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Media type:
E-Article
Title:
Inhibition of Krebs Cycle Enzymes by Hydrogen Peroxide: A Key Role of α-Ketoglutarate Dehydrogenase in Limiting NADH Production under Oxidative Stress
Description:
<jats:p>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 H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>proved to be aconitase, being completely blocked at 50 μ<jats:sc>m</jats:sc>H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>. α-Ketoglutarate dehydrogenase (α-KGDH) was also inhibited but only at higher H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>concentrations (≥100 μ<jats:sc>m</jats:sc>), 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 H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, and the effect exerted at small concentrations (≤50 μ<jats:sc>m</jats:sc>) of the oxidant was completely prevented by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. BCNU-insensitive decline by H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>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 H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>at concentrations inhibiting aconitase. It is concluded that (1) aconitase is the most sensitive enzyme in the Krebs cycle to inhibition by H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, (2) at small H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>concentrations (≤50 μ<jats:sc>m</jats:sc>) when aconitase is inactivated, glutamate fuels the Krebs cycle and NADH generation is unaltered, (3) at higher H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>concentrations (≥100 μ<jats:sc>m</jats:sc>) 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 H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>-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.</jats:p>