Measuring Drosophila Complex I Activity and Deactivation

All activity measurements were measured on a 96-well Spectramax 384 plate reader at 32°C. For NADH:decylubiquinone (dQ) oxidoreductase activities, NADH (200 µM final concentration) was used to initiate catalysis by complex I (0.2 µg mL^–1) with 200 µM dQ, 0.15% (w/v) asolectin, and 0.15% (w/v) CHAPS in 20 mM Tris-HCl (pH 7.55). NADH oxidation was monitored at 340–380 nm (ε = 4.81 mM^–1 cm^–1), and was confirmed to be sensitive to rotenone and piericidin A. The cryo-EM sample had an activity of 7.3 ± 0.3 µmol min^–1 mg^–1 (mean ± SD; n = 4).
For evaluation of the active/deactive state ratio of Drosophila complex I using the N-ethylmaleimide (NEM) assay (Galkin et al., 2008 (link); Yin et al., 2021 (link)), 4 mg mL^–1 mitochondria were incubated with 2 mM NEM or the equivalent volume of DMSO on ice for 20 min., before determining the NADH:O₂ oxidoreductase activity. The mitochondria had been frozen for storage before measurement. To attempt to deactivate the complex, the mitochondria were incubated at 37°C for 30 min (equivalent to, or longer than, the treatments used to deactivate complex I in mammalian mitochondrial membranes [Agip et al., 2018 (link); Blaza et al., 2018 (link)]). NADH:O₂ oxidoreductase activities were measured in 20 mM Tris-HCl (pH 7.55) using 10 µg mL^–1 mitochondria and 10 µg mