Modulation of mitochondrial function by hydrogen peroxide induced glutathionylation

During normal cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion ($O2\dot{}$) and subsequently hydrogen peroxide (H₂O ₂). Because H₂O₂ increases in concentration under certain physiologic and pathophysiologic conditions and can oxidatively modify cellular components, it is critical to understand the response of mitochondria to H₂O₂. In the present study, treatment of isolated rat heart mitochondria with H₂O₂ resulted in a decline and subsequent recovery of state 3 NADH-linked respiration. NADH levels closely paralleled H₂O₂ induced alterations in state 3 respiration. Assessment of electron transport chain complexes and Kreb's Cycle enzymes revealed that α-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and aconitase were susceptible to H₂O₂ inactivation. Of particular importance, KGDH and SDH activity returned to control levels, concurrent with the recovery of state 3 respiration. Inactivation is not due to direct interaction of H₂O₂ with KGDH and SDH. In addition, removal of H₂O₂ alone is not sufficient for reactivation. Enzyme activity does not recover unless mitochondria remain intact. Inactivation is not simply due to direct interaction of H₂O ₂ with KGDH. In addition, incubation of mitochondria with deferroxamine, an iron chelator, or 1,3-dimethyl-2-thiourea, an oxygen radical scavenger, prior to addition of H₂O₂ did not alter the rate or extent of inactivation. Thus, inactivation does not appear to involve a more potent oxygen radical formed upon metal catalyzed oxidation. Inactive KGDH from H₂O₂-treated mitochondria was reactivated with dithiothreitol, implicating oxidation of a protein sulfhydryl(s). However, the thioredoxin system had no effect, indicating that enzyme inactivation is not due to the formation of intra- or inter-molecular disulfide(s) or a sulfenic acid. Upon incubation of mitochondria with H₂O₂, reduced glutathione (GSH) levels fell rapidly prior to enzyme inactivation but recovered concurrent with enzyme activity. Importantly, treatment of inactive KGDH with glutaredoxin facilitated the GSH-dependent recovery of KGDH activity. Glutaredoxin is characterized as a specific and efficient catalyst of protein deglutathionylation. Thus, KGDH activity appears to be modulated through enzymatic glutathionylation and deglutathionylation. These studies demonstrate a novel mechanism for the regulation of KGDH and overall mitochondria) function by redox status of the mitochondria.