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

In this work, the topology of mitochondrial and H 2 O 2 generation and their interplay with matrix GSH in isolated heart mitochondria were examined. We observed that complex I releases 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 into the matrix and into the intermembrane space. Antimycin, which increases steady-state levels of (ubisemiquinone at the Qo site) in complex III, enhanced both H 2 O 2 generation from the matrix and production from the intermembrane space. On the other hand, myxothiazol, which inhibits formation, completely inhibited antimycin induced 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 can cause generation toward the matrix. As expected, mitochondrial GSH was found to modulate H 2 O 2 production from the matrix but not 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.