NADH and NADPH as electron donors to respiratory and photosynthethic electron transport in the blue-green alga,Aphanocapsa

In the blue-green alga, Aphanocapsa, light inhibits respiration. This can be observed with spheroplasts when O₂ uptake is measured with NADH or NADPH as electron donor. However, NAD(P)H oxidation is unaffected by illumination. Furthermore, it was possible to demonstrate electron transfer from NAD(P)H to Photosystem I. Thus, the inhibition of respiratory oxygen uptake by light is explained by a competition of cytochrome oxidase and Photosystem I for reduction equivalents. Based on studies with inhibitors, electron transfer from NAD(P)H to Photosystem I involves the chloroplast cytochrome b₆-f complex.     

NADH dehydrogenase of Corynebacterium glutamicum. Purification of an NADH dehydrogenase II homolog able to oxidize NADPH

NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH.     

Properties of the oxidation of exogenous NADH and NADPH by plant mitochondria. Evidence against a phosphatase or a nicotinamide nucleotide transhydrogenase being responsible for NADPH oxidation

(1) The optimum pH for the oxidation of exogenous NADH by mitochondria from both Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices was 7.0–7.1. NADPH oxidation had a lower optimum pH of 6.6 in Arum and 6.0 in Jerusalem artichoke mitochondria. In both types of mitochondria the rates of NADH and NADPH oxidation were identical below pH 6.0–5.5. (2) It is shown conclusively that neither a phosphatase converting NADPH to NADH nor a nicotinamide nucleotide transhydrogenase was involved in the oxidation of NADPH by these mitochondria. (3) Palmitoyl-CoA, an inhibitor of transhydrogenase activity in mammalian mitochondria, inhibits both NADH and NADPH oxidation by plant mitochondria with a K_i of about 10 μM. (4) It is concluded that the known properties of NAD(P)H oxidation are best explained by assuming the presence of a second dehydrogenase specific for NADPH. At low pH, electron flow from the two dehydrogenases to oxygen shares a common rate-limiting step.     

The activities of several detoxication enzymes are differentially induced by juices of garden cress, water cress and mustard in human HepG2 cells

It has been previously demonstrated in a human-derived hepatoma cell line (HepG2) that juices from cruciferous vegetables protect against the genotoxicity caused by dietary carcinogens. HepG2 cells possess different enzymes involved in the biotransformation of xenobiotics. Therefore, we investigated the effect of cruciferous juices on the activities of CYP 1A and several phase II enzymes in this cell model. For each experiment, 1 × 10⁶ cells were seeded on Petri dishes. After 2 days, the juices (0.5–8 μl/ml of culture medium) were added for 48 h prior to cell harvesting. The addition of juice from water cress (Nasturtium officinalis R. Br) significantly increased the activities of ethoxyresorufin-O-deethylase at high doses only and NAD(P)H-quinone reductase in a dose-dependent manner (1.8- and 5-fold, respectively). The addition of juice from garden cress (Lepidum sativum L.) significantly increased the activities of NAD(P)H-quinone reductase and UDP-glucuronosyl-transferase with a maximal effect around the dose of 2 μl/ml juice (1.4- and 1.2-fold, respectively) while the other enzymes were not altered. Mustard (Sinapis alba L.) juice increased the activities of NAD(P)H-quinone reductase (2.6-fold at the dose of 8 μl/ml), and N-acetyl-transferase (1.4-fold at the dose of 8 μl/ml) in a dose-dependent manner while a maximal induction of UDP-glucuronosyl-transferase was obtained with a dose of 2 μl/ml (1.8-fold). These observations show that the three juices have different induction profiles: only water cress acted as a bifunctional inducer by enhancing both phase I and phase II enzymes. As a consequence, each juice may preferentially inhibit the genotoxicity of specific compounds.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na⁺/H⁺ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na⁺-resistance on an antiporter-deficient E. coli strain, nor did they confer Na⁺/H⁺ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Interaction of respiratory and photosynthetic electron transport, and evidence for membrane-bound pyridine-nucleotide dehydrogenases in Anabaena variabilis

