Generation of reactive oxygen species in the reaction catalyzed by α-ketoglutarate dehydrogenase

alpha-Ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H2O2 and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H2O2 after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized(NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H2O2 formation, switched the H2O2 forming mode of the enzyme to the catalytic [ nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H2O2 formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H2O2 production. The higher the NADH/NAD(+) ratio the higher the rate of H2O2 production. H2O2 production as well as the catalytic function of the enzyme was activated by Ca2+. In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H2O2 production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H2O2 in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H2O2 production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.