Characterization of oxidation of glutathione by cytochrome c

Abstract

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.

Keywords: Cytochrome c; Glutathione; Mitochondria.

Fig. 1

Cyt c reduction by GSH at differently pH. 100 µM bovine cytochrome c, 1 mM GSH, 1 mM EDTA at 25 °C. A pH = 7.0, 7.4 and 8.0 (40 mM Tris/HCl). B pH = 6.0, 6.5 (40 mM NaHCO₃) and 7.0 (40 mM Tris/HCl). The reaction was started by adding cytochrome c (n = 6 ± SD)

Fig. 2

Cyt c reduction by GSH at differently concentrations of Ca²⁺. 100 µM bovine cytochrome c, 1 mM GSH, Ca²⁺ concentrations 0–10 mM, 40 mM Tris/HCl (pH = 8.0), at 25 °C. The reaction was started by adding cytochrome c (n = 6 ± SD)

Fig. 3

Cyt c reduction by GSH at differently concentrations of Mg²⁺. 100 µM bovine cytochrome c, 1 mM GSH, Mg²⁺ concentrations 0–10 mM 40 mM Tris/HCl (pH = 8.0), at 25 °C. The reaction was started by adding cytochrome c (n = 6 ± SD)

Fig. 4

Effect of thiols on the reduction of cytochrome c. 100 µM bovine cytochrome c, 1 mM GSH, 1 mM cysteine (Cys), 0.5 mM (1 mM thiol) dithiothreitol (DTT), 40 mM Tris/HCl (pH = 8.0), at 25 °C. The reaction was started by adding cytochrome c (n = 6 ± SD)

Fig. 5

Effect of phospholipids on the reduction rate of cytochrome c by GSH. 100 µM bovine cytochrome c, 40 mM Tris/HCl (pH = 8.0); 0.1 mM EDTA, at 25 °C; 250 µg/ml phosphatidic acid (PA); 250 µg/ml phosphatidyl-serine (PS); 250 μg/ml cardiolipin (CL) 1 mM GSH; 5 v/v% ethanol (phospholipid solvent). The reaction was started by adding cytochrome c

Fig. 6

Effect of Ca²⁺, Mg²⁺ and phospholipids on the reduction rate of cytochrome c by GSH. 100 µM bovine cytochrome c; 40 mM Tris/HCl (pH = 8.0); 5 v/v% ethanol (cardiolipin solvent); 1 mM GSH; at 25 °C. 250 μg/ml cardiolipin (CL); 25 μg/ml cardiolipin and 1 mM Ca²⁺ (Ca, CL); control (cont) was without cardiolipin, Ca²⁺, Mg²⁺. The reaction was started by adding cytochrome c

Fig. 7

Effect of Mg²⁺ on O₂ consumption of mitochondria. 80 mM KCl; 20 mM TRIS/HCl; 0,1 mM EGTA; 10 mM KH₂PO₄; pH 8.0; 37 °C; 5 mM GSH; 1 mg mitochondrial protein/ml

Fig. 8

Effect of Ca²⁺ on O₂ consumption of mitochondria. 80 mM KCl; 20 mM TRIS/HCl; 0,1 mM EGTA; 10 mM KH₂PO₄; pH 8.0; 37 °C; 5 mM GSH; 1 mg mitochondrial protein/ml

Fig. 9

Summary. Putative mechanism of action. GSH: glutathione; GSSG: oxidized glutathione