Purified cytochrome c

Purified bovine liver complex IV was diluted to a concentration of 3 µM, and cardiolipin (Sigma-Aldrich) was added to a 40:1 cardiolipin:complex IV molar ratio. The enzyme was dialyzed (molecular mass cutoff: 12,000 to 14,000 Da) in dialysis buffer (10 mM K-Hepes, pH 7.4, 40 mM KCl, and 1% Tween 20, supplemented with 0.2 mM ATP). Oxygen consumption was measured via Clark-type oxygen electrode (Oxygraph system; Hansatech) at a final concentration of 45 nM liver complex IV in a total volume of 200 µL of measuring buffer (10 mM K-Hepes, 40 mM KCl, and 1% Tween 20) at pH 7.4 or 7.9 as indicated. Ascorbate (20 mM) was added as an electron donor for cytochrome c. Purified cytochrome c (Sigma-Aldrich; 10 µM) was injected into the air-tight oxygen electrode chamber. Each drug was titrated into the chamber at the concentrations indicated.