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

Publication date: Available online 12 May 2017
Source:Biochimica et Biophysica Acta (BBA) - General Subjects
Author(s): Liam Slade, Julia Chalker, Nidhi Kuksal, Adrian Young, Danielle Gardiner, Ryan J. Mailloux
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 (H2O2). 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 O2^●−/H2O2 formation. 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 O2^●−/H2O2 formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H2O2. 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 O2^●−/H2O2 production 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 O2^●−/H2O2 formation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating 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 O2^●−/H2O2 production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H2O2.



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