Type II NAD(P)H dehydrogenases are targeted to mitochondria and chloroplasts or peroxisomes in Arabidopsis thaliana

We found that four type II NAD(P)H dehydrogenases (ND) in Arabidopsis are targeted to two locations in the cell; NDC1 was targeted to mitochondria and chloroplasts, while NDA1, NDA2 and NDB1 were targeted to mitochondria and peroxisomes. Targeting of NDC1 to chloroplasts as well as mitochondria was shown using in vitro and in vivo uptake assays and dual targeting of NDC1 to plastids relies on regions in the mature part of the protein. Accumulation of NDA type dehydrogenases to peroxisomes and mitochondria was confirmed using Western blot analysis on highly purified organelle fractions. Targeting of ND proteins to mitochondria and peroxisomes is achieved by two separate signals, a C-terminal signal for peroxisomes and an N-terminal signal for mitochondria.     

Disruption of alternative NAD(P)H dehydrogenases leads to decreased mitochondrial ROS in Neurospora crassa

Mitochondria are a main providers of high levels of energy, but also a major source of reactive oxygen species (ROS) during normal oxidative metabolism. The involvement of Neurospora crassa alternative NAD(P)H dehydrogenases in mitochondrial ROS production was evaluated. The growth responses of a series of respiratory mutants to several stress conditions revealed that disrupting alternative dehydrogenases leads to an increased tolerance to the redox cycler paraquat, with a mutant devoid of the external NDE1 and NDE2 enzymes being significantly more resistant. The nde1nde2 mutant mitochondria show a significant decrease in ROS generation in the presence and absence of paraquat, regardless of the respiratory substrate used, and an intrinsic increase in catalase activity. Analysis of ROS production by a complex I mutant (nuo51) indicates that, as in other organisms, paraquat-derived ROS in Neurospora mitochondria occur mainly at the level of complex I. We propose that disruption of the external NAD(P)H dehydrogenases NDE1 and NDE2 leads to a synergistic effect diminishing ROS generation by the mitochondrial respiratory chain. This, in addition to a robust increase in scavenging capacity, provides the mutant strain with an improved ability to withstand paraquat treatment.     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.     

New insights into type II NAD(P)H:quinone oxidoreductases.

Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2.     

NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

Submitochondrial particles (SMP) were isolated from potato ( Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O₂, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O₂ as acceptor) with optimum activity at pH 8 and a very low K_m(NADH) of 3 μ M . It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high K_m(NADPH) of more than 1 m M . This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher K_m(NADH) of 14 μ M . It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low K_m(NADPH) of 25 μ M . This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.     

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.     

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

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 gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of synechocystis PCC6803

The physiological function of the type 1 NAD(P)H dehydrogenase (Ndh-1) of Synechocystis sp. PCC6803 has been investigated by inactivating the gene ndhH encoding a subunit of the complex. Molecular analysis of independent transformants revealed that all clones were heteroploid, containing both wild-type and mutant ndhH copies, whatever the metabolic conditions used during genome segregation, including high CO(2) concentration. By replacing the chromosomal copy of the ndhH gene by a plasmidial copy under the control of a temperature-controlled promoter, we induce a conditional phenotype, growth being only possible at high temperature. This clearly shows for the first time that an ndh gene is indispensable to the survival of Synechocystis sp. PCC6803.     

The chloroplast NAD(P)H dehydrogenase complex interacts with photosystem I in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I (PSI) cyclic and chlororespiratory electron transport in higher plants. Although biochemical and genetic evidence for its subunit composition has accumulated, it is not enough to explain the complexes putative activity of NAD(P)H-dependent plastoquinone reduction. We analyzed the NDH complex by using blue native PAGE and found that it interacts with PSI to form a novel supercomplex. Mutants lacking NdhL and NdhM accumulated a pigment-protein complex with a slightly lower molecular mass than that of the NDH-PSI supercomplex; this may be an intermediate supercomplex including PSI. This intermediate is unstable in mutants lacking NdhB, NdhD, or NdhF, implying that it includes some NDH subunits. Analysis of thylakoid membrane complexes using sucrose density gradient centrifugation supported the presence of the NDH-PSI supercomplex in vivo. Although the NDH complex exists as a monomer in etioplasts, it interacts with PSI to form a supercomplex within 48 h during chloroplast development.     

