NADH Oxidation Research Articles

Effect of Water Supplementation on Oxidant/Antioxidant Activities and Total Phenol Content in Growing Olives of the Morisca and Manzanilla Varieties.

The objective of this work was to analyse, using a time series analysis, the effect of water regime for two cultivars at three stages of ripeness, during three consecutive years. Fruit and oil yield; O2− production and NADH oxidation activities; polyphenol oxidase (PPO), superoxide dismutase (SOD) and peroxidase (POX) activities; total phenols, flavonoid and phenylpropanoid glycoside content; and total antioxidant capacity (FRAP) were determined. All these parameters were found to depend on variety, irrigation and year. The results showed that the fruit and oil yields were strongly dependent on both irrigation and variety. The DW/FW ratio was practically constant during ripening, with small variety-dependent changes due to irrigation. Total amino acid and protein contents increased with ripening, with a close dependence on variety but not on irrigation. The SOD and POX activities appeared closely related, and related to the NADH oxidation and the amount of O2−. The evolution of phenols and FRAP during ripening was complementary to that of NADH oxidation, O2− production as well as SOD and POX activities. The determining factors of the SOD, POX and PPO activities were the variety and the ripening; the determining factor of the yield, ROS production, total phenols and antioxidant capacity was the water regime. Inverse correlations were observed between maximum temperature and total phenols (−0.869), total flavonoids (−0.823), total PPGs (−0.801) and FRAP (−0.829); and between DW/FW and irrigation (−0.483). The remaining significant correlations were positive.

Reverse Electron Transfer by Respiratory Complex I Catalyzed in a Modular Proteoliposome System.

Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature.

Electrochemical Detection of Glucose and Lactate Dehydrogenase Using a Zwitterionic Phenazine Compound as an Electron Mediator for NADH Oxidation

AbstractPhenazine compounds have been used as simple organic catalysts for the rapid oxidation of NADH. Although positively charged phenazine compounds are highly water‐soluble, their uncharged, reduced forms are not. We report the use of a zwitterionic phenazine compound, 3‐(1‐methoxyphenazin‐5‐ium‐5‐yl)propane‐1‐sulfonate (mPPS), as an organic catalyst and electron mediator for the electrochemical oxidation of NADH and detection of glucose and lactate dehydrogenase (LDH). mPPS is highly stable in phosphate buffered saline (pH 7.4). Furthermore, the negatively charged, reduced form of mPPS is highly water‐soluble. The detection limits of glucose and LDH in artificial serum were approximately 0.02 mM and 2 ng/mL, respectively.

Mitochondrial Respiratory Complexes as Targets of Drugs: The PPAR Agonist Example.

Mitochondrial bioenergetics are progressively acquiring significant pathophysiological roles. Specifically, mitochondria in general and Electron Respiratory Chain in particular are gaining importance as unintentional targets of different drugs. The so-called PPAR ligands are a class of drugs which not only link and activate Peroxisome Proliferator-Activated Receptors but also show a myriad of extrareceptorial activities as well. In particular, they were shown to inhibit NADH coenzyme Q reductase. However, the molecular picture of this intriguing bioenergetic derangement has not yet been well defined. Using high resolution respirometry, both in permeabilized and intact HepG2 cells, and a proteomic approach, the mitochondrial bioenergetic damage induced by various PPAR ligands was evaluated. Results show a derangement of mitochondrial oxidative metabolism more complex than one related to a simple perturbation of complex I. In fact, a partial inhibition of mitochondrial NADH oxidation seems to be associated not only with hampered ATP synthesis but also with a significant reduction in respiratory control ratio, spare respiratory capacity, coupling efficiency and, last but not least, serious oxidative stress and structural damage to mitochondria.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 μW cm−2, and good operational stability.

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H2O2) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H2O2 around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H2O2-alone treatment kills catalase-deficient (katEG) mutants similar to H2O2+NO treatment. However, the Fenton reaction, in addition to H2O2, requires Fe(II), which H2O2 excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H2O2 is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H2O2-alone and H2O2+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H2O2-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H2O2 and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.

How many molecules of mitochondrial complex I are in a cell?

Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD+ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis.Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 105 complex I molecules/cell. Our method can be used for accurate quantification of complex I content.

