simultaneous-measurement-superoxidehydrogen-peroxide-nadh-production

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Memorial University of Newfoundland

Here, we report that choline and dimethylglycine can stimulate reactive oxygen species (ROS) production in liver mitochondria. Choline stimulated O˙/HOformation at a concentration of 5 μm. We also observed that Complex II and III inhibitors, atpenin A5 and myxothiazol, collectively induced a 95% decrease in O˙/HOproduction indicating both sites serve as the main sources of ROS during choline oxidation. Dimethylglycine, an intermediate of choline oxidation, was a more effective ROS generator. Rates of production were ~ 43% higher than choline-mediated O˙/HOproduction. The main site for dimethylglycine-mediated ROS production was via reverse electron transfer to Complex I. Our results demonstrate that metabolism of essential metabolites involved in methionine and folic acid biosynthesis can stimulate mitochondrial ROS production.

Choline and dimethylglycine produce superoxide/hydrogen peroxide from the electron transport chain in liver mitochondria.

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (HO). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O/HOformation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O/HOformation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial HO. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O/HOproduction by KGDHC, induced a ~86% and ~84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O/HOformation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O/HOproduction in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial HO.

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria.

Here, we found that formate, an essential one-carbon metabolite, activates superoxide (O2·-)/hydrogen peroxide (HO) release from mitochondria. Sodium formate (30 μm) induces a significant increase in O2·-/HOproduction in liver mitochondria metabolizing pyruvate (50 μm). At concentrations deemed to be toxic, formate does not increase O2·-/HOproduction further. It was observed that the formate-mediated increase in O2·-/HOproduction is not associated with cytochrome c oxidase (COX) inhibition or changes in membrane potential and NAD(P)H levels. Sodium formate supplementation increases phosphorylating respiration without altering proton leaks. Finally, it was observed that the 2-oxoglutarate dehydrogenase (OGDH) inhibitors 3-methyl-2-oxovaleric acid (KMV) and CPI-613 inhibit the formate-induced increase in pyruvate-driven ROS production. The importance of these findings in one-carbon metabolism and physiology are discussed herein.

Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

Article	Year
Choline and dimethylglycine produce superoxide/hydrogen peroxide from the electron transport chain in liver mitochondria. FEBS letters\| PubMed ID: 27761911	2016
Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria. Biochimica et biophysica acta\| PubMed ID: 28506882	2017
Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria. FEBS letters\| PubMed ID: 28771687	2017
Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake. PloS one\| PubMed ID: 29444156	2018

Adrian Young

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