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• W2898849930 abstract "We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant (“clamped”) at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed. We recently reported a previously unrecognized mitochondrial respiratory phenomenon. When [ADP] was held constant (“clamped”) at sequentially increasing concentrations in succinate-energized muscle mitochondria in the absence of rotenone (commonly used to block complex I), we observed a biphasic, increasing then decreasing, respiratory response. Here we investigated the mechanism. We confirmed decades-old reports that oxaloacetate (OAA) inhibits succinate dehydrogenase (SDH). We then used an NMR method to assess OAA concentrations (known as difficult to measure by MS) as well as those of malate, fumarate, and citrate in isolated succinate-respiring mitochondria. When these mitochondria were incubated at varying clamped ADP concentrations, respiration increased at low [ADP] as expected given the concurrent reduction in membrane potential. With further increments in [ADP], respiration decreased associated with accumulation of OAA. Moreover, a low pyruvate concentration, that alone was not enough to drive respiration, was sufficient to metabolize OAA to citrate and completely reverse the loss of succinate-supported respiration at high [ADP]. Further, chemical or genetic inhibition of pyruvate uptake prevented OAA clearance and preserved respiration. In addition, we measured the effects of incremental [ADP] on NADH, superoxide, and H2O2 (a marker of reverse electron transport from complex II to I). In summary, our findings, taken together, support a mechanism (detailed within) wherein succinate-energized respiration as a function of increasing [ADP] is initially increased by [ADP]-dependent effects on membrane potential but subsequently decreased at higher [ADP] by inhibition of succinate dehydrogenase by OAA. The physiologic relevance is discussed. Understanding mitochondrial electron transport is fundamental to our knowledge of a myriad of metabolic, neurologic, neoplastic, and other physiologic and pathophysiologic states. Complex II (succinate dehydrogenase, SDH) 3The abbreviations used are: SDHsuccinate dehydrogenaseOAAoxaloacetic acidRETreverse electron transportROSreactive oxygen speciesΔΨmembrane potentialFCCPp-trifluoromethoxyphenylhydrazoneEDLextensor digitorum longus2DOG2-deoxyglucoseMPCmitochondrial pyruvate carrierODoptical densityANOVAanalysis of variance. is a major site for electron input into the mitochondrial respiratory chain. However, the control of complex II (succinate) -energized respiration is still not well-understood. succinate dehydrogenase oxaloacetic acid reverse electron transport reactive oxygen species membrane potential p-trifluoromethoxyphenylhydrazone extensor digitorum longus 2-deoxyglucose mitochondrial pyruvate carrier optical density analysis of variance. Mitochondrial respiration is commonly studied by adding substrates to generate electrons, most often using donors to complex I and/or complex II and under state 4 4For the purposes of this manuscript, we refer to state 4 as respiration in the presence of substrate but absence of added ADP, slightly at variance with the original description by Chance and Williams in 1955 (37Chance B. Williams G.R. Respiratory enzymes in oxidative phosphorylation. III. The steady state.J. Biol. Chem. 1955; 217 (13271404): 409-427Abstract Full Text PDF PubMed Google Scholar) as respiration after depletion of added ADP. (no ATP production) and state 3 (ADP present in high amounts to generate maximal ATP production) conditions. However, when ADP is added to succinate-energized mitochondria in amounts expected to induce state 3 respiration, O2 flux drops to low levels (1Chance B. Hagihara B. Activation and inhibition of succinate oxidation following adenosine diphosphate supplements to pigeon heart mitochondria.J. Biol. Chem. 1962; 237 (14019996): 3540-3545Abstract Full Text PDF PubMed Google Scholar, 2Schollmeyer P. Klingenberg M. Oxaloacetate and adenosinetriphosphate levels during inhibition and activation of succinate oxidation.Biochem. Biophys. Res. Commun. 