Measurement of electron transport complex and TCA cycle enzyme activities

All enzymatic activities were measured as previously described (8, 50) in cells (50–100 μg of protein) after undergoing three cycles of freeze/thaw. Briefly, citrate synthase activity was measured by monitoring the rate of conversion of acetyl coenzyme A (CoA) (100 μM) and oxaloacetate (200 μM) to citrate spectrophotometrically at 412 nm by coupling CoA production with the colorimetric indicator dithionitrobenzoic acid (200 μM). Complex I activity was measured using NADH (100 μM) and ubiquinone (100 μM) as substrates and monitoring the rotenone (10 μM)-sensitive decrease in absorbance of NADH at 340 nm. Complex II activity was measured using succinate (1.2 mM) and ubiquinone (50 μM) as substrates, coupling the (i) complex II catalyzed transfer of an electron from succinate to ubiquinone and the (ii) ubiquinol reduction of the dye 2,6-dichlorophenolindophenol (150 μM), which is monitored by a decrease in its absorbance at 600 nm and inhibited by thenoyltrifluoroacetone (50 μM). Complex IV activity was measured using reduced cytochrome c (50 μM) as the substrate and monitoring the oxidation of cytochrome c at 550 nm that was inhibitable by potassium cyanide (50 μM). All complex activities were normalized to protein concentration as determined by the Bradford protein assay method.