A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

Effect of NAD recirculation on the mechanism of ATP stabilization in cytoplasm. Mathematical models]

A mathematical model of the glycolytic system with the cytoplasmic coenzymes NAD+ and NADH as essential variables is proposed. It has been shown that any increase in the steady-state concentration of NADH will reduce the range of activity of the "generalized" ATPase, wherein the level of ATP is stabilized. Such a reduction in the range of ATP stabilization may be caused by an increasing rate of the pyruvate loss into non-glycolytic pathways, in particular, into mitochondria. This effect may be compensated by increasing oxidation of NADH by the dehydrogenases of H+-transferring cytosol-mitochondrial shuttles (malate-aspartate or alpha-glycerophosphate). The properties of the complete model were compared with those of its simplified version, which takes account only of the phosphotransferase reactions of glycolysis. The effects of various factors, which do not alter the level of NADH in the system, may be studied within the scope of the simplified model.     

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.     

Isolated durum wheat and potato cell mitochondria oxidize externally added NADH mostly via the malate/oxaloacetate shuttle with a rate that depends on the carrier-mediated transport

We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH. We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle. In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane. No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the alpha-glycerophosphate/dihydroxyacetonphosphate shuttles. We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 microm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively). The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.     

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 NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

Inhibition of NADH-dehydrogenase by low concentrations of NAD+]

Low concentrations of NAD+ inhibit the NADH: acceptor reductase reactions catalyzed by soluble NADH dehydrogenase from bovine heart mitochondria. The degree of incomplete inhibition of the enzyme depends on the nature and concentration of artificial electron acceptors and is manifested only at low concentrations of the latter. Marked inhibition was demonstrated for the 2.6-dichlorophenolindophenol-, ferricyanide- and O2-reductase reactions, being weakly pronounced during the measurement of the NADH: cytochrome c reductase activity. The inhibition of the above reactions by oxidized NAD+ isn't competitive towards NADH. A kinetic scheme is proposed, which postulates NADH: acceptor reductase reactions occurrence via two mechanisms, namely, a ping-pong mechanism and oxidation of the product-enzyme complex by the acceptor. It was shown that low concentrations of NAD+ also inhibit the NADH oxidase reaction catalyzed by complex I.     

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.     

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.     

Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography.

In order to isolate the mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase EC 1.6.99.3) from wheat, we developed a one-step immunoaffinity procedure using antibodies raised against the NAD9 subunit. By native electrophoresis we showed that the antibodies are able to recognize the NAD9 subunit on the complex in its native form, therefore allowing the immunoaffinity chromatography. The complex retained on the column proved to be a functional complex I, since the preparation showed NADH:duroquinone and NADH:FeK3(CN)6 reductase activities which were inhibited by rotenone. The pattern of the protein subunits (about 30) eluted from the purified complex showed a high level of similarities with complex I purified from potato and broad bean by conventional techniques. Twelve subunits were identified by cross-reactions with antibodies against heterologous complex I subunits including mitochondrial- and nuclear-encoded proteins. In order to study the genetic origin of the subunits, we purified wheat complex I after in organello labelling of mitochondrial-encoded polypeptides. We found that no other complex I subunit than those corresponding to the nine mitochondrial nad genes sequenced so far, is encoded in the mitochondria of wheat.     

Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD+ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD+ carrier protein (Ndt1p) is described. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not alpha-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD+-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The delta ndt1 delta ndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.     

The oxidation of cytosolic NAD(P)H by external NAD(P)H dehydrogenases in the respiratory chain of plant mitochondria

The respiratory chain of plant mitochondria differs from that in mammalian mitochondria by containing several rotenone-insensitive NAD(P)H dehydrogenases. Two of these are located on the outer, cytosolic surface of the inner membrane. One is specific for NADH, the other for NADPH. Only the latter is inhibited by diphenyleneiodonium (DPI). Both of these enzymes are normally dependent upon Ca²⁺ for activity and this constitutes a potentially important mechanism by which the cell can regulate the oxidation of cytosolic NAD(P)H via the concentration of free Ca²⁺. This and other potential regulatory mechanisms such as the substrate concentration and polyamines are discussed.     

Ubiquinone-1 Induces External Deamino-NADH Oxidation in Potato Tuber Mitochondria

The addition of ubiquinone-1 (UQ-1) induced Ca2+-independent oxidation of deamino-NADH and NADH by intact potato (Solanum tuberosum L. cv Bintje) tuber mitochondria. The induced oxidation was coupled to the generation of a membrane potential. Measurements of NAD+-malate dehydrogenase activity indicated that the permeability of the inner mitochondrial membrane to NADH and deamino-NADH was not altered by the addition of UQ-1. We conclude that UQ-1-induced external deamino-NADH oxidation is due to a change in specificity of the external rotenone-insensitive NADH dehydrogenase. The addition of UQ-1 also induced rotenone-insensitive oxidation of deamino-NADH by inside-out submitochondrial particles, but whether this was due to a change in the specificity of the internal rotenone-insensitive NAD(P)H dehydrogenase or to a bypass in complex I could not be determined.     

Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+

1-methyl-4-phenylpyridine (MPP+), a major product of the oxidation of the neurotoxic amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been postulated to be the compound responsible for destruction of nigrostriatal neurons in man and primates and for inhibition of mitochondrial NADH oxidation which leads to cell death. We have confirmed that 0.5 mM MPP+ inhibits extensively the oxidation of NAD+-linked substrates in intact liver mitochondria in State 3 and after uncoupling, while succinate oxidation is unaffected. However, in inverted mitochondria, inner membrane preparations, and Complex I NADH oxidation is not significantly affected at this concentration of MPP+, nor are malate and glutamate dehydrogenases or the carriers of these substrates inhibited. We report here the discovery of an uptake system for MPP+ in mitochondria which is greatly potentiated by the presence of malate plus glutamate and inhibited by respiratory inhibitors, suggesting an energy-dependent carrier. A 40-fold concentration of MPP+ in the mitochondria occurs in ten minutes. This might account for the inhibition of malate and glutamate oxidation in intact mitochondria.     

Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: central role of mitochondrial NADH redox state

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonadotropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by L-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.     

The flavoprotein subcomplex of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria: insights into the mechanisms of NADH oxidation and NAD+ reduction from protein film voltammetry

Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.     

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD⁺ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD⁺ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.