Generation of Reactive Oxygen Species in the Reaction Catalyzed by α-Ketoglutarate Dehydrogenase

Abstract

α-Ketoglutarate dehydrogenase (α-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 α-KGDH is able to generate H 2 O 2 and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated α-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H 2 O 2 after addition of α-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD+). NAD+, which proved to be a powerful inhibitor of α-KGDH-mediated H 2 O 2 formation, switched the H 2 O 2 forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H 2 O 2 formation by α-KGDH, and for this, neither α-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 H 2 O 2 production. The higher the NADH/NAD+ratio the higher the rate of H 2 O 2 production. H 2 O 2 production as well as the catalytic function of the enzyme was activated by Ca 2+. In synaptosomes, using α-ketoglutarate as respiratory substrate, the rate of H 2 O 2 production increased by 2.5-fold, and aconitase activity decreased, indicating that α-KGDH can generate H 2 O 2 in in situ mitochondria. Given the NADH/NAD+ratio as a key regulator of H 2 O 2 production by α-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 α-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.