Respiration-dependent Removal of Exogenous H sub(2)O sub(2) in Brain Mitochondria: Inhibition by Ca super(2+)

In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H sub(2)O sub(2) removal. Omitting EGTA decreases the H sub(2)O sub(2) removal rate by almost 80%. The decrease depends on the influx of contaminating Ca super(2+), being prevented by the Ca super(2+) uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect [~]40%, IC sub(50), 12 mu M). The H sub(2)O sub(2) removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60-70% in fully uncoupled conditions. H sub(2)O sub(2) removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca super(2+). Glutathione reductase is decreased by 70% with an IC sub(50) of about 0.9 mu M, and glutathione peroxidase is decreased by 38% with a similar IC sub(50). The highest Ca super(2+) effect with glutathione reductase is observed in the presence of low concentrations of H sub(2)O sub(2). With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H sub(2)O sub(2) by reverse electron flow at NADH dehydrogenase competing with exogenous H sub(2)O sub(2) for removal. Succinate-dependent H sub(2)O sub(2) is inhibited by rotenone, decreased [Delta][Psi], as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H sub(2)O sub(2) removal with succinate. Succinate-dependent H sub(2)O sub(2) generation is also inhibited by contaminating Ca super(2+). Therefore, Ca super(2+) acts as an inhibitor of both H sub(2)O sub(2) removal and the succinate-supported H sub(2)O sub(2) production. It is concluded that mitochondria function as intracellular Ca super(2+)-modulated peroxide sinks.