Inhibition of Krebs Cycle Enzymes by Hydrogen Peroxide: A Key Role of α-Ketoglutarate Dehydrogenase in Limiting NADH Production under Oxidative Stress

摘要

In this study we addressed the function of the Krebs cycle to determine which enzyme(s) limits the availability of reduced nicotinamide adenine dinucleotide (NADH) for the respiratory chain under H2O2-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H2O2 proved to be aconitase, being completely blocked at 50 μmH2O2. α-Ketoglutarate dehydrogenase (α-KGDH) was also inhibited but only at higher H2O2 concentrations (≥100 μm), and only partial inactivation was achieved. The rotenone-induced increase in reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] fluorescence reflecting the amount of NADH available for the respiratory chain was also diminished by H2O2, and the effect exerted at small concentrations (≤50 μm) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNU-insensitive decline by H2O2 in the rotenone-induced NAD(P)H fluorescence correlated with inhibition of α-ketoglutarate dehydrogenase. Decrease in the glutamate content of nerve terminals was induced by H2O2 at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H2O2, (2) at small H₂O₂ concentrations (≤50 μm) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H₂O₂concentrations (≥100 μm) inhibition of α-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H₂O₂-induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of α-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.