Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O₂⁻). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O₂⁻ generation, besides complex III, stimulated NO degradation. Larger amounts of O₂⁻ were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O₂⁻ production were stimulated by free Ca²⁺ concentration. Together, these results indicate that Ca²⁺-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O₂⁻ production that favors NO degradation in potato tuber mitochondria.     

Platanetin and 7-iodo-acridone-4-carboxylic acid are not specific inhibitors of respiratory NAD(P)H dehydrogenases in potato tuber mitochondria

Progress in understanding the role of NAD(P)H oxidation in plant respiration is restricted by the lack of access to specific inhibitors of each of the unknown number of NAD(P)H dehydrogenases in the inner mitochondrial membrane. Platanetin (3,5,7,8-tetrahydroxy-6-isoprenyl flavone) is known to be an inhibitor of extermal NADH oxidation by plant mitochondria, while 7-iodo-acridone-4-carboxylic acid (IACA) is an inhibitor of an internal, rotenone-insensitive NAD(P)H dehydrogenase isolated from yeast mitochondria.
Here we show that platanetin inhibits external NAD(P)H oxidation by intact potato ( Solanum tuberosum L. cv. Bintje) tuber mitochondria, deamino-NADH oxidation by Complex I assayed using inside-out submitochondrial particles from these mitochondria, and rotenone-insensitive NAD(P)H oxidation by these submitochondrial particles. IACA was found to inhibit the oxidation of external NADH and succinate by intact mitochondria with similar efficiency. However, IACA also inhibited NADPH and duroquinol oxidation by intact mitochondria as well as deamino-NADH and NAD(P)H oxidation by inside-out submitochondrial particles. This indicates that IACA has several sites of inhibition in the electron transport chain. The lack of specificity of both platanetin and IACA prevents these inhibitors from being used to shed more light on the identity of the NAD(P)H dehydrogenases in plant mitochondria.     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.     

Rotenone-insensitive NAD(P)H dehydrogenases in plants: Immunodetection and distribution of native proteins in mitochondria

Antisera produced against peptides deduced from potato nda1 and ndb1, homologues of yeast genes for mitochondrial rotenone-insensitive NADH dehydrogenases, recognise respective proteins upon expression in Escherichia coli. In western blots of potato (Solanum tuberosum L.) mitochondrial proteins, the NDB and NDA antibodies specifically detect polypeptides of 61 and 48 kDa, respectively. The proteins are found in mitochondria of flowers, leaves and tubers. Different signal intensities are seen relative to other respiratory chain components when organs are compared, indicating variations in relative abundance of dehydrogenases within the plant. The antibodies detect single polypeptides, of similar size as in potato, in mitochondria from several plant species. No specific cross-reaction was found in chloroplasts, but a weak NDA signal of 50 kDa was found in microsomes, possibly associated with peroxisomes. Two-dimensional native/SDS-PAGE analyses indicate that both NDA and NDB proteins reside as higher molecular mass forms, possibly oligomeric. The NDB immunoreactive protein is released by sonication of mitochondria, but is resistant to extraction by digitonin and partially to Triton X-100. In comparison, the NDA protein remains bound to the inner membrane at sonication or digitonin treatment, but can be solubilised with Triton. Investigation of a beetroot (Beta vulgaris L.) induction system for external NADH dehydrogenase indicates that the NDB antibody does not recognise the induced external NADH dehydrogenase in this species, but possibly an external NADPH dehydrogenase.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

After incubation at 42°C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (ΔndhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of ΔndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42°C for 6 h, the photochemical activity declined more markedly in ΔndhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (ΔpH), while the consumption of ΔpH was highly inhibited in ΔndhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO₂ assimilation by providing extra ΔpH, thus reducing the photooxidative damage.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

The environmental temperature is one of the mainfactors affecting plant growth and development. Insummer, plants are frequently influenced by hightemperature. In recent years, global temperature wasremarkably elevated accompanied with the climaticchanges,…     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

Dihydrodipicolinate reductase-like protein, CRR1, is essential for chloroplast NAD(P)H dehydrogenase in Arabidopsis

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in cyclic electron transport around photosystem I. In higher plants, 14 subunits of the NDH complex have been identified. The subunit that contains the electron donor-binding site or an electron donor to NDH has not been determined. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 is homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the dihydrodipicolinate-binding motif was not conserved in CRR1, and the crr1 defect was specific to accumulation of the NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. Similarly to other nuclear-encoded genes for NDH subunits, CRR1 was expressed only in photosynthetic tissue. CRR1 contained a NAD(P)H-binding motif and was a candidate electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable in crr2-2 lacking the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate.     

