Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Abstract

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.

Targeting of mitochondrial reactive oxygen species production does not avert lipid-induced insulin resistance in muscle tissue from mice

“…Briefly, mitochondria (50 μg) were added to respiration buffer (100 mmol/l sucrose, 50 mmol/l KCl, 20 mmol/l TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), 1 mmol/l EDTA, 4 mmol/l KH 2 PO 4 , 2 mmol/ l MgCl 2 , 3 mmol/l malic acid and 0.1% [wt/vol.] fatty acid-free BSA, pH 7.2) fuelled by succinate (10 mmol/l) in the presence of complex III inhibitor antimycin (1 μmol/l) to maximise ROS production [27]. The reaction started upon the addition of Amplex Red reagent (100 μmol/l) and horseradish peroxidase (2 U/ml).…”

Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions

“…The majority of the work on site-specific mitochondrial O 2 . /H 2 O 2 production has focused on measurement of the maximum capacities of these sites under conditions in which concentrations of substrates were optimal (usually saturating) and inhibitors of electron transport were used to induce optimal (usually maximal) reduction of redox centers (16,34,35). However, maximum capacities, which are mainly dependent on the abundance of the relevant proteins or complexes in the mitochondria, are not necessarily related to the importance of particular mitochondrial sites in physiological or pathological O 2 .…”

Proteomic responses to environmentally induced oxidative stress

“…A comprehensive picture of the sources of ROS in mitochondria needs to also consider the role of fatty acids in uncoupling the inner mitochondrial membrane, interacting with ETS complexes and the transfer of electrons from fatty acid β-oxidation via flavin adenine dinucleotide (reduced form; FADH) to the ETS (Schönfeld and Wojtczak, 2008;Tahara et al, 2009). Importantly, recent evidence suggests that a transfer of electrons (as FADH) to CoQ via electron transfer flavoprotein, originating from β-oxidation, does not cause RET and therefore ROS production from complex I, opposite to the action of succinate dehydrogenase (SDH), which transfers electrons (as FADH) from succinate, emphasizing that the substrate-specific entry of electrons to the ETS affects ROS production (Chouchani et al, 2014;Schönfeld et al, 2010).…”