Characterization of oxidation of glutathione by cytochrome c

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca(2+), Mg(2+) and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca(2+) and Mg(2+). The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca(2+) level and Mg(2+)—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O(2) consumption at pH = 8.0, in the presence of ADP either 1 mM Mg(2+) or 1 mM Ca(2+). Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O(2) is depleted only in the presence of ADP, so the O(2) depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.