Highly sensitive voltammetric determination of NADH based on N-CQDs decorated SnO2/ionic liquid/carbon paste electrode

A highly sensitive and selective modified electrode was successfully developed for the monitoring of nicotinamide adenine dinucleotide (NADH) in the presence of folic acid. In this regard, a carbon paste electrode (CPE) was functionalized by the nitrogen-doped carbon quantum dots/tin oxide (N-CQDs/SnO2) nanocomposite and 1-butyl-2,3-dimethyl imidazolium hexafluorophosphate ([C4DMIM][PF6]) ionic liquid (IL). The structure and surface morphology of the nanocomposite were characterized by various methods, including field emission scanning electron microscopy, energy dispersive spectroscopy (EDS), high-resolution transmission electron microscopy (HR-TEM), and x-ray diffraction (XRD). The modified electrode displayed powerful and long-lasting electron mediating activity, with well-separated NADH and folic acid oxidation peaks. The sensing response of the developed [C4DMIM][PF6]/N-CQDs/SnO2/CPE platform was evaluated by determining NADH via the voltammetric technique under the optimized operating conditions. The current peaks of the square wave voltammograms of NADH and folic acid increased linearly with enhancing its concentrations within the ranges of 0.003–275 μM NADH and 0.4–380 μM folic acid. The detection limits for NADH and folic acid were obtained at 0.8 nM and 0.1 μM, respectively. Interference species such as glucose, urea, tryptophan, glycine, methionine, and vitamin B12 had no influence on the ability of the fabricated modified electrode to detect the target species. The low detection limit, high sensitivity, excellent selectivity, superior stability, and cost-effectiveness made it suitable for the quantification of NADH in the real biological samples with the recovery percent values in the range of 97.5%–103%.

Electrochemical Oscillatory Baffled Reactors Fabricated with Additive Manufacturing for Efficient Continuous-Flow Oxidations.

Electrochemical continuous-flow reactors offer a great opportunity for enhanced and sustainable chemical syntheses. Here, we present a novel application of electrochemical continuous-flow oscillatory baffled reactors (ECOBRs) that combines advanced mixing features with electrochemical transformations to enable efficient electrochemical oxidations under continuous flow at a millimeter distance between electrodes. Different additive manufacturing techniques have been employed to rapidly fabricate reactors. The electrochemical oxidation of NADH, a very sensitive substrate key for the regeneration of enzymes in biocatalytic transformations, has been employed as a benchmark reaction. The oscillatory conditions improved bulk mixing, facilitating the contact of reagents to electrodes. Under oscillatory conditions, the ECOBR demonstrated improved performance in the electrochemical oxidation of NADH, which is attributed to improved mass transfer associated with the oscillatory regime.

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.

Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone – giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

Wash-Free, Sandwich-Type Protein Detection Using Direct Electron Transfer and Catalytic Signal Amplification of Multiple Redox Labels.

Direct electron transfer (DET) between a redox label and an electrode has been used for sensitive and selective sandwich-type detection without a wash step. However, applying DET is still highly challenging in protein detection, and a single redox label per probe is insufficient to obtain a high electrochemical signal. Here, we report a wash-free, sandwich-type detection of thrombin using DET and catalytic signal amplification of multiple redox labels. The detection scheme is based on (i) the redox label-catalyzed oxidation of a reductant, (ii) the conjugation of multiple redox labels per probe using a poly-linker, (iii) the low nonspecific adsorption of the conjugated poly-linker due to uncharged, reduced redox labels, and (iv) a facile DET using long, flexible poly-linker and spacer DNA. Amine-reactive phenazine ethosulfate and NADH were used as the redox label and reductant, respectively. N3-terminated polylysine was used as the poly-linker for the conjugation between an aptamer probe and multiple redox labels. Approximately 11 redox labels per probe and rapid catalytic NADH oxidation enable high signal amplification. Thrombin in urine could be detected without a wash step with a detection limit of ∼50 pM, which is practically promising for point-of-care testing of proteins.

MITODYN: An Open Source Software for Quantitative Modeling of Mitochondrial and Cellular Energy Metabolic Flux Dynamics in Health and Disease.

Mitochondrial respiratory chain (RC) transforms the reductive power of NADH or FADH2 oxidation into a proton gradient between the matrix and cytosolic sides of the inner mitochondrial membrane, that ATP synthase uses to generate ATP. This process constitutes a bridge between carbohydrates' central metabolism and ATP-consuming cellular functions. Moreover, the RC is responsible for a large part of reactive oxygen species (ROS) generation that play signaling and oxidizing roles in cells. Mathematical methods and computational analysis are required to understand and predict the possible behavior of this metabolic system. Here we propose a software tool that helps to analyze individual steps of respiratory electron transport in their dynamics, thus deepening understanding of the mechanism of energy transformation and ROS generation in the RC. This software's core is a kinetic model of the RC represented by a system of ordinary differential equations (ODEs). This model enables the analysis of complex dynamic behavior of the RC, including multistationarity and oscillations. The proposed RC modeling method can be applied to study respiration and ROS generation in various organisms and naturally extended to explore carbohydrates' metabolism and linked metabolic processes.