Membranes isolated from vegetative cells of Anabaena variabilis (ATCC 29413) oxidine NADPH and NADH in a cyanide-sensitive reaction, NADPH being the better donor. In addition, both act as reductants for photosystem I in the light and in the presence of DCMU. An NADH-regenerating system has been introduced experimentally, functioning as donor system for photosystem I of Anabaena membranes. This NADH dehydrogenation is strictly light-dependent, sensitive to DBMIB, establishes a transhydrogenase system with NADP⁺ was hydrogen acceptor and competes with oxygen. Kinetic analysis of oxygen uptake with reference to nucleotide concentration exhibited different K_m- and V_max-values for NADPH and NADH in the light and in the dark.     

Kinetic and structural features of betaine aldehyde dehydrogenases: Mechanistic and regulatory implications

The betaine aldehyde dehydrogenases (BADH; EC 1.2.1.8) are so-called because they catalyze the irreversible NAD(P)⁺-dependent oxidation of betaine aldehyde to glycine betaine, which may function as (i) a very efficient osmoprotectant accumulated by both prokaryotic and eukaryotic organisms to cope with osmotic stress, (ii) a metabolic intermediate in the catabolism of choline in some bacteria such as the pathogen Pseudomonas aeruginosa, or (iii) a methyl donor for methionine synthesis. BADH enzymes can also use as substrates aminoaldehydes and other quaternary ammonium and tertiary sulfonium compounds, thereby participating in polyamine catabolism and in the synthesis of γ-aminobutyrate, carnitine, and 3-dimethylsulfoniopropionate. This review deals with what is known about the kinetics and structural properties of these enzymes, stressing those properties that have only been found in them and not in other aldehyde dehydrogenases, and discussing their mechanistic and regulatory implications.     

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.     

Oscillations of the NAD(P)H pool size and of the redox state of a cytochrome b during dark respiration of the blue-green alga, Anacystis nidulans

Oscillations of the oxygen uptake rate of the blue-green alga (cyanobacterium) Anacystis nidulans were induced by light pulses. The pool size of NAD(P)H and the redox state of a cytochrome b showed oscillations of similar shape and frequency. Phase diagrams revealed that these three oscillations were presumably linked. The cytochrome b should be a part of the respiratory chain of this blue-green alga. The oscillations were inducible only in a limited physiological state of the alga.     

Solubilization and spectral characteristics of chlorophyll-protein complexes isolated from the thermophilic blue-green alga Synechococcus lividus

The thermophilic blue-green alga Synechococcus lividus was grown at 38 and 55°C. The reaction center chlorophyll-protein complexes (CP) of Photosystem (PS I) and PS II, CP a_I and CP a_II, were isolated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis at 4°C. SDS solubilization of thylakoids was performed in the temperature range 0–65°C. The low-temperature absorption and fluorescence emission spectral properties of the isolated chlorophyll-protein complexes were analyzed. Only traces of CP a_I were solubilized at temperatures below the lipid phase transition temperature. Instead, a minor PS I component, CP a′_I, was obtained that had absorption and fluorescence characteristics similar to those of CP a_I. CP a′_I had a slightly lower mobility than CP a_I in SDS-polyacrylamide gel electrophoresis. The amount of CP a_I in the gel scan profile increased dramatically when solubilization was carried out above the phase transition temperatures, but started to decrease above 60°C. CP a_II, on the other hand, could be efficiently extracted even at 0°C and was stable in the scan profile up to extraction temperatures of 30–40°C. Low-temperature absorption and fluorescence emission spectra were typical for CP a_I and CP a_II and no specific effects of the two growth temperatures on these properties were observed. The phase transition temperature was considered to be critical for the solubilization of CP a_I, either because of the difficulties of SDS (especially as it forms micelles at low temperatures) in penetrating the solidified membrane lipids at temperatures below that of the phase transition or because the CP a_I monomers of the PS I antennae are so strongly bound to each other that they cannot be dissociated by SDS before thermal agitation has reached a certain level that is achieved above the phase transition temperature. We consider both the difficulties in solubilizing CP a_I at sub-transition temperatures and the heat stability of the two complexes as adaptations which enable Synechococcus to grow under extreme high-temperature regimes.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02–0.8 μ g/plate (38–1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in “classical” nitroreductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 μg NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP₆, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Effects of Photoperiod and Plant Developmental Stage on NADH Dehydrogenase and Photosystem Activities of Isolated Chloroplasts

