Reversible inhibition of α-ketoglutarate dehydrogenase by hydrogen peroxide: Glutathionylation and protection of lipoic acid

We have previously demonstrated that when cardiac mitochondria were challenged with H₂O₂, NADH production and oxidative phosphorylation declined. Upon consumption of H₂O₂, mitochondrial function was restored. These alterations were due, in large part, to reversible glutathionylation and inhibition of the Krebs cycle enzyme α-ketoglutarate dehydrogenase. The current study was undertaken to identify the site and consequences of α-ketoglutarate dehydrogenase glutathionylation. Mitochondria were treated with H₂O₂ for varying periods of time. Protein sulfhydryls that had undergone H ₂O₂ mediated glutathionylation were specifically derivatized with N-ethylmaleimide-biotin. Subsequent purification of biotin labeled (glutathionylated) protein and Western blot analysis revealed that the E2 subunit of α-ketoglutarate dehydrogenase was reversibly glutathionylated. Further analysis revealed that lipoic acid, a required cofactor covalently attached to the E2 subunit, was the site of glutathionylation. The relative level of glutathionylated lipoic acid closely paralleled the degree of enzyme inhibition and reactivation. Glutathionylation of α-ketoglutarate dehydrogenase protected lipoic acid from modification by the electrophilic lipid peroxidation product 4-hydroxy-2-nonenal. Glutathionylation of α-ketoglutarate dehydrogenase can therefore be viewed as an antioxidant response protecting the enzyme from oxidative damage.