1961; 4 (13748457): 43-4710.1016/0006-291X(61)90252-2Crossref PubMed Scopus (20) Google Scholar). By adding incremental amounts of ADP rather than large amounts and by clamping the concentrations of added ADP, we recently observed a phenomenon not reported previously (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar). The phenomenon is that respiration energized by succinate (without rotenone, commonly used with succinate to prevent reverse electron transport to complex I) actually increases over state 4 at low [ADP], reaches a peak at an intermediate concentration, and then sharply decreases to very low levels at high [ADP]. The loss of respiration at high ADP was clearly not because of simple loss of function over time of incubation, lower O2 tension, or opening of the mitochondrial permeability transition pore, because the loss was rapidly reversible by complex I substrates or downstream electron donors and there was no evidence of pore opening (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar). We were surprised to find no prior report indicating that succinate-energized respiration peaks at low [ADP] before declining at higher [ADP]. One reason may be that, as stated above, most past studies of respiration by isolated mitochondria have been carried out in the absence and/or presence of substantial amounts of ADP rather than at intermediate concentrations. It is not physiologic to study respiration only under state 3 or state 4 conditions. Mitochondria in vivo do not function at either extreme, but rather in between (4Brand M.D. Nicholls D.G. Assessing mitochondrial dysfunction in cells.Biochem. J. 2011; 435 (21726199): 297-31210.1042/BJ20110162Crossref PubMed Scopus (1617) Google Scholar). Another reason may be that the large portion of early studies of mitochondrial metabolism used liver mitochondria wherein we found that this biphasic succinate-energized effect is barely evident (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar). Moreover, an additional reason this phenomenon had not received past attention may be that, as above, it has become common practice to carry out studies of succinate-energized respiration in the presence of rotenone. However, rotenone is also not physiologic. In the current report, we describe the mechanism underlying the above-mentioned phenomenon. To do this, we used ADP clamp methodology, 2-deoxyglucose plus hexokinase (5Yu L. Fink B.D. Sivitz W.I. Simultaneous quantification of mitochondrial ATP and ROS production.Methods Mol. Biol. 2015; 1264 (25631011): 149-15910.1007/978-1-4939-2257-4_14Crossref PubMed Scopus (9) Google Scholar), combined with sensitive NMR technology to detect TCA cycle metabolites. In particular, this includes quantification of oxaloacetic acid (OAA), a metabolite that is very difficult to measure by MS because of instability (6Al Kadhi O. Melchini A. Mithen R. Saha S. Development of a LC-MS/MS method for the simultaneous detection of tricarboxylic acid cycle intermediates in a range of biological matrices.J. Anal. Methods Chem. 2017; 2017 (29075551)539183210.1155/2017/5391832Crossref PubMed Scopus (50) Google Scholar, 7Zimmermann M. Sauer U. Zamboni N. Quantification and mass isotopomer profiling of α-keto acids in central carbon metabolism.Anal. Chem. 2014; 86 (24533614): 3232-323710.1021/ac500472cCrossref PubMed Scopus (44) Google Scholar). Here we provide compelling evidence for the role of OAA in regulating biphasic complex II–supported respiration. Further, we describe how this process is related to changes in reverse electron transport (RET), reactive oxygen species (ROS), membrane potential (ΔΨ), and the oxidation/reduction state of NAD+ and NADH. These findings advance our understanding of complex II–energized respiration and, moreover, suggest that we may need to re-think how complex II respiration is studied, i.e. that we might best do this at intermediate degrees of ADP availability without rotenone. The physiologic relevance of our findings is discussed. Mitochondria were incubated for 20 min and the ADP concentration was clamped at the desired level (Fig. 1). On a given day separate 20-min incubations were carried out at all ADP concentrations shown on the x axis and the experiment was repeated on 6 different days. The O2 tension in the Oxygraph drops with time but respiration is not affected until O2 levels become very low. Nonetheless, because incubations were carried out for 20 min, it was necessary to periodically open the chamber to prevent marked deterioration in the oxygen content of the medium. O2 flux (Fig. 1A) is depicted as the average respiration over time assuming that respiration, which cannot be measured while the chamber is open, remains at the average of the value between the time of chamber opening and closing (see “Experimental procedures”). As shown in Fig. 1A, succinate-energized respiration was [ADP]-dependent, reaching a peak at 8 μm ADP before decreasing at higher concentrations. Metabolite and nucleotide concentrations were measured in the respiratory medium obtained at the end of each run (Fig. 1, B–G). Notably, the increase in OAA corresponded closely with the decrease in O2 flux whereas precursors (malate and fumarate) paralleled O2 flux and succinate utilization varied as expected dependent on consumption of the added substrate. NADH began to decrease as respiration peaked. Measurements of NAD+ were more variable but trended upward as NADH decreased. In these experiments (Fig. 2), incubations were performed on succinate-energized mitochondria with or without a low (0.5 mm) concentration of pyruvate or carried out in the presence of pyruvate alone (no succinate). In each individual incubation, ADP was sequentially added at intervals to increase clamped [ADP] to the levels indicated. Respiration and ΔΨ were measured as the plateau values at each ADP concentration. These data show that pyruvate prevented the loss in succinate-energized respiration (Fig. 2A) and potential (Fig. 2B) at higher [ADP]. This was the case even though pyruvate was not sufficient to drive respiration on its own (Fig. 2A). Succinate-energized mitochondria were incubated for 20 min in the presence of ADP clamped at 32 μm. Table 1 depicts respiration determined in the presence or absence of pyruvate and in the presence of pyruvate plus UK5099, a chemical inhibitor of the mitochondrial pyruvate carrier (8Hildyard J.C. Ammälä C. Dukes I.D. Thomson S.A. Halestrap A.P. Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier.Biochim. Biophys. Acta. 2005; 1707 (15863100): 221-23010.1016/j.bbabio.2004.12.005Crossref PubMed Scopus (79) Google Scholar). Metabolite and NADH concentrations were measured in the respiratory medium obtained at the end of each run. These data show that pyruvate rescue of respiration (otherwise very low at 32 μm ADP) was blocked by UK5099 and the clearance of OAA to citrate was prevented. Changes in the OAA precursors (malate and fumarate) and succinate utilization were as expected based on respiration. The lower concentrations of NADH in the absence of pyruvate or in the presence of pyruvate carrier inhibition are consistent with what was observed in Fig. 1F at high [ADP].Table 1Respiration and metabolite and NADP concentrations in succinate (10 mm) -energized mouse hind limb skeletal muscle mitochondria incubated in the presence of 32 μm ADPSuccinate aloneSuccinate + pyruvateSuccinate + pyruvate + UK5099Respiration (pmol O2/mg/min)652 ± 19**††6406 ± 2381393 ± 37**OAA (μm)8.63 ± 0.65**†0 ± 06.68 ± 0.29**Citrate (μm)0 ± 0**5.60 ± 0.420 ± 0**Malate (μm)215.6 ± 6.2**1641 ± 66351.3 ± 7.2**Fumarate (μm)10.2 ± 0.40**††40.0 ± 1.717.7 ± 0.19**Succinate (mm)9.53 ± 0.11**†7.43 ± 0.208.87 ± 0.12**NADH (pmol/mg)335 ± 30**†1019 ± 44541 ± 57**NADH/NAD+0.083 ± 0.006**0.263 ± 0.0260.146 ± 0.025* Open table in a new tab In further experiments (Table 2), succinate-energized mitochondria were isolated from mice deficient in the mitochondrial pyruvate carrier 1 (MPC1) or littermate controls in presence or absence of 0.5 mm pyruvate. Again, mitochondria were incubated for 20 min in the presence of ADP clamped at 32 μm. Consistent with the data for chemical inhibition of the pyruvate carrier (Table 1), pyruvate rescue of respiration required the carrier protein whereas the variations in metabolite concentrations were consistent with that observed for chemical inhibition.Table 2Respiration and metabolite concentrations in succinate (10 mm) -energized mouse hind limb skeletal muscle mitochondria isolated from the mitochondrial pyruvate carrier 1 knock out (MCP1 KO) and littermate control mice incubated in the presence of 32 μm ADPControlMCP1 KOp value (interaction)No pyruvate0.5 mm pyruvateNo pyruvate0.5 mm pyruvateRespiration (pmol O2/mg/min)832 ± 876553 ± 185***697 ± 411335 ± 45**p < 0.0001OAA (μm)8.70 ± 0.530 ± 0***10 ± 0.938.98 ± 1.2p < 0.001Citrate (μm)0 ± 06.58 ± 0.78***0 ± 00 ± 0p < 0.0001Malate (μm)274 ± 331538 ± 51***207 ± 10342 ± 12*p < 0.0001Fumarate (μm)14.2 ± 1.241.0 ± 1.3***13.9 ± 1.318.6 ± 0.69*p < 0.0001Succinate (mm)9.04 ± 0.167.74 ± 0.16***8.97 ± 0.058.95 ± 0.09p < 0.001 Open table in a new tab SDH activity of the extracted complex was first determined in the absence of ADP or in the presence of ADP at a low or high concentration (Fig. 3A). As opposed to the effect of ADP on succinate-driven respiration in the presence of functioning mitochondria (Fig. 