Bioluminescence analysis of NAD(P)H and NAD(P)+ preventing mutual interference by selective nucleotide destruction and enhanced specific light emission.

Improved bioluminescence analysis of pyridine nucleotides has been designed based on the fact that the luminescence intensity expresses the velocity of the light formation. The bacterial luciferase system is, in principle, composed of two reactions with two different velocities, one for energy supply by the oxidation of NAD(P)H and the other for the subsequent light generation. The rate setting can be arranged such that an emission maximum is produced 30 to 40 s after mixing the sample with the light-yielding solution, hence providing for a convenient analytical performance. The maximal intensity which is easily recorded, e.g., by a tracking volt-meter, is proportional to the concentration of the reduced nucleotide. Discriminative analysis of the various pyridine nucleotides is facilitated by selective destruction of the oxidized forms with alkali and the reduced forms with acid. Erroneous conversion of NAD(P)H to NAD(P)+ may be induced by haemoglobin in a tissue sample but this is prevented by the presence of 2 mM ascorbic acid at the instant of the acidification. Simultaneous coupling of the ongoing reduction of a pyridine nucleotide to the oxidation in the bacterial luciferase system generates a light-yielding cycle which offers important advantages. With NAD(P)+ as the analytic target compound, direct measurement replaces a preceding separate conversion to NAD(P)H. The four nucleotide forms become determinable in a sample by combining selective destruction of either the reduced or oxidized species with a nucleotide-specific reduction in the cycle. Discriminative analyses are furthermore facilitated by the enhanced emission which is due to the energy derived from the continuous specific reduction, whereas initial light signals from side reactions fade out. It is often possible to suppress disturbing analytical errors by the design of the light-yielding cycle. If the rate of the dehydrogenase reactions is kept low compared with the overall rate of the luciferase system, moderately impaired function of some of its components may only give rise to a slight and tolerable decrease in emission intensity. Kinetic evaluations and model experiments are presented and supplemented with applications to tissue samples.     

Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control

An NAD(P)H oxidase has been hypothesized to be the main source of reactive oxygen species (ROS) in vessels; however, questions remain about its function and similarity with the neutrophil oxidase. Therefore, vascular superoxide generation was measured by electron paramagnetic resonance spectroscopy using the spin-trap 5,5'-dimethly-pyrroline-N-oxide in aortas from wild-type (WT) and gp91(phox)-deficient mice (gp91(phox)-/-), which do not have a functioning neutrophil NADPH oxidase. There was no significant difference between radical adduct formation by WT or gp91(phox)-/- mouse aortas either at baseline or after stimulation with NADPH or NADH. Also, spin-adduct formation was identical in the 100,000-g pellets obtained from WT and gp91(phox)-/- mouse aortas. SOD mimetics and the flavoenzyme inhibitor diphenyleneiodonium blocked spin-adduct formation from both intact vessels and particulate fractions. Other pharmacological inhibitors of metabolic pathways involved in ROS generation had no effect on this phenomenon. To examine the role of this enzyme in vascular tone control, aortic rings were suspended in organ chambers and preconstricted with phenylephrine to reach half-maximal contraction. Exposure to NADPH elicited a 20% increase in vascular tone, which was decreased by SOD mimetics in a concentration-dependent manner, suggesting that superoxide was responsible for this phenomenon. NADH had no effect on vascular tone. Thus superoxide is generated in the vessel wall by an NAD(P)H-dependent oxidase, which modulates vascular contractile tone. This enzyme is structurally and genetically distinct from the neutrophil NADPH oxidase.     

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.