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.     

Activation of the external pathway of NADH oxidation in liver mitochondria of cold-adapted rats.

15 min cold exposure of rats adapted to cold results in switching on a pathway of the fast oxidation of extramitochondrial NADH in the isolated liver mitochondria. This pathway is sensitive to mersalyl and cyanide, resistant to amytal and antimycin A, and can be stimulated by dinitrophenol. A portion of the endogenous cytochrome c pool can easily be removed by washing mitochondria of the cold-exposed rats. A scheme is discussed, postulating desorption of the inner membrane-bound cytochrome c into intermembrane space of mitochondria, resulting in formation of a link between the non-phosphorylating NADH-cytochrome c reductase in the outer mitochondrial membrane and cytochrome c oxidase in the inner membrane. It is suggested that such an oxidative pathway is involved in the urgent heat production in liver in response to the cold treatment.     

Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes

Addition of exogenous NADH to rotenone- and antimycin A-treated mitochondria, in 125 mM KCl, results in rates of oxygen uptake of 0.5-1 and 10-12 nanoatoms of oxygen X mg protein-1 X min-1 in the absence and presence of cytochrome c, respectively. During oxidation of exogenous NADH there is a fast and complete reduction of cytochrome b5 while endogenous or added exogenous cytochrome c become 10-15% and 100% reduced, respectively. The reoxidation of cytochrome b5, after exhaustion of NADH, precedes that of cytochrome c. NADH oxidation is blocked by mersalyl, an inhibitor of NADH-cytochrome b5 reductase. These observations support the view of an electron transfer from the outer to the inner membrane of intact mitochondria. Both the rate of exogenous NADH oxidation and the steady state level of cytochrome c reduction increase with the increase of ionic strength, while the rate of succinate oxidation undergoes a parallel depression. These observations suggest that the functions of cytochrome c as an electron carrier in the inner membrane and as an electron shuttle in the intermembrane space are alternative. It is concluded that aerobic oxidation of exogenous NADH involves the following pathway: NADH leads to NADH-cytochrome b5 reductase leads to cytochrome b5 leads to intermembrane cytochrome c leads to cytochrome oxidase leads to oxygen. It is suggested that the communication between the outer and inner membranes mediated by cytochrome c may affect the oxidation-reduction level of cytosolic NADH and the related oxidation-reduction reactions.     

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.     

Disappearance of the cyanide-insensitive pathway of oxidation in mitochondria of MI-1 mutant of Neurospora crassa in vitro.

Oxidation of exogenous NADH in mitochondria isolated from wild type and mi-1 mutant of Neurospora crassa decreases rapidly in vitro. In mi-1 mutant mitochondria the inactivation concerns the alternate pathway of oxidation whereas in the wild type it involves an unknown component of the respiratory chain. The activity of the primary NADH dehydrogenase is constant within the time of the experiments (2-4 h). NADH oxidase is not inactivated if oxygen is removed from the incubation medium by nitrogen bubbling. Succinate oxidase does not show any remarkable changes in activity within 2-3 h. In fresh mitochondria of the mi-1 mutant reduced ubiquinone is completely reoxidized by cytochrome oxidase but only 80% reoxidized by the alternate oxidase. In aged mitochondria of the mi-1 mutant in the presence of cyanide, ubiquinone is reduced to the level characteristic for fresh mitochondria in which respiration is completely inhibited by cyanide plus salicylhydroxamic acid. In these mitochondria the reoxidation of the reduced ubiquinone proceeds only via the cytochrome pathway. It is supposed that a labile component(s) of the respiratory chain present in the mi-1 mutant and the wild type mitochondria may, in mi-1 mutant, act as an alternate oxidase.     

Activation of the external pathway of NADH oxidation in liver mitochondria of cold-adapted rats

15 min cold exposure of rats adapted to cold results in switching on a pathway of the fast oxidation of extramitochondrial NADH in the isolated liver mitochondria. This pathway is sensitive to mersalyl and cyanide, resistant to amytal and antimycin A, and can be stimulated by dinitrophenol. A portion of the endogenous cytochrome c pool can easily be removed by washing mitochondria of the cold-exposed rats.A scheme is discussed, postulating desorption of the inner membrane-bound cytochrome c into intermembrane space of mitochondria, resulting in formation of a link between the non-phosophorylating NADH-cytochrome c reductase in the outer mitochondrial membrane and cytochrome c oxidase in the inner membrane. It is suggested that such an oxidative pathway is involved in the urgent heat production in liver in response to the cold treatment.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

Modulation of the Access of Exogenous NAD(P)H to the Alternative Pathway in Potato Tuber Callus Mitochondria with Triton X-100

Alternative oxidase activity in potato tuber (Solanum tuberosum L. cv Bintje) callus mitochondria with exogenous NAD(P)H as substrate is inhibited by low concentrations of the detergent Triton X-100. Alternative oxidase activity with succinate or malate as substrate is not affected by these low concentrations of Triton X-100. Cytochrome pathway activity was not influenced under these conditions, neither with endogenous nor with exogenous substrate. Washing of Triton X-100-treated mitochondria did partially restore both uninhibited and CN-resistant NADH oxidation, indicating that under these conditions Triton X-100 does not permanently remove major components from the mitochondrial membrane. Apparently, it is possible to manipulate mitochondria in such a way that the access of exogenous NADH to the alternative pathway is blocked while access to the cytochrome pathway is uninhibited. It is suggested that membrane conditions have a regulatory function (possibly via influencing the diffusion path) in the oxidation of exogenous NADH via the alternative pathway.     

