The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

A novel phenomenon of burst of oxygen uptake during decavanadate-dependent oxidation of NADH

Oxidation of NADH by decavanadate, a polymeric form vanadate with a cage-like structure, in presence of rat liver microsomes followed a biphasic pattern. An initial slow phase involved a small rate of oxygen uptake and reduction of 3 of the 10 vanadium atoms. This was followed by a second rapid phase in which the rates of NADH oxidation and oxygen uptake increased several-fold with a stoichiometry of NADH: O₂ of 11. The burst of NADH oxidation and oxygen uptake which occurs in phosphate, but not in Tris buffer, was prevented by SOD, catalase, histidine, EDTA, MnCl₂ and CuSO₄, but not by the hydroxyl radical quenchers, ethanol, methanol, formate and mannitol. The burst reaction is of a novel type that requires the polymeric structure of decavanadate for reduction of vanadium which, in presence of traces of H₂O₂, provides a reactive intermediate that promotes transfer of electrons from NADH to oxygen.     

The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commerical NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.Abbreviations EDTA = ethylenediaminetetracetate - HQNO = 2–n-heptyl-4-hydroxy-quinoline-N-oxide - DEAE = diethylaminoethyl     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Effectiveness of phenoxyl radicals generated by peroxidase/H2O2-catalyzed oxidation of caffeate, ferulate, and p-coumarate in cooxidation of ascorbate and NADH

The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation by peroxidase/H₂O₂-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from soybean (Glycine max L.) seed coat, maize (Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed both in the rate of H₂O₂-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn²⁺ and Al³⁺) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation systems can function as mechanisms to scavenge H₂O₂ and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of high-performance liquid chromatography to detect hydroxyl and superoxide radicals generated from mitomycin C

Distinguishing between short-lived reactive oxygen species like hydroxyl and superoxide radicals is difficult; the most successful approaches employ electron spin resonance (ESR) spin-trapping techniques. Using the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO) to selectively trap various radicals in the presence and absence of ethanol, an HPLC system which is capable of separating the hydroxyl- and superoxide-generated DMPO adduct species has been developed. The radical-generated DMPO adducts were measured with an electrochemical detector attached to the HPLC system and confirmed by spin-trapping techniques. The HPLC separation was carried out on an ODS reverse-phase column with a pH 5.1 buffered 8.5% acetonitrile mobile phase. The advantage of the HPLC system described is that it permits the separation and detection of hydroxyl and superoxide radicals without requiring ESR instrumentation. The antineoplastic bioreductive alkylating agent mitomycin C, when activated by NADPH-cytochrome c reductase, was shown to generate both hydroxyl and superoxide radicals.     

How to deal with oxygen radicals stemming from mitochondrial fatty acid oxidation

Oxygen radical formation in mitochondria is an incompletely understood attribute of eukaryotic cells. Recently, a kinetic model was proposed, in which the ratio between electrons entering the respiratory chain via FADH₂ or NADH determines radical formation. During glucose breakdown, the ratio is low; during fatty acid breakdown, the ratio is high (the ratio increasing—asymptotically—with fatty acid length to 0.5, when compared with 0.2 for glucose). Thus, fatty acid oxidation would generate higher levels of radical formation. As a result, breakdown of fatty acids, performed without generation of extra FADH₂ in mitochondria, could be beneficial for the cell, especially in the case of long and very long chained ones. This possibly has been a major factor in the evolution of peroxisomes. Increased radical formation, as proposed by the model, can also shed light on the lack of neuronal fatty acid oxidation and tells us about hurdles during early eukaryotic evolution. We specifically focus on extending and discussing the model in light of recent publications and findings.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.     

The oxidation of exogenous NADH by mitochondria of Euglena gracilis

A novel oxidase activity of external NADH was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external NADH in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of NADH. Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of NADH does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting.     

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Among various metal ions of physiological interest, Cu²⁺ is uniquely capable of catalyzing the oxidation of NADH by H₂O₂. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu²⁺-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H₂O₂ utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD⁺. D₂O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding¹O₂ as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H₂O₂.     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.     

