Absence Of Oxidative Phosphorylation Research Articles

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

The flexibility of metabolic interactions between chloroplasts and mitochondria in Nicotiana tabacum leaf.

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the 'P700 oxidation capacity' of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700+ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast-mitochondrion interactions able to overcome lesions in energy metabolism.

The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases.

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.

Connexin-43 channels are a pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells

Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H+-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H+ ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 μm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore, lactate entry into the better-perfused recipient cells has a favourable alkalinizing effect and supplies substrate for oxidative phosphorylation. Cx43 is thus a novel target for influencing metabolite handling in junctionally-coupled tumours.

Hypoxia and classical activation limits Mycobacterium tuberculosis survival by Akt-dependent glycolytic shift in macrophages.

Cellular reactive oxygen species (ROS) is a major antibacterial defense mechanism used by macrophages upon activation. Exposure of Mycobacterium tuberculosis (Mtb)-infected macrophages to hypoxia is known to compromise the survival of the pathogen. Here we report that the hypoxia-induced control of intracellular Mtb load in RAW 264.7 macrophages was mediated by regulating the cellular ROS levels. We show that similar to classical activation, hypoxia incubation of macrophages resulted in decreased mitochondrial outer membrane potential (MOMP) and a concomitant increase in the cellular ROS levels. Mitochondrial depolarization and consequently higher ROS could be blocked by knocking down Akt using siRNAs, which acted by inhibiting the switch to glycolytic mode of metabolism, an essential adaptive response upon classical activation or hypoxic incubation of macrophages. Moreover, in the classically activated macrophages or in the macrophages under hypoxia incubation, supplementation with additional glucose had similar effects as Akt knockdown. Interestingly, in both the cases, the reversal of phenotype was linked with the ability of the mitochondrial F0–F1 ATP synthase activity to maintain the MOMP in the absence of oxidative phosphorylation. Both Akt knockdown and glucose supplementation were also able to rescue Mtb survival in these macrophages upon classical activation or hypoxia incubation. These results provide a framework for better understanding of how the interplay between oxygen supply, which is limiting in the human tubercular granulomas, and nutrient availability could together direct the outcome of infections in vivo.

Mitochondrial diaphorases as NAD+donors to segments of the citric acid cycle that support substrate‐level phosphorylation yielding ATP during respiratory inhibition

Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD(+) supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD(+) pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD(+) derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.

Which way does the citric acid cycle turn during hypoxia? The critical role of α‐ketoglutarate dehydrogenase complex

The citric acid cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the α-ketoglutarate dehydrogenase complex dictating their directionalities.

Stage specific developmental changes in the mitochondrial and surface membrane associated redox systems of Leishmania donovani promastigote and amastigote

Energy metabolism of Leishmania donovani parasite has been investigated under conditions imitating intralysosomal-like environment in the host organism. Trans-plasma membrane electron transport and oxygen uptake were inhibited progressively when promastigote cells were exposed to pH 5.5 and 37 degrees C. A special feature of the respiratory chain in amastigote was the absence of complex I, II, and IV. When L. donovani was grown at pH 5.5 and 37 degrees C, the acid excretory product succinate was increased in comparison to cells grown at pH 7.5 and 24 degrees C. The findings of this study showed that the amastigote form catabolized fatty acid to excrete succinic acid when oxidative phosphorylation was impaired. Amastigote mitochondria failed to generate membrane potential by oxidizable substrates. On the other hand, the amastigote cell showed absorbance change of safranine O when fatty acid was the oxidizable substrate. The safranine signal was completely reversed by valinomycin, carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone, malonate, and oxaloacetate. Our data suggest that the generation of metabolic energy from succinate/H+ efflux will contribute to energy requiring process of amastigote significantly. On the basis of these results, we conclude that due to absence of oxidative phosphorylation in amastigotes, energy linked functions in amastigotes might occur through fumarate reduction leading to DeltapH generation by succinate excretion.

Ecophysiology of neuronal metabolism in transiently oxygen-depleted environments: Evidence that GABA is accumulated pre-synaptically in the cerebellum

Interactions between coral reef topography, tide cycles, and photoperiod provided selection pressure for adaptive physiological changes in sheltered hypoxic niches to be exploited by specialized tropical reef fish. The epaulette shark Hemiscyllium ocellatum withstands cyclic hypoxia in its natural environment, many hours of experimental hypoxia, and anoxia for up to 5 h. It shows neuronal hypometabolism in response to 5% oxygen saturation. Northern-hemisphere hypoxia- and anoxia-tolerant vertebrates that over-winter under ice alter their inhibitory to excitatory neurotransmitter balance to forestall brain ATP depletion in the absence of oxidative phosphorylation. GABA immunochemistry, HPLC analysis and receptor binding studies in H. ocellatum cerebellum revealed a heterogeneous regional accumulation of neuronal GABA despite no change in its overall concentration, and a significant increase in GABA A receptor density without altered binding affinity. Increased GABA A receptor density would protect the cerebellum during reoxygenation when transmitter release resumes. While all hypoxia- and anoxia-tolerant teleosts examined to date respond to low oxygen levels by elevating brain GABA, the phylogenetically older epaulette shark did not, suggesting that it uses an alternative neuroprotective mechanism for energy conservation. This may reflect an inherent phylogenetic difference, or represent a novel ecophysiological adaptation to cyclic variations in the availability of oxygen.

