Production of Reactive Oxygen Species by Mitochondria

Abstract

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H 2 O 2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H 2 O 2 . Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H 2 O 2 . Rotenone prevented antimycin A-induced H 2 O 2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H 2 O 2 . In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H 2 O 2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

“…Normalized density values of each individual carbonylated protein are plotted as a percentage of the young heart protein density for all five ETC complex subunits. (B) Densitometry for modified proteins found in CI (1 & 2), CII (3-5) and CIII (6). (C) Densitometry for modified proteins found in CIV (7-10) and CV (11).…”

“…Complex I (CI) and complex III (CIII) are the major sites for ROS production in aging and ischemia-reperfusion injury of the heart [5][6][7]. The NADH dehydrogenase site of CI, which is also the site of electron leakage, is located in the matrix side of the inner mitochondrial membrane [8].…”

“…Complex III (CIII), also a key site of ROS generation, [6,[10][11][12] has been shown to release superoxide to both sides of the inner mitochondrial membrane [10,[13][14][15] The sites of release of ROS suggest that the protein components most proximal to these sites may also be at risk for oxidative damage. Thus, the identification of oxidatively modified proteins of the ETC complexes may contribute to further understanding of the molecular mechanisms of mitochondrial dysfunction in aging and age-associated cardiomyocytes injury.…”

“…Mitochondrial ROS production plays a central role in the age-associated decline in tissue function [18][19][20]. Mitochondrial generated ROS, produced by in vivo electron leakage from ETC CI and CIII play a key role in the modification of mitochondrial proteins [18,[21][22][23][24][25]. These modifications have served as molecular markers of oxidative stress [26,27].…”

A complex dietary supplement augments spatial learning, brain mass, and mitochondrial electron transport chain activity in aging mice

“…Mitochondrial complex I dysfunction is strongly implicated in PD (Navarro and Boveris 2009), and complex III is also associated with generation of reactive oxygen species (ROS) relevant to aging (Chen et al 2004;Muller et al 2004). Complex IV deficiency was recently identified in AD (Lin and Beal 2006;Luques et al 2007).…”