Barley (Hordeum vulgare L. cv. Hassan) leaves were used to study the effects of developmental stage and photoperiod on the NAD(P)H-ferricyanide oxidoreductase (NAD(P)H-FeCNR) and on the photosystem (PS) 1 and 2 activities of isolated chloroplasts. From day 6 to day 12, both the PS 1 and NADH-FeCNR activities decreased while NADPH-FeCNR activity remained almost unchanged. Methyl jasmonate had no significant effect on the NAD(P)H-FeCNR activity changes. In 6- to 7- and 14- to 15-d-old plants, the NADH-FeCNR activity was higher during the photoperiod than during the dark period and, in the 14- to 15-d-old plants, the PS 1 activity increased during photoperiod in the same way, but to a lesser extent. The PS 1 activity of plants during a dark-accelerated senescence was low. The simultaneous changes in chloroplast PS 1 and NADH-FeCNR activities support the role proposed for the chloroplast NADH dehydrogenase complex in the cyclic electron transport.     

Different enzymes involved in NADH- and NADPH-dependent respiration in the cyanobacterium Anabaena variabilis

Abstract Filaments of N₂-grown Anabaena variabilis exhibit soluble NADPH- and membrane-bound NADH-oxidizing activities. The NADPH-specific enzyme has been identified as ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) by the thionicotinamide-NADP transhydrogenase test, a ferredoxin-dependent hydrogenase assay, and by diaphorase systems. The FNR is easily removed by washing of French-press-prepared membranes. Concurrently, a loss of NADPH-dependent respiration is apparent, which is not reconstitutable by addition of Anabaena cytochrome c -553. The NADH-oxidizing activity, however, is only slightly affected by the washing procedure, and is completely reconstituted by cytochrome c -553. NADPH-dependent oxygen uptake is strongly inhibited by NADP, whereas inhibition of NADH-dependent oxygen uptake by NAD is less pronounced. The data give evidence that NADH and NADPH oxidations linked to the respiratory chain are mediated by two different enzymes.     

Enhanced production of glutaric acid by NADH oxidase and GabD-reinforced bioconversion from l-lysine

Glutaric acid is a promising alternative chemical to phthalate plasticizer since it can be produced by the bioconversion of lysine. Though, recent studies have enabled the high-yield production of its precursor, 5-aminovaleric acid (AMV), glutaric acid production via the AMV pathway has been limited by the need for cofactors. Introduction of NAD(P)H oxidase (Nox) with GabTD enzyme remarkably diminished the demand for oxidized nicotinamide adenine dinucleotide (NAD⁺). Supply of oxygen through vigorous shaking had a significant effect on the conversion of AMV with a reduced requirement of NAD ⁺. A high conversion rate was achieved in Nox coupled GabTD reaction under optimized expression vector, terrific broth (TB), and pH 8.5 at high cell density. Supplementary expression of GabD resulted in the production of 353 ± 35 mM glutaric acid with 88.3 ± 8.7% conversion from 400 mM AMV. Moreover, the reaction with a higher concentration of AMV could produce 528 ± 21 mM glutaric acid with 66.0 ± 2.7% conversion. In addition, the co-biotransformation strategy of GabTD and DavBA whole cells could produce 282 mM glutaric acid with 70.8% conversion from lysine, compared to the 111 mM glutaric acid yield from the combined GabTD–DavBA system.     

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate,permeabilized cells,and tissue slices

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.     

Modification of substrate specificity in single point mutants of Agrobacterium tumefaciens type II NADH dehydrogenase

Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.     

Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)