Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Abstract

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H 2 O 2 levels, we were able to quantitatively measure the contributions of potential systems toward H 2 O 2 removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H 2 O 2 removal (9 -12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H 2 O 2 removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H 2 O 2 removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H 2 O 2 removal. Inhibition of the thioredoxin system exacerbated mitochondrial H 2 O 2 production by the redox cycling agent, paraquat. Additionally, decreases in H 2 O 2 removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration-and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.Since the discovery that electron leak and incomplete reduction of oxygen occurs in the respiration chain (1, 2), mitochondria have been considered a major contributor to cellular oxidative damage through the generation of reactive oxygen species (ROS), 2 including superoxide (O 2 . ), H 2 O 2 , and hydroxyl radical (HO ⅐ ). Mitochondrial ROS production has been implicated in numerous pathological processes, including the etiology of various acute and chronic neuronal disorders (3), as well as aging (4). More recent studies suggest that ROS, such as H 2 O 2 , also serve an important role in cell signaling pathways and thereby regulate a diverse set of physiological processes (5). Therefore, maintaining the delicate balance between pathological and physiological levels of H 2 O 2 is critical for proper cell function and survival. The biological significance of mitochondrial ROS are highlighted by targeted deletion or overexpression of antioxidant enzymes: 1) thioredoxin 2 (Trx2) knock-out mice present an embryonic lethal phenotype (6), 2) manganese superoxide dismutase knock-out...

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