Mitochondrial respiratory chain complex activity assay

Mitochondrial respiratory chain complex activities were measured as previously reported^73,74 with slight modifications¹⁵. In brief, assays were performed in 200 µl/well in a 96-well plate at 37 °C (except citrate synthase, which was performed at 30 °C) using a Tecan Infinite 200 PRO series spectrophotometer (Tecan Group Ltd., Crailsheim, Germany). Frozen mitochondria samples were disrupted by two cycles of freezing in liquid nitrogen and thawing at room temperature. Complex I activity was measured at 340 nm as the rotenone-sensitive rate of NADH oxidation and complex I-dependent electron transfer to decylubiquinone. Complex III activity was assayed at 550 nm as the antimycin A-sensitive rate of reduction of cytochrome c by electron transfer from decylubiquinol through complex III. Complex IV activity was measured at 550 nm as the cyanide-sensitive oxidation of cytochrome c. Complex V activity was assayed at 340 nm as oligomycin-sensitive ATP hydrolysis by complex V coupled to pyruvate kinase and lactate dehydrogenase activities and the corresponding oxidation of NADH. Citrate synthase activity was measured as the condensation reaction of oxaloacetic acid and acetyl-CoA to citrate and the subsequent reduction of DTNB (5,5′-dithiobis (2-nitrobenzoic acid)) by coenzyme A as detected at 412 nm.