Reversible inactivation of α-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status

In a previous study, we found that treatment of rat heart mitochondria with H₂O₂ resulted in a decline and subsequent recovery in the rate of state 3 NADH-linked respiration. These effects were shown to be mediated by reversible alterations in NAD(P)H utilization and in the activities of specific Krebs cycle enzymes α-ketoglutarate dehydrogenase (KGDH) and succinate dehydrogenase. The purpose of the current study was to examine potential mechanism(s) by which H₂O₂ reversibly alters KGDH activity. We report here that inactivation is not simply due to direct interaction of H₂O₂ 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 intermolecular disulfide(s) or a sulfenic acid. Upon incubation of mitochondria with H₂O₂, reduced GSH levels fell rapidly prior to enzyme inactivation but recovered at the same time as 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, the results of the current study indicate that KGDH activity appears to be modulated through enzymatic glutathionylation and deglutathionylation. These studies demonstrate a novel mechanism by which KGDH activity and mitochondrial function can be modulated by redox status.