Synthesis, physicochemical characterization and antiproliferative activity of phosphino Ru(II) and Ir(III) complexes.

Herein, we present the synthesis of new complexes based on ruthenium(II) (Ru(η6-p-cymene)Cl2PPh2CH2OH (RuPOH) and Ru(η6-p-cymene)Cl2P(p-OCH3Ph)2CH2OH (RuMPOH)) and iridium(III) (Ir(η5-Cp*)Cl2P(p-OCH3Ph)2CH2OH (IrMPOH) and Ir(η5-Cp*)Cl2PPh2CH2OH (IrPOH)) containing phosphine ligands with/without methoxy motifs on phenyl rings (P(p-OCH3Ph)2CH2OH (MPOH) and PPh2CH2OH (POH)). The complexes were characterized by mass spectrometry, NMR spectroscopy (1D: 1H, 13C{1H}, and 31P{1H} and 2D: HMQC, HMBC, and COSY NMR) and elemental analysis. All the complexes were structurally identified by single-crystal X-ray diffraction analysis. The Ru(II) and Ir(III) complexes have a typical piano-stool geometry with an η6-coordinated arene (RuII complexes) or η5-coordinated (IrIII compounds) and three additional sites of ligation occupied by two chloride ligands and the phosphine ligand. Oxidation of NADH to NAD+ with high efficiency was catalyzed by complexes containing P(p-OCH3Ph)2CH2OH (IrMPOH and RuMPOH). The catalytic property might have important future applications in biological and medical fields like production of reactive oxygen species (ROS). Furthermore, the redox activity of the complexes was confirmed by cyclic voltamperometry. Biochemical assays demonstrated the ability of Ir(III) and Ru(II) complexes to induce significant cytotoxicity in various cancer cell lines. Furthermore, we found that RuPOH and RuMPOH selectively inhibit the proliferation of skin cancer cells (WM266-4; IC50, after 24 h: av. 48.3 μM; after 72 h: av. 10.2 μM) while Ir(III) complexes were found to be moderate against prostate cancer cells (DU145).

PYRUVATE DEHYDROGENASE COMPLEX ACTIVITY IN THE RAT`S KIDNEYS UNDER CONDITIONS OF TOXIC ACETAMINOPHEN DAMAGE WITH PROTEIN DEFICIENCY

The aim of this work was to study the research the enzymatic activity of pyruvate dehydrogenase complex and lac-tate dehydrogenase and determination of the lactate/pyruvate ratio in rat kidneys under conditions of toxic damage by acetaminophen against the background of protein deficiency.The activity of the pyruvate dehydrogenase complex activity was determined according to a method based on the reaction of oxidative decarboxylation of pyruvate with simultaneous reduction of NAD+, which is measured spectrophotometrically at 340 nm. LDH activity was studied by an optimized optical method, which is based on the reaction of conversion of pyruvate to lactate with concomitant oxidation of NADH, which was measured using a spectrophotometer at λ = 340 nm. Lactate content was measured spectrophotometrically by the ability of FeCl3 to interact with lactate ions, resulting in the formation of a bright yellow iron lactate solution, which is recorded at a wavelength of 390 nm. The concentration of pyruvate was deter-mined by the modified Umbright method, which is based on the reaction of the interaction of pyruvate with 2,4-Dinitrophenylhydrazine (2,4-DNPH) in an alkaline medium, during which hydrazine is formed, which was recorded using a photoelectrocolorimeter at λ = 440 nm. The study was conducted on 4 groups of experimental animals: I - control animals (K); II – rats kept on a low-protein diet (LPD); ІІІ – animals in which acute toxic damage was caused by acetaminophen (TI); IV – rats on a low-protein diet, simulated acute toxic damage with acetaminophen (LPD/TI). It is demonstrated that under the conditions of dietary protein deficiency in the kidneys, there is a decrease in the activi-ty of the pyruvate dehydrogenase complex while maintaining the control indicators of the lactate/pyruvate ratio and lactate dehydrogenase activity. However, under the conditions of the toxic effect of acetaminophen against the back-ground of dietary protein deficiency in the kidneys, an intensification of the anaerobic pathway of energy supply is observed, as evidenced by a significant decrease in the activity of the pyruvate dehydrogenase complex, an increase in the lactate/pyruvate ratio against the background of lactate dehydrogenase activation. The obtained results can be used to justify approaches to correct energy deficit in animals under the conditions of acetaminophen overdose against the background of protein deficiency.

Accelerating NADH Oxidation and Hydrogen Production with Mid-Gap States of Nitrogen-Rich Carbon Nitride Photocatalyst

Accelerating NADH Oxidation and Hydrogen Production with Mid-Gap States of Nitrogen-Rich Carbon Nitride Photocatalyst

Photodynamic therapy with mitochondria-targeted biscyclometallated Ir(III) complexes. Multi-action mechanism and strong influence of the cyclometallating ligand.

