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The Journal of Neuroscience, December 15, 2000, 20(24):8972-8979
Inhibition of Krebs Cycle Enzymes by Hydrogen Peroxide: A Key
Role of  -Ketoglutarate Dehydrogenase in Limiting NADH Production
under Oxidative Stress
Laszlo
Tretter and
Vera
Adam-Vizi
Department of Medical Biochemistry, Neurochemical Group, Semmelweis
University of Medicine, Budapest, H-1444, Hungary
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 µM H2O2.  -Ketoglutarate dehydrogenase
(-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 -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 H2O2
concentrations (100 µM) inhibition of -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 -KGDH in impaired mitochondrial function under
oxidative stress, with implications for neurodegenerative diseases and
cell damage induced by ischemia/reperfusion.
Key words:
hydrogen peroxide; oxidative stress; mitochondria; Krebs
cycle; -ketoglutarate dehydrogenase; aconitase; NADH
Copyright © 2000 Society for Neuroscience 0270-6474/00/20248972-08$05.00/0
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