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, Ca²⁺, Mg²⁺ 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 Ca²⁺ and Mg²⁺. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis-elevated pH, Ca²⁺ level and Mg²⁺-increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O₂ consumption at pH = 8.0, in the presence of ADP either 1 mM Mg²⁺ or 1 mM Ca²⁺. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O₂ is depleted only in the presence of ADP, so the O₂ 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.