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Effect of Glutathione Depletion on Sites and Topology of Superoxide and Hydrogen Peroxide Production in Mitochondria

2003; American Society for Pharmacology and Experimental Therapeutics; Volume: 64; Issue: 5 Linguagem: Inglês

10.1124/mol.64.5.1136

1521-0111

Derick Han, Raffaella Canali, Daniel Rettori, Neil Kaplowitz,

Biochemical effects in animals

In this work, the topology of mitochondrial \(\mathrm{O}_{{\dot{2}}}^{-}\) and H 2 O 2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases \(\mathrm{O}_{{\dot{2}}}^{-}\) into the matrix (where it is converted to H 2 O 2 by Mn-SOD) but not into the intermembrane space. No free radical generation was observed from complex II, but succinate treatment caused H 2 O 2 generation from the matrix through a reverse electron flow to complex I. Complex III was found to release \(\mathrm{O}_{{\dot{2}}}^{-}\) into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of \(\mathrm{UQ}_{{\dot{\mathrm{O}}}}^{-}\) (ubisemiquinone at the Qo site) in complex III, enhanced both H 2 O 2 generation from the matrix and \(\mathrm{O}_{{\dot{2}}}^{-}\) production from the intermembrane space. On the other hand, myxothiazol, which inhibits \(\mathrm{UQ}_{{\dot{\mathrm{O}}}}^{-}\) formation, completely inhibited antimycin induced \(\mathrm{O}_{{\dot{2}}}^{-}\) toward the intermembrane space and inhibited H 2 O 2 generation from the matrix by 70%. However, myxothiazol alone enhanced H 2 O 2 production from complex III, suggesting that other components of complex III besides the \(\mathrm{UQ}_{{\dot{\mathrm{O}}}}^{-}\) can cause \(\mathrm{O}_{{\dot{2}}}^{-}\) generation toward the matrix. As expected, mitochondrial GSH was found to modulate H 2 O 2 production from the matrix but not \(\mathrm{O}_{{\dot{2}}}^{-}\) generation from the intermembrane space. Low levels of GSH depletion (from 0—40%, depending on the rate of H 2 O 2 production) had no effect on H 2 O 2 diffusion from mitochondria. Once this GSH depletion threshold was reached, GSH loss corresponded to a linear increase in H 2 O 2 production by mitochondria. The impact of 50% mitochondrial GSH depletion, as seen in certain pathological conditions in vivo, on H 2 O 2 production by mitochondria depends on the metabolic state of mitochondria, which governs its rate of H 2 O 2 production. The greater the rate of H 2 O 2 generation the greater the effect 50% GSH depletion had on enhancing H 2 O 2 production.