Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of [alpha]-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress.

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(2)O(2)-induced oxidative stress, in intact isolated nerve terminals. The enzyme that was most vulnerable to inhibition by H(2)O(2) proved to be aconitase, being completely blocked at 50 microm H(2)O(2). alpha-Ketoglutarate dehydrogenase (alpha-KGDH) was also inhibited but only at higher H(2)O(2) concentrations (>/=100 microm), 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(2)O(2), and the effect exerted at small concentrations (/=100 microm) inhibition of alpha-ketoglutarate dehydrogenase limits the amount of NADH available for the respiratory chain, and (4) increased consumption of NADPH makes a contribution to the H(2)O(2)-induced decrease in the amount of reduced pyridine nucleotides. These results emphasize the importance of alpha-KGDH in impaired mitochondrial function under oxidative stress, with implications for neurodegenerative diseases and cell damage induced by ischemia/reperfusion.