Respiration-dependent Removal of Exogenous H2O2 in Brain Mitochondria

• Published in: The Journal of biological chemistry 2004-02, Vol.279 (6), p.4166-4174
• Main Authors: Zoccarato, Franco, Cavallini, Lucia, Alexandre, Adolfo
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M308143200

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 H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect ∼40%, IC50, 12 μm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60–70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 μm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased ΔΨ, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks.