Renata L.S. Goncalves

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Background: At the redox potential of NADH/NAD+, at least four mitochondrial sites produce superoxide/H2O2. Results: We compare their capacities in situ in isolated mitochondria. Conclusion: Maximum capacities of complexes were 2-oxoglutarate dehydrogenase > pyruvate dehydrogenase > branched-chain 2-oxoacid dehydrogenase > complex I. Significance: H2O2 production from 2-oxoacid dehydrogenases can be considerable but may previously have been misattributed to complex I. Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.

Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

Background: Ten mitochondrial sites of superoxide/H2O2 generation are known, but their contributions in vivo are undefined. Results: We assessed their rates ex vivo in conditions mimicking rest and exercise. Conclusion: Sites IQ and IIF generated half the signal at rest. During exercise, rates were lower, and site IF dominated. Significance: Contributing sites ex vivo probably reflect those in vivo. The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.

The contributions of respiration and glycolysis to extracellular acid production

BACKGROUND The rate at which cells acidify the extracellular medium is frequently used to report glycolytic rate, with the implicit assumption that conversion of uncharged glucose or glycogen to lactate(-)+H(+) is the only significant source of acidification. However, another potential source of extracellular protons is the production of CO2 during substrate oxidation: CO2 is hydrated to H2CO3, which then dissociates to HCO3(-)+H(+). METHODS O2 consumption and pH were monitored in a popular platform for measuring extracellular acidification (the Seahorse XF Analyzer). RESULTS We found that CO2 produced during respiration caused almost stoichiometric release of H(+) into the medium. With C2C12 myoblasts given glucose, respiration-derived CO2 contributed 34% of the total extracellular acidification. When glucose was omitted or replaced by palmitate or pyruvate, this value was 67-100%. Analysis of primary cells, cancer cell lines, stem cell lines, and isolated synaptosomes revealed contributions of CO2-produced acidification that were usually substantial, ranging from 3% to 100% of the total acidification rate. CONCLUSION Measurement of glycolytic rate using extracellular acidification requires differentiation between respiratory and glycolytic acid production. GENERAL SIGNIFICANCE The data presented here demonstrate the importance of this correction when extracellular acidification is used for quantitative measurement of glycolytic flux to lactate. We describe a simple way to correct the measured extracellular acidification rate for respiratory acid production, using simultaneous measurement of oxygen consumption rate. SUMMARY STATEMENT Extracellular acidification is often assumed to result solely from glycolytic lactate production, but respiratory CO2 also contributes. We demonstrate that extracellular acidification by myoblasts given glucose is 66% glycolytic and 34% respiratory and describe a method to differentiate these sources.

Suppressors of superoxide production from mitochondrial complex III

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

The Determination and Analysis of Site-Specific Rates of Mitochondrial Reactive Oxygen Species Production

Mitochondrial reactive oxygen species (ROS) are widely implicated in physiological and pathological pathways. We propose that it is critical to understand the specific sites of mitochondrial ROS production and their mechanisms of action. Mitochondria possess at least eight distinct sites of ROS production in the electron transport chain and matrix compartment. In this chapter, we describe the nature of the mitochondrial ROS-producing machinery and the relative capacities of each site. We provide detailed methods for the measurement of H2O2 release and the conditions under which maximal rates from each site can be achieved in intact skeletal muscle mitochondria.

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria.

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.

Dependence of Brown Adipose Tissue Function on CD36-Mediated Coenzyme Q Uptake

Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.

Sources of superoxide/H2O2 during mitochondrial proline oxidation.

p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex.

In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H2O2 at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H2O2 at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H2O2 production. Rates increased at higher NAD(P)H/NAD(P)(+) ratios and were higher at each NAD(P)H/NAD(P)(+) ratio when 2-oxoadipate was present, showing that superoxide/H2O2 was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H2O2 is comparable to that of site IF of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H2O2 production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site AF of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H2O2 associated with the NADH isopotential pool in mitochondria.

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.