The oxidation of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

The oxidation of NADH by tetravalent vanadium

Vanadyl (V_(IV)) salts autoxidize in neutral aqueous solution yielding O₂⁻ plus vanadate (V_(V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H₂O₂ was present V_(IV)) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H₂O₂ to OH⁻ plus HO., by V_(IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V_(V)) to V_(IV), these reactions are likely to contribute to the toxicity of vanadate.     

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.     

Interactions of Adriamycin aglycones with mitochondria may mediate Adriamycin cardiotoxicity

Adriamycin and related anthracyclines are potent oncolytic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone metabolites of Adriamycin (5–20 μM) induce a Ca²⁺-dependent increase in the permeability of the inner mitochondrial membrane of both heart and liver mitochondria to small (< 1500 Da) solutes; this phenomenon is accompanied by release of mitochondrial Ca²⁺, mitochondrial swelling, collapse of the membrane potential, oxidation of mitochondrial pyridine nucleotides [NAD(P)H], uncoupling, and a transition from the condensed to the orthodox conformation and is inhibited by ATP, dithiothreitol, the immunosuppressant cyclosporin A, and the ubiquitous polyamine spermine. Aglycones also modify mitochondrial sulfhydryl groups and induce a Ca²⁺ independent oxidation of mitochondrial NAD(P)H which appears to reflect electron transport from NADH to oxygen, mediated by the aglycones and resulting in the production of Superoxide (O₂⁻). Selenium deficiency and butylated hydroxytoluene inhibit aglycone-induced Ca²⁺ release from liver, but not heart, mitochondria, suggesting that the interactions of the aglycones with mitochondria diner in these two tissues. It can be proposed that the effects of Adriamycin aglycones on heart mitochondria are responsible for the cardiotoxicity of the parent drug.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

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.     

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)     

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD⁺ or NADH at about the same operating redox potential as the NADH/NAD⁺ pool and comprise the NADH/NAD⁺ isopotential enzyme group. Complex I (specifically the flavin, site I_F) is often regarded as the major source of matrix superoxide/H₂O₂ production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H₂O₂ production. To differentiate the superoxide/H₂O₂-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H₂O₂ production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H₂O₂ production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)⁺ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H₂O₂ at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H₂O₂ was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site I_F of complex I. Depending on the substrates present, the dominant sites of superoxide/H₂O₂ production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.     

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.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

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.     

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.     

NAD(P)H oxidase V from Lactobacillus plantarum (NoxV) displays enhanced operational stability even in absence of reducing agents

Active pharmaceutical ingredients (APIs) such as l-sugars and keto acids are favorably accessed through selective oxidation of sugar alcohols and amino acids, respectively, catalyzed by NAD(P)-dependent dehydrogenases. Cofactor regeneration from NAD(P)H conveniently is achieved via water-forming NAD(P)H oxidases (nox2), which only need molecular oxygen as co-substrate. Turnover-dependent overoxidation of the conserved cysteine residue in the active site of water-forming NADH oxidases is the presumed cause of the limited nox2 stability.We present a novel NAD(P)H oxidase, NoxV from Lactobacillus plantarum, with specific activity of 167 U/mg and apparent kinetic constants at air saturation and 25 °C of k_cat,app = 212 s⁻¹ and K_M,app = 50.2 μM in the broad pH optimum from 5.5 to 8.0. The enzyme features a higher stability than other NAD(P)H oxidases against overoxidation, as is evidenced by a higher total turnover number, in the presence (168,000) and, most importantly, also in the absence (128,000) of exogenously added reducing agents. While the native enzyme shows exclusively activity on NADH, we engineered the substrate binding pocket to generate variants, G178K,R and L179K,R,H that accommodate and oxidize both NADH and NADPH as substrates.     

Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria

This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca²⁺, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca²⁺, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca²⁺ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.     

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

Oxidation of External NAD(P)H by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria : A Kinetic and Inhibitor Study

The functional interaction between the externally located NAD(P)H dehydrogenase and the Q-pool acceptor site(s) in Percoll-purified mitochondria from Jerusalem artichoke (Helianthus tuberosus L. cv OB1) mitochondria has been investigated. Oxidation of exogenous NADH is stimulated by ubiquinone (UQ₁) with a parallel decrease of the apparent K_m for NADH. In the presence of saturating amounts of UQ₁ as electron acceptor, the K_m (NADH) is not affected by variations of the ionic strength. Conversely, the K_m for UQ₁ is decreased by the screening effect of negative charges on the outer membrane surface. Under low-ionic strength, the hydroxyflavone platanetin progressively inhibits NADH oxidation with a mean inhibition dose of approximately 3 nanomoles of inhibitor per milligram of protein. Interestingly, under high-ionic strength, oxidation of NADH proceeds through two platanetin binding sites, one of which has a lower affinity for the inhibitor (mean inhibition dose = 20 nanomoles per milligram protein), because it is located near the outer surface of the membrane. This latter site is the one involved in the oxidation of external NADPH and, possibly, also affected by spermine and spermidine. Similarly to NADH, oxidation of NADPH is fully sensitive to micromolar concentrations of free Ca²⁺ ions; in addition, similar concentrations of the sulfhydryl reagent mersalyl are required to inhibit both NADH and NADPH oxidative activities. The results are interpreted as evidence for the presence of a single nonspecific NAD(P)H dehydrogenase.     

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O₂-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.