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho + cells rho − mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho − mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho − cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho −. It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function ( rho −).

The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function

Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasite's life cycle. Here we report that silencing expression of the alpha subunit of the mitochondrial F(1)-ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the gamma subunit of F(1) that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.

Escherichia coli proton-translocating F 0F 1-ATP synthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation–reduction under fermentation

The Escherichia coli proton-translocating F 0F 1-ATP synthase has a priority in H + circulation through the membrane in maintaining proton-motive force in the context of ATP synthesis and hydrolysis. Recent advances in the study of this complex under fermentative growth have led to hypothesis that, in the absence of oxidative phosphorylation, F 0F 1 is implicated as an essential part of H + movement and ATP hydrolysis, associated with solute secondary transporters and/or enzymes of anaerobic oxidation–reduction. These associations can result from a protein–protein interaction by dithiol–disulfide interchange. In such associations F 0F 1 has novel functions in bacterial cell physiology.

A bioenergetic model of gossypol action: effects of gossypol on adult rat spermatogenic cells.

Rat spermatogenic cells have glycolytic rates of 124 +/- 36 nmol lactate.mg protein-1.h-1. Maximum glycolytic rates in the absence of oxidative phosphorylation do not exceed the basal rates by greater than 40%. From the rates of oxygen consumption and lactate production it can be calculated that less than 8% of the total ATP produced by spermatogenic cells is provided by glycolysis. These properties of spermatogenic cells would render them highly susceptible to cytotoxicity due to the action of agents like gossypol that impair mitochondrial oxidative phosphorylation or to conditions that produce testicular hypoxia. The main effect of gossypol on spermatogenic cell energy metabolism is to uncouple mitochondrial oxidative phosphorylation. In the absence of glucose, this uncoupling action of gossypol would change the NAD+/NADH redox coupling and, consequently, could modify metabolic fluxes through the lactic dehydrogenase catalyzed reaction.

The role of state 4 electron transport in the activation of state 3 respiration in potato mitochondria.

The initial state 3 respiration rate of potato mitochondria is markedly depressed, or attenuated. With several consecutive state 3/state 4 cycles the state 3 rate rises to a maximum, while the state 4 rate remains essentially unchanged. The development of state 3 respiration has been termed conditioning. An analysis of the process has indicated that state 4 is a better conditioner than state3 per se. Conditioning is also attained by preincubation in state 2, or under conditions designated pseudostate 2, wherein ADP is present, with or without oligomycin, and inorganic phosphate is absent. ADP implements the conditioning process in the absence of oxidative phosphorylation. The action of ADP in its secondary or modulator role appears to be positively cooperative, the kinetics of ADP involvement being second-order. S0.5 for ADP as a modulator of the conditioning process is approximately 62 μM, a value in excess of the Ks for ADP in oxidative phosphorylation. Electron transport is indispensable for conditioning, and it is suggested that conditioning and ATP synthesis represent alternative uses of respiratory energy. It is further suggested that to some extent state 4 underlies state 3.

Control of Phosphoenolpyruvate Synthesis by Substrate Level Phosphorylation in Guinea Pig Liver Mitochondria

Control of P-enolpyruvate synthesis has been studied in guinea pig liver mitochondria with respect to the relative rates of GTP generation directly from substrate level phosphorylation or indirectly from ATP via nucleoside diphosphate kinase. With malate or pyruvate plus malate as substrates, P-enolpyruvate production was greatly decreased in State 3 by addition of fluorocitrate (an inhibitor of aconitase) to prevent flux through substrate level phosphorylation. On the other hand, when ATP production by the electron transport chain was prevented by use of an uncoupling agent (together with oligomycin to inhibit ATPase activity), rates of P-enolpyruvate production from α-ketoglutarate were the same as in State 3, and were also unaffected by addition of fluorocitrate. In the absence of oxidative phosphorylation and with fluorocitrate present together with malate or pyruvate plus malate, addition of exogenous ATP was much less effective than α-ketoglutarate in promoting P-enolpyruvate synthesis. Intramitochondrial GTP levels and rates of P-enolpyruvate formation were consistently lower in State 4, State 3, and the uncoupled plus oligomycin state with pyruvate plus malate than with α-ketoglutarate plus malate as substrate when fluorocitrate was present, indicating a limitation in the rate of phosphorylation of GDP by intramitochondrial ATP for optimal rates of P-enolpyruvate synthesis. The maximal rate of conversion of ATP to GTP by nucleoside diphosphate kinase in the intact mitochondria was estimated to be 2 to 3 nmoles per min per mg of protein compared with a maximal rate of P-enolpyruvate formation of about 22 nmoles per min per mg of protein obtained with α-ketoglutarate plus malate as substrate. It is concluded that provision of GTP by substrate level phosphorylation is essential to support rates of P-enol-pyruvate formation by guinea pig liver mitochondria greater than 3 nmoles per min per mg of protein.