Cyclic AMP Enhancement of Depolarization-Induced NAD(P)H Changes in Brain Cortical Slices

The increased levels of NAD(P)H effected by electrical depolarization are markedly augmented in the presence of cyclic AMP, isoproterenol, or RO 20-1724, agents known to elevate cyclic AMP in rat brain slices. The data presented indicate that the cyclic AMP effect on an important component of intermediate metabolism is not an enhancement of a basal response but a separate response that is activated by depolarization, is Ca2+-dependent, regulates cytochrome a-a3 independently of its effects on NAD(P)H levels, and is dependent on a substrate other than glucose.     

Metabolic monitoring by using the rate of change of NAD(P)H fluorescene

The amino acid fermentation by Corynebacterium glutamicum was monitored with an new technique that uses the first derivative of the NAD(P)H fluorescene signal. The rate of change of NAD(P)H pools is indicative of intracellular redox balance variations that correspond to metabolic changes. The profile of this signal showed several characteristics that coincided with major metabolic events during fermentation. We show here that the derivative fluorescence signal can accurately estimate points of threonine depletion, viable cell count, and the end of amino acid formation. Furthermore, on-line optimization strategies can be developed by using the derivative fluorescene signal. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiae

Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole‐cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed‐batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95 mM adenine and 9.5 mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed‐batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95 mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl‐4‐chloro acetoacetate (CAAE) to the corresponding (S)‐4‐chloro‐3‐hydroxybutanoate (S‐CHBE) was evaluated with S. cerevisiae FasB His6 as an example. An intracellular threshold concentration above 0.07 mM NADP(H) was sufficient to increase the biocatalytic S‐CHBE productivity by 25 % compared to lower intracellular NADP(H) concentrations.     

Three novel subunits of Arabidopsis chloroplastic NAD(P)H dehydrogenase identified by bioinformatic and reverse genetic approaches

Chloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially in adaptation to environmental changes. Although the NDH complex contains 11 subunits that are homologous to NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), recent genetic and biological studies have indicated that NDH also comprises unique subunits. We describe here an in silico approach based on co-expression analysis and phylogenetic profiling that was used to identify 65 genes as potential candidates for NDH subunits. Characterization of 21 Arabidopsis T-DNA insertion mutants among these ndh gene candidates indicated that three novel ndf (NDH-dependent cyclic electron flow) mutants ( ndf1 , ndf2 and ndf4 ) had impaired NDH activity as determined by measurement of chlorophyll fluorescence. The amount of NdhH subunit was greatly decreased in these mutants, suggesting that the loss of NDH activity was caused by a defect in accumulation of the NDH complex. In addition, NDF1, NDF2 and NDF4 proteins co-migrated with the NdhH subunit, as shown by blue native electrophoresis. These results strongly suggest that NDF proteins are novel subunits of the NDH complex. Further analysis revealed that the NDF1 and NDF2 proteins were unstable in the mutants lacking hydrophobic subunits of the NDH complex, but were stable in mutants lacking the hydrophilic subunits, suggesting that NDF1 and NDF2 interact with a hydrophobic sub-complex. NDF4 protein was predicted to possess a redox-active iron–sulfur cluster domain that may be involved in the electron transfer.     

集胞蓝藻PCC6803含疏水亚基的NAD（P）H脱氢酶亚复合体的分离

利用离子交换与凝胶过滤层析 ,从n dodecylβ D maltoside(DM)处理的集胞蓝藻SynechocystisPCC6 80 3细胞粗提液中 ,首次分离到两个包含NDH疏水亚基NdhA的亚复合体。酶活性分析表明 ,分离到的NDH亚复合体具有NADPH 氮蓝四唑 (NBT)氧化还原酶活性 ,以NADPH为电子供体可以还原铁氰化钾、二溴百里香醌 (DBMIB)、二氯酚靛酚 (DCPIP)、duroquinone以及UQ 0等质醌类电子受体。     

Cyclic electron flow around photosystem I via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice

The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO(2) assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 μmol photons m(-2) sec(-1)), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The F(v)/F(m) ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

NAD(P)H: quinone oxidoreductase 1 inducer activity of novel 4-aminoquinazoline derivatives

Fourteen novel 4-aminoquinazoline derivatives 2–15 were designed and synthesized. The structure of the newly synthesized compounds was established on the basis of elemental analyses, IR, ¹H-NMR, ¹³C-NMR, and mass spectral data. The compounds were evaluated for their potential cytoprotective activity in murine Hepa1c1c7 cells. All of the synthesized compounds showed concentration-dependent ability to induce the cytoprotective enzyme NAD(P)H: quinone oxidoreductase (NQO1) with potencies in the low- to sub-micromolar range. This approach offers an encouraging framework which may lead to the discovery of potent cytoprotective agents.     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.