1A), ADP, as expected, had no direct effect on the activity of the isolated complex. In further experiments, we found that OAA as low as 5 μm strongly inhibited SDH (Fig. 3B). To assess the specificity of the antibody employed in these assays, SDH was extracted by immune immobilization. The eluted protein was then run on a gradient gel and stained with Coomassie Blue (Fig. 3C). Visualization revealed bands at kDa values consistent with those expected for the four subunits of the SDH complex with little to suggest nonspecific extraction of other proteins and with a pattern very similar to that reported by the manufacturer of the antibody. Mitochondria were incubated in microplate wells with shaking within a plate reader for 20 min at different concentrations of clamped ADP in the presence or absence of pyruvate (0.5 mm). Mitochondria were energized by succinate under the same conditions used for the Oxygraph incubations of Fig. 1. H2O2 production under these conditions is largely generated at complex I through conversion from superoxide by MnSOD and serves as a marker for reverse electron transport (9Lambert A.J. Brand M.D. Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2004; 279 (15262965): 39414-3942010.1074/jbc.M406576200Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 10Liu Y. Fiskum G. Schubert D. Generation of reactive oxygen species by the mitochondrial electron transport chain.J. Neurochem. 2002; 80 (11948241): 780-78710.1046/j.0022-3042.2002.00744.xCrossref PubMed Scopus (972) Google Scholar11Votyakova T.V. Reynolds I.J. ΔΨm-Dependent and -independent production of reactive oxygen species by rat brain mitochondria.J. Neurochem. 2001; 79 (11677254): 266-27710.1046/j.1471-4159.2001.00548.xCrossref PubMed Scopus (509) Google Scholar). As shown in Fig. 4A, H2O2 production with or without pyruvate decreased markedly in incubations performed at higher [ADP]. In other experiments (Fig. 4, B–D), we measured ROS production by EPR spectroscopy, a specific signal for superoxide. Although superoxide may be generated at complex III or other sites beyond complex I, the marked decrease in superoxide at high [ADP] confirms the overall loss of ROS. In our prior report (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar), we showed that the effect of decreasing potential with incremental ADP additions on O2 flux could be mimicked using the chemical uncoupler, p-trifluoromethoxyphenylhydrazone (FCCP). However, we have become aware that the amount of incremental FCCP in our prior report was high compared with what has been used to titrate potential in other reports. Therefore, we reexamined this issue, using both FCCP and 2,4-dinitrophenol (DNP) to titrate potential. As shown in Fig. S1, when these uncouplers were employed, the relationship between potential and O2 flux was similar to that observed for ADP titration. We noted that the amount of FCCP needed to titrate potential to a given degree was less in this current study than in our above-mentioned past report. Mitochondria isolated from extensor digitorum longus (EDL), soleus, and gastrocnemius muscle from normal Sprague-Dawley rats (rats were used instead of mice for ease in isolating specific muscle types) were energized with 10 mm succinate and incubated with sequential increments in clamped ADP. Fig. S2A shows that respiration reached much higher levels in mitochondria from gastrocnemius (mixed fiber type) compared with soleus (oxidative) or EDL (glycolytic). However, when respiration was expressed as a percent of state 4 (Fig. S2B), there was no difference in peak respiration between these muscle types. Interestingly, however, for the oxidative soleus mitochondria, respiration maintained a greater fractional increase over state 4 through higher concentrations of ADP. As expected, mitochondrial membrane potential decreased for all muscle types with sequential additions of ADP (Fig. S2C), while appearing initially higher in gastrocnemius mitochondria presumably because of the initial higher respiratory rate (Fig. S2A). Studies dating back decades describe OAA inhibition of SDH (1Chance B. Hagihara B. Activation and inhibition of succinate oxidation following adenosine diphosphate supplements to pigeon heart mitochondria.J. Biol. Chem. 1962; 237 (14019996): 3540-3545Abstract Full Text PDF PubMed Google Scholar, 2Schollmeyer P. Klingenberg M. Oxaloacetate and adenosinetriphosphate levels during inhibition and activation of succinate oxidation.Biochem. Biophys. Res. Commun. 1961; 4 (13748457): 43-4710.1016/0006-291X(61)90252-2Crossref PubMed Scopus (20) Google Scholar, 12Azzone G.F. Ernster L. Klingenberg M. Energetic aspects of the mitochondrial oxidation of succinate.Nature. 1960; 188 (13685479): 552-55510.1038/188552a0Crossref PubMed Scopus (9) Google Scholar13Chance B. Hollunger G. Energy-linked reduction of mitochondrial pyridine nucleotide.Nature. 1960; 185 (13809106): 666-67210.1038/185666a0Crossref PubMed Scopus (69) Google Scholar, 14Panov A.V. Kubalik N. Zinchenko N. Ridings D.M. Radoff D.A. Hemendinger R. Brooks B.R. Bonkovsky H.L. Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2011; 300 (21248309): R844-R85410.1152/ajpregu.00528.2010Crossref PubMed Scopus (35) Google Scholar15Panov A.V. Vavilin V.A. Lyakhovich V.V. Brooks B.R. Bonkovsky H.L. Effect of bovine serum albumin on mitochondrial respiration in the brain and liver of mice and rats.Bull. Exp. Biol. Med. 2010; 149 (21113488): 187-19010.1007/s10517-010-0904-5Crossref PubMed Scopus (20) Google Scholar). However, those studies did not address the role of OAA in regulating O2 flux in intact respiring mitochondria. An older report showed that pyruvate prevented the loss of succinate-supported state 3 respiration, presumably by clearing OAA (16Panov A. Schonfeld P. Dikalov S. Hemendinger R. Bonkovsky H.L. Brooks B.R. The neuromediator glutamate, through specific substrate interactions, enhances mitochondrial ATP production and reactive oxygen species generation in nonsynaptic brain mitochondria.J. Biol. Chem. 2009; 284 (19304986): 14448-1445610.1074/jbc.M900985200Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). That report did not actually demonstrate OAA clearance, however. Also, until our recent report (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar), the biphasic dependence of succinate-supported respiration on [ADP] had not been reported. Here we clarify the mechanism underlying this biphasic phenomenon (Fig. 5). The initial increase in respiration with incremental but lower amounts of ADP is easily understandable because of the reduction in ΔΨ as ADP phosphorylation consumes potential (4Brand M.D. Nicholls D.G. Assessing mitochondrial dysfunction in cells.Biochem. J. 2011; 435 (21726199): 297-31210.1042/BJ20110162Crossref PubMed Scopus (1617) Google Scholar). It has long been known, that respiration increases with consumption of potential because of reduced downstream resistance to electron flow (17Sivitz W.I. Yorek M.A. Mitochondrial dysfunction in diabetes: From molecular mechanisms to functional significance and therapeutic opportunities.Antioxid. Redox Signal. 2010; 12 (19650713): 537-57710.1089/ars.2009.2531Crossref PubMed Scopus (503) Google Scholar). In fact, in our past report we documented that ATP production, along with respiration, rises and falls with respiration as [ADP] is sequentially incremented and that the phenomenon can be mimicked by sequential addition of a chemical uncoupler (3Bai F. Fink B.D. Yu L. Sivitz W.I. Voltage-dependent regulation of complex II energized mitochondrial oxygen flux.PloS One. 2016; 11 (27153112)e015498210.1371/journal.pone.0154982Crossref PubMed Scopus (10) Google Scholar). On the other hand, it is more difficult to explain the decrease in respiration despite continued reduction in ΔΨ at higher ADP concentrations (Figs. 1A and 2, A and B). Our current data strongly imply that this is because of inhibition of SDH by OAA. First, OAA accumulation closely paralleled the decrease in respiration (Fig. 1B) as well as the decrease in malate and fumarate precursors (Fig. 1, C and D) consistent with OAA feedback inhibition of SDH. Second, we show that metabolism of OAA to citrate by a low concentration of added pyruvate, in uninhibited and genetically normal mitochondria, prevents the inhibition of respiration and the decline in the succinate metabolites, malate and fumarate (Table 1, Table 2). Further, this effect of pyruvate was not because of pyruvate-energized respiration because the concentration of pyruvate required for reversal was too low to generate meaningful O2 flux on its own (Fig. 2A). Third, we show that the reversal effect of pyruvate was prevented by chemical or genetic inhibition of pyruvate uptake (Table 1, Table 2), indicating dependence on direct mitochondrial pyruvate utilization. Finally, we used modern methods to confirm decades-old reports that OAA, in fact, inhibits the extracted SDH complex and to add information indicating that it does so well within the range of the metabolite concentrations measured herein (Fig. 3B). Further, it is important to note that the activity of the extracted complex is not directly affected by ADP (Fig. 3A). This is as expected because any such effect would be dissociated from the effect of ADP to consume potential. Although OAA inhibition can explain the post-peak decrease in succinate-supported respiration, other processes come into play. It is important to consider the following. ADP additions decrease ΔΨ. Higher ΔΨ impairs forward electron transport and favors RET (18Brand M.D. Buckingham J.A. Esteves T.C. Green K. Lambert A.J. Miwa S. Murphy M.P. Pakay J.L. Talbot D.A. Echtay K.S. Mitochondrial superoxide and aging: Uncoupling-protein activity and superoxide production.Biochem. Soc. Symp. 2004; 71 (15777023): 203-21310.1042/bss0710203Crossref PubMed Scopus (146) Google Scholar, 19Skulachev V.P. Uncoupling: New approaches to an old problem of bioenergetics.Biochim. Biophys. Acta. 1998; 1363 (9507078): 100-12410.1016/S0005-2728(97)00091-1Crossref PubMed Scopus (817) Google Scholar). These conditions maintain NADH in the reduced state (9Lambert A.J. Brand M.D. Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2004; 279 (15262965): 39414-3942010.1074/jbc.M406576200Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 20Scialò F. Fernández-Ayala D.J. Sanz A. Role of mitochondrial reverse electron transport in ROS signaling: Potential roles in health and disease.Front. Physiol. 2017; 8 (28701960): 42810.3389/fphys.2017.00428Crossref PubMed Scopus (254) Google Scholar), in other words, impair electron donation at complex I. Conversely, lowering ΔΨ enhances forward electron transport and electron donation by NADH at complex I. Such electron donation allows NADH cycling back to NAD+, whereas the consequent reduction in [NADH] favors a shift in the malate < > OAA equilibrium toward OAA. RET generates ROS at complex I, and H2O2 production is a known marker of RET (9Lambert A.J. Brand M.D. Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2004; 279 (15262965): 39414-3942010.1074/jbc.M406576200Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 21O'Malley Y. Fink B.D. Ross N.C. Prisinzano T.E. Sivitz W.I. Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria.J. Biol. Chem. 2006; 281 (17060316): 39766-3977510.1074/jbc.M608268200Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), although some degree of RET might remain in the absence of detectable ROS. With these factors in mind, the sequence of events (over increasing [ADP]) would be as follows. As ADP is incremented to low concentrations ΔΨ drops mildly and, therefore, respiration increases. ΔΨ is still high and RET is active, however, so [NADH] is maintained, limiting electron donation by NADH at complex I and limiting NADH/NAD+ cycling. Hence, malate conversion to OAA will be impaired and SDH is not inhibited. As ADP is further incremented to intermediate concentrations, conditions begin to change. Although, RET (as detected by H2O2) decreases, OAA is still absent (or too low to detect) either because potential is still high enough or RET is still active enough that electron donation by NADH to CoQ at complex I is minimal. Given the leftward nature of the malate < > OAA equilibrium (22McEvily A.J. Mullinax T.R. Dulin D.R. Harrison J.H. Regulation of mitochondrial malate dehydrogenase: Kinetic modulation independent of subunit interaction.Arch. Biochem. Biophys. 1985; 238 (3985618): 229-23610.1016/0003-9861(85)90160-2Crossref PubMed Scopus (15) Google Scholar), it is not surprising that these conditions need to change substantially to enable OAA formation. However, as ADP is further incremented to higher concentrations, the greater drop in ΔΨ will prevent RET and favor forward electron transport, now enabling NADH electron donation and NADH/NAD+ cycling. OAA can now be generated in sufficient amounts to inhibit SDH and respiration. The concentrations of OAA are still small compared with malate, again consistent with the leftward nature of the malate dehydrogenase reaction, but only small concentrations of OAA are needed to inhibit SDH (Fig. 3B). The above processes at low and high [ADP] are depicted in Fi" @default.
• W2898849930 created "2018-11-09" @default.
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• W2898849930 date "2018-12-01" @default.
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• W2898849930 title "Oxaloacetic acid mediates ADP-dependent inhibition of mitochondrial complex II–driven respiration" @default.
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