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.     

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).     

Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH

Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.     

Fine structural localization of alkaloid synthesis in endoplasmic reticulum of submergedClaviceps purpurea

Cyanide-insensitive mitochondria from Saccharomycopsis lipolytica possess an exogenous NADH dehydrogenase, located outside the inner mitochondrial membrane, and linked to coupling site II. These mitochondria are able to oxidize exogenous NADH via two pathways: (1) a cyanide- and antimycin-sensitive pathway, or cytochrome pathway, and (2) a cyanide- and antimycin-insensitive pathway, or alternative pathway. Although the oxidation of exogenous NADH through the cytochrome pathway occurs with an ATP/0 ratio tending to 2, it proceeds, per molecule of NADH oxidized, with the apparent ejection in the outer medium of only 3 protons instead of 4 protons, as is the case with glycerol 3-phosphate as control substrate, but leaves 1 hydroxyl ion in the outer medium after decay of the protonmotive force. These properties were used to demonstrate the non electrogenic function of the alternative pathway. Indeed, the oxidation of exogenous NADH via the alternative pathway proceeds without apparent ejection of protons, although this oxidation generates an electron flux in the alternative pathway as demonstrated by the net appearance in the outer medium of 1 hydroxyl ion per atom of oxygen reduced, appearance which proves sensitive to benhydroxamic acid, a specific inhibitor of the alternative pathway. The non electrogenicity of the alternative pathway is accompanied by the absence of ATP synthesis as expected from Mitchell's chemiosmotic model. The absence of energy conservation when the electron transfer proceeds via the alternative pathway is not the result of an uncoupling property of an active alternative pathway, as the oxidation of malate plus pyruvate via coupling site I and the alternative pathway occurs with an ATP/0 ratio tending to 1.     

Ceramide-induced activation of cytosolic NADH/cytochrome c electron transport pathway: An additional source of energy for apoptosis

We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

The mechanism of action of a synthetic derivative of 1,4-naphthoquinone on the respiratory chain of liver and heart mitochondria

It was found that the 1.4-naphthoquinone derivative AK-135 (2-methyl-3-piperidine-methyl-1.4-naphthoquinone hydrochloride) possesses a marked acceptor capacity during succinate and glutamate oxidation by rat liver and rabbit heart mitochondria. AK-135 fully restores the rate of glutamate (but not succinate) oxidation by liver and heart mitochondria catalyzed by rotenone, antimycin A and cyanide. In non-phosphorylating preparations of liver and heart mitochondria, AK-135 eliminates the inhibition of respiration on exogenous NADH induced by the same electron transport inhibitors. In liver mitochondria, the stimulation of succinate oxidation is due to a reverse electron transfer, whereas in the heart it proceeds via the rotenone-insensitive pathway. The experimental results suggest that in the liver and heart AK-135 accepts electrons from NADH-dehydrogenase oxidizing endogenous NADH. Besides, in the liver this compound is also capable of accepting electrons from NADH-cytochrome b5 reductase.     

Evidence for an alternative and non-phosphorylating pathway for NADH reoxidation in a yeast strain resistant to glucose repression

A yeast strain (SP1) resistant to glucose repression modified simultaneously in the fermentative and in the oxidative pathways (loss of alcohol dehydrogenase I and over production of cytochrome a + a3, being insensitive to the glucose effect) developed a secondary mitochondrial hydrogen pathway. Oxidative phosphorylation was measured with exogenous NADH as substrate on mitochondria derived from repressed or derepressed cells. In this strain, antimycin A promotes a partial inhibition of NADH oxidation but a complete inhibition of phosphorylation. Amytal partially inhibits oxidation of NADH but not phosphorylation. KCN inhibits NADH oxidation in a biphasic way (first level 0.1 mM, second level 5 mM) but phosphorylation was fully inhibited by 0.1 mM KCN. This alternative but non-phosphorylating pathway is insensitive to salicyl hydroxamate. The external NADH dehydrogenase, like cytochrome c oxidase is partially insensitive to catabolite repression. These results provide evidence for the presence in strain SP1 of an alternative mitochondrial pathway, going from the external NADH dehydrogenase to an oxidase, different from the normal NADH dehydrogenase ubiquinone pathway.     

External Pathway of NADH Oxidation in the Liver of the River Lamprey Lampetra fluviatilis

A possibility of exogenous NADH oxidation via the external pathway has been shown on homogenates and isolated liver cells of the lamprey Lampetra fluviatilis in the presence of rotenone and antimycin A. The homogenates were incubated in isotonic and hypotonic sucrose media, while cells, in isotonic salt medium. At incubating the tissue preparations in isotonic media, digitonin was used to enhance membrane permeability to NADH and cytochrome c. In homogenates, the maximal rate of NADH oxidation via the external pathway in the presence of cytochrome c and digitonin was 5.3 nmol O₂/min/10 mg wet weight. This value in the cells amounted to 12.6, while without addition of exogenous NADH and cytochrome c, to 11.0 nmol O₂/min/10 million cells. Cyanide inhibited completely the NADH oxidation via the external pathway both in homogenates and in cells. The intact lamprey hepatocytes, unlike homogenates, are suggested to contain sufficient concentrations of cytochrome c and extramitochondrial NADH to provide maximal NADH oxidation rate in mitochondria through external pathway. This allows thinking that potential possibilities of NADH oxidation via the external pathway in Cyclostomata and mammals are qualitatively and quantitatively close.