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.     

Reactive oxygen species generation through NADH oxidation by 6-formylpterin derivatives in the dark

6-formylpterin (6FP) has been reported to produce reactive oxygen species (ROS) such as *O2- and H2O2 from O2 in the presence of NADH under light condition. In the present study, we prepared a variety of 6FP derivatives and found that 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridin-4-one and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-methylpteridin-4-one, in which the 2-amino groups are modified by a dimethylaminomethylene group and the 3-positions by pivaloyl and methyl groups and 2-amino-6-formyl-3-methylpteridin-4-one in which the amino group at the 2-position is free and the 3-position is modified by a methyl group generated H2O2 from O2 on oxidation of NADH to NAD+ in the dark. However, 6FP and 2-(N,N-dimethylaminomethyleneamino)-6-formylpteridin-4-one, in which the 3-position is free did not yield H2O2. These results indicate that modification of the 3-position is essential to make the activities of 6FP available in the dark and would be suggestive for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.     

Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis

A new NADH oxidase, useful for the regeneration of NAD⁺, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg^–1. After purification by four chromatographic steps, an activity of 116 U mg^–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K _m value for NADH was 24 M.     

Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria

Mitochondria from Vicia faba L. exhibit two pathways for the oxidation of endogenous NADH. One pathway is rotenone-sensitive and includes three phosphorylation sites, the other one is rotenone-insensitive and by-passes site I. This by-pass is located in the area of flavoproteins, but several lines of evidence suggest that the rotenone-insensitive oxidation of matrix-NADH is not mediated by the external NADH dehydrogenase. With saturating substrate concentrations the two pathways are superimposed in state 3 while in state 4 the electrons are transferred only via the by-pass. The rotenone-resistant electron flux is obviously regulated from the substrate side. We suggest that the function of the by-pass is to regulate the NADH/NAD⁺ ratio of the matrix space.Abbreviations BSA bovine serum albumin - CCCP carbonylcyanide-m-chlorophenylhydrazone - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxy-ethylpiperazine-ethanesulphonic acid - PVP polyvinylpyrrolidone - TPP thiaminepyrophosphate     

Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH

Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.     

The involvement of oxygen-derived free radicals in the resistant response of potato tubers toErwinia carotovora

Summary The absolute requirement of oxygen for a potato tuber (cv. Pentland Crown) to display a resistant response toErwinia carotovora has been demonstrated in EPR measurements. These show that a relatively stable free radical is formed in inoculated hosts only after exposure to air. In an attempt to isolate and identify unstable free radical precursors, experiments have been conducted in which the chemical spin trap -(4-pyridyl 1-oxide)-N-tert-butyl-nitrone (POBN) was incorporated into the tissue either at the time of inoculation or upon exposure to air. A single radical adduct was observed with spectral parameters that resemble those of a lipid-derived radical.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

Interplay between bax, reactive oxygen species production, and cardiolipin oxidation during apoptosis

Bax/Bak activation and cardiolipin peroxidation are essential for cytochrome c release during apoptosis, yet, the link between them remains elusive. We report that sequence of events after exposure of mouse embryonic fibroblast (MEF) cells to actinomycin D followed the order: Bax translocation → superoxide production → cardiolipin peroxidation. Genetic ablation of Bax/Bak inhibited actinomycin D induced superoxide production and cardiolipin peroxidation. Rotenone caused robust superoxide generation but did not trigger cardiolipin peroxidation in Bax/Bak double knockout MEF cells. This suggests that, in addition to participating in ROS generation, Bax/Bak play another specific role in cardiolipin oxidation. In isolated mitochondria, recombinant Bax enhanced succinate induced cardiolipin oxidation and cytochrome c release. Mitochondrial peroxidase activity, likely involved in cardiolipin peroxidation, was enhanced upon incubation with recombinant Bax. Thus, cardiolipin peroxidation may be causatively and time-dependently related to Bax/Bak effects on ROS generation and peroxidase activation of cytochrome c.