ and pH‐sensitive CdTe quantum dots as fluorescence probes for the detection of glucose

A novel fluorescence assay system for glucose was developed with thioglycollic acid (TGA)‐capped CdTe quantum dots (QDs) as probes. The luminescence quantum yield of the TGA‐capped CdTe QDs was highly sensitive to H₂O₂ and pH. In the presence of glucose oxidase, glucose is oxidized to yield, gluconic acid and H₂O₂. H₂O₂ and H⁺ (dissociated from gluconic acid) intensively quenched the fluorescence of QDs. The experimental results showed that the quenched fluorescence was proportional to the glucose concentration within the range of 0.01–5.0 mm under optimized experimental conditions. Compared with most of the existing methods, this newly developed system possesses many advantages, including simplicity, low cost, high flexibility, and good sensitivity. Furthermore, no complicated chemical modification of QDs and enzyme immobilization was needed in this system. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo detection of protein cysteine sulfenylation in plastids

Protein cysteine thiols are post‐translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H₂O₂) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation‐sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C‐trapping technology to the plastids of light‐grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H₂O₂ pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H₂O₂ treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle‐tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Triosephosphate isomerase tyrosine nitration induced by heme–NaNO2–H2O2 or peroxynitrite: Effects of different natural phenolic compounds

Peroxynitrite and heme peroxidases (or heme)–H₂O₂–NaNO₂ system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways‐induced protein nitration and oxidation are controversial. Both nitrating systems can dose‐dependently induce triosephosphate isomerase (TIM) nitration, however, heme–H₂O₂–NaNO₂ was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3‐morpholinosydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme–H₂O₂–NaNO₂ and SIN‐1 and protein oxidation induced by SIN‐1, but promoted heme–H₂O₂–NaNO₂‐induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN‐1‐induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme–H₂O₂–NaNO₂‐induced nitration. Our results indicated that peroxynitrite and heme–H₂O₂–NaNO₂‐induced protein nitration was different, and the later one could be a better model for anti‐nitration compounds screening.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (˙NO) are key reactive species in signal transduction pathways leading to activation of plant defense against biotic or abiotic stress. Here, we investigated the effect of pre‐treating citrus plants (Citrus aurantium L.) with either of these two molecules on plant acclimation to salinity and show that both pre‐treatments strongly reduced the detrimental phenotypical and physiological effects accompanying this stress. A proteomic analysis disclosed 85 leaf proteins that underwent significant quantitative variations in plants directly exposed to salt stress. A large part of these changes was not observed with salt‐stressed plants pre‐treated with either H₂O₂ or sodium nitroprusside (SNP; a ˙NO‐releasing chemical). We also identified several proteins undergoing changes either in their oxidation (carbonylation; 40 proteins) and/or S‐nitrosylation (49 proteins) status in response to salinity stress. Both H₂O₂ and SNP pre‐treatments before salinity stress alleviated salinity‐induced protein carbonylation and shifted the accumulation levels of leaf S‐nitrosylated proteins to those of unstressed control plants. Altogether, the results indicate an overlap between H₂O₂‐ and ˙NO‐signaling pathways in acclimation to salinity and suggest that the oxidation and S‐nitrosylation patterns of leaf proteins are specific molecular signatures of citrus plant vigour under stressful conditions.     

Immobilization of the hyperthermophilic hydrogenase from Aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient H2 oxidation

The electrochemistry of membrane-bound [NiFe] hydrogenase I ([NiFe]-hase I) from the hyperthermophilic bacterium Aquifex aeolicus was investigated at gold and graphite electrodes. Direct and mediated H₂ oxidation were proved to be efficient in a temperature range of 25–70 °C, describing a potential window for H₂ oxidation similar to that of O₂-tolerant hydrogenases. Search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. We report high catalytic currents for H₂ oxidation up to 1 mA cm⁻², 10 times higher than at the bare electrode. Interestingly, high stability of the direct catalytic process was observed when encapsulating A. aeolicus [NiFe]-hase I into a carboxylic functionalized single walled carbon nanotube network. This suggests a peculiar interaction between the enzyme and the electrode material. The parameters that governed the orientation of the enzyme before electron transfer were thus investigated using self-assembled-monolayer gold electrodes. No control of the orientation by the charge or the hydrophobicity of the interface was demonstrated. This behavior was explained on the basis of a structural comparison between A. aeolicus [NiFe]-hase I and Desulfovibrio fructosovorans [NiFe] hydrogenase, which revealed the absence of acidic residues and an additional loop in the environment of the [4Fe–4S] distal cluster in A. aeolicus [NiFe]-hase I. Finally, the effect of inhibitors on the direct oxidation of H₂ by A. aeolicus [NiFe]-hase I encapsulated in a single walled carbon nanotube network was investigated. No inhibition by CO and tolerance toward O₂ were observed. Discussion of the reasons for such tolerance was undertaken on the basis of structural comparison with hydrogenases from aerobic bacteria.