Generation of reactive oxygen species in the reaction catalyzed by alpha-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 H(2)O(2) 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 H(2)O(2) 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 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 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 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 alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-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 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.
Tretter
Laszlo
L
Department of Medical Biochemistry, Semmelweis University, and Neurochemistry Group, Hungarian Academy of Sciences, Budapest H-1444, Hungary.
Adam-Vizi
Vera
V
D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002540:Cerebral Cortex / Q000201:enzymology; D006168:Guinea Pigs; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013574:Synaptosomes / Q000201:enzymology*
D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D009243:NAD; D006861:Hydrogen Peroxide; D007655:Ketoglutarate Dehydrogenase Complex; D000154:Aconitate Hydratase; D002118:Calcium