Photodynamic therapy is an alternative to classical chemotherapy due to its potential to reduce side effects by a controlled activation of a photosensitizer through local irradiation with light. The photosensitizer then interacts with oxygen and generates reactive oxygen species. Iridium biscyclometallated complexes are very promising photosensitizers due to their exceptional photophysical properties and their ability to target mitochondria. Four Ir(III) biscyclometallated complexes of formula [Ir(C^N)2(N^N')]Cl, where N^N' is a ligand containing a benzimidazolyl fragment, have been synthesized and characterized. The C^N ligands were 2-phenylpyridinate (ppy) and 2-(2,4-difluorophenyl)pyridinate (dfppy). The complexes exhibited high photostability. The electrochemical and photophysical properties were modulated by both the cyclometallating and the ancillary ligands. The dfppy derivatives yielded the highest emission energy values, quantum yields of phosphorescence and excited state lifetimes. All complexes generated 1O2 in aerated solutions upon irradiation. Biological studies revealed that these complexes have a moderate cytotoxicity in the dark against different human cancer cell lines: prostate (PC-3), colon (CACO-2) and melanoma (SK-MEL-28), and against non-malignant fibroblasts (CCD-18Co). However, derivatives with ppy ligands ([1a]Cl, [2a]Cl) yielded a relevant photodynamic activity upon light irradiation (450 nm, 24.1 J cm-2), with phototoxicity indexes (EC50,dark/EC50,light) of 20.8 and 17.3, respectively, achieved in PC-3 cells. Mechanistic studies showed that these complexes are taken up by the cells through endocytosis and preferentially accumulate in mitochondria. Upon photoactivation, the complexes induced mitochondrial membrane depolarization and DNA damage, thus triggering cell death, mainly by apoptosis. Complex [1a]Cl is also able to oxidize NADH. This mitochondria-targeted photodynamic mechanism greatly inhibited the reproductive capacity of cancer cells and provides a valuable alternative to traditional chemotherapy for the controlled treatment of cancer.

Vitamin A intake forms resistance to hypervitaminosis A and affects the functional activity of the liver

Vitamin A intake forms resistance to hypervitaminosis A and affects the functional activity of the liver

Hierarchical porous MoS2 particles: excellent multi-enzyme-like activities, mechanism and its sensitive phenol sensing based on inhibition of sulfite oxidase mimics

It is important to exploit highly efficient methods for detecting pollutants selectively and sensitively. Artificial enzymes are promising to replace natural enzymes with diverse functions for sustainable developments and various applications. However, it remains the challenge to develop novel mimic enzymes or multi-enzyme mimics for pollutant detection. Herein we report hierarchical porous MoS2 particles prepared by a simple hydrothermal method, which demonstrated excellent sulfite oxidase (SuOx)-, nicotinamide adenine dinucleotide (NADH) oxidase- and superoxide dismutase-mimicking activities. In addition, the catalytic conditions for SuOx-like and NADH oxidase-like activities of MoS2 were optimized. The catalytic mechanism of the NADH oxidase mimics is that O2 involves in the oxidation of NADH, to generate O2.- intermediate and finally turn to H2O2, while SuOx mimics comes from that MoS2 particles can effectively catalyze sulfite to reduce [Fe(CN)6]3-. Based on the excellent SuOx-like activity of MoS2 particles, while phenol can inhibit the oxidation of sulfite, a phenol colorimetric sensor was explored with the dynamic range of 2–1000 μM and the limit of detection of 0.72 μM, applicable to detect phenol in effluents. Therefore, MoS2 particles with the SuOx-like, NADH oxidase-like and SOD-like activities has broad application prospects in environmental monitoring and bio-analysis.

Controlled power: how biology manages succinate-driven energy release.

Oxidation of succinate by mitochondria can generate a higher protonmotive force (pmf) than can oxidation of NADH-linked substrates. Fundamentally, this is because of differences in redox potentials and gearing. Biology adds kinetic constraints that tune the oxidation of NADH and succinate to ensure that the resulting mitochondrial pmf is suitable for meeting cellular needs without triggering pathology. Tuning within an optimal range is used, for example, to shift ATP consumption between different consumers. Conditions that overcome these constraints and allow succinate oxidation to drive pmf too high can cause pathological generation of reactive oxygen species. We discuss the thermodynamic properties that allow succinate oxidation to drive pmf higher than NADH oxidation, and discuss the evidence for kinetic tuning of ATP production and for pathologies resulting from substantial succinate oxidation in vivo.