Demonstration of the existence of an organo-specific NADH dehydrogenase in heart mitochondria

Experimental evidence is presented showing the existence of an NADH-consuming enzyme in heart mitochondria, in addition to the NADH--ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo-specific. The "exogenous NADH dehydrogenase" of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy-dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin.     

The nuclear origin of the non-phosphorylating NADH dehydrogenases of plant mitochondria.

The oxidation of matrix and cytosolic NADH by isolated beetroot and wheat leaf mitochondria was investigated to determine whether the rotenone-insensitive NADH dehydrogenases of plant mitochondria were the products of nuclear or mitochondrial genes. After aging beetroot tissue (slicing and incubating in a CaSO4 solution), the induction of the level of matrix NADH oxidation in the presence of rotenone was greatly reduced in mitochondria isolated from tissue treated with cycloheximide, a nuclear protein synthesis inhibitor. This was also true for the oxidation of cytosolic NADH. Mitochondria isolated from chloramphenicol-treated tissue exhibited greatly increased levels of both matrix and external rotenone-insensitive NADH oxidation when compared to the increase due to the aging process alone. This increase was not accompanied by an increase in matrix NAD-linked substrate dehydrogenases such as malic enzyme nor intra-mitochondrial NAD levels. Possible explanations for this increase in rotenone-insensitive NADH oxidation are discussed. Based on these results we have concluded that the matrix facing rotenone-insensitive NADH dehydrogenase of plant mitochondria is encoded by a nuclear gene and synthesis of the protein occurs in the cytosol.     

The effect of rotenone on respiration in pea cotyledon mitochondria

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of α-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.     

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase.     

Modelling NADH turnover in plant mitochondria

NADH is central to the functioning of mitochondrial respiration. It is produced by enzymes in, or associated with, the tricarboxylic acid cycle in the matrix, and it is oxidized by two respiratory chain enzymes in the inner membrane, the rotenone-sensitive complex I and the rotenone-insensitive internal NADH dehydrogenase (ND_in). A simplified kinetic model for NADH turnover in the matrix of plant mitochondria is presented. Only the two main NADH-producing enzymes, NAD-malate dehydrogenase [EC 1.1.1.37] (MDH) and NAD-malic enzyme [EC 1.1.1.39] (ME), are considered. This model reproduces the complex behaviour of malate oxidation by isolated mitochondria in response to additions of ADP (state 3/state 4), NAD⁺ and/or rotenone, as well as to changes in pH. It is found that MDH always operates at or close to equilibrium. Changes in the activity of complex I, ND_in, or ME are predicted to cause clear changes in the pattern of malate oxidation. In general, the model predicts high sensitivity to changes in the ME activity. In contrast, MDH activity can be reduced 100-fold without detectable changes in malate oxidation. It is demonstrated that it is not the high activity, but the equilibrium properties of MDH that are important for the redox-buffering function of MDH in the mitochondrial matrix. Binding of NAD⁺ and NADH in the matrix reduces the concentrations of free NAD⁺ and NADH, depending on the concentration of binding sites and the binding strength. On the basis of the modelling results it is estimated that a significant proportion of the mitochondrial NAD is bound.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Characteristics of External NADH Oxidation by Beetroot Mitochondria

Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with O₂ as the electron acceptor. These mitochondria have a rotenone- and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO₄ solutions, and is present in the mitochondria isolated from aged tissue.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

The effect of Ca2+ on the oxidation of exogenous NADH by rat liver mitochondria.

The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl₂ (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca²⁺ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca²⁺ is also observed in mitochondria partially swollen in a hypotonic medium.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.     

The effect of halothane on mitochondrial permeability to NADH

The effect of halothane on oxidation of exogenous NADH was evaluated in intact and disrupted rat liver mitochondria. A change in oxygen uptake was observed which represented an interplay between inhibition of NADH dehydrogenase (tending to decrease respiratory rate) and loss of impermeability to NADH (tending to increase respiratory rate).     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

ATP synthesis during exogenous NADH oxidation. A reappraisal

This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c.     

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.     

Oxidation of NADH via an "external" pathway in skeletal-muscle mitochondria and its possible role in the repayment of lactacid oxygen debt

Mitochondria isolated from skeletal muscle of rat catalyse oxidation of the external NADH (in the presence of rotenone, antimycin A and cytochrome c) at a rate of 15 natoms O2/min/mg protein by a pathway sensitive to mersalyl. In a medium supplemented with commercial lactate dehydrogenase, or when mitochondria were incubated in the presence of a cytoplasm, the NADH oxidation could be arrested by pyruvate. The inhibitory effect of pyruvate could be released by lactate. In the presence of NAD and cytochrome c, the reconstructed system containing skeletal muscle mitochondria plus cytoplasmic fraction was active in oxidation of L-lactate despite of the presence of rotenone and antimycin A. The lactate oxidation was sensitive to mersalyl and cyanide.     

Rotenone-Insensitive Malate Oxidation by Isolated Plant Mitochondria

The inhibitory effect of rotenone, and the variability of theinhibition, on malate oxidation by isolated plant mitochondriahas been investigated. Under conditions where oxaloacetate removalis enhanced (by transamination with glutamate) the rotenoneinhibition is less severe. Where oxaloacetate is allowed toaccumulate, inhibition by rotenone is marked. Similarly, underconditions which would tend to increase the steady state levelof reduction of the NAD pool (restricting coupled electron flowwith oligomycin) rotenone inhibition is severe. It is concludedthat the degree of rotenone inhibition is related to factorsaffecting the equilibrium poise of malic dehydrogenase, anddoes not necessarily reflect the interaction between rotenoneand the respiratory chain. There appears to be no correlationbetween the effectiveness of rotenone inhibition and mitochondrialmalic enzyme activity. Although there may be separate pathwaysof intramitochondrial NADH oxidation, it appears to be prematureto postulate separation or compartmentation of intramitochondrialmalate oxidation pathways.     

Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.     

Inhibition of NADH oxidation by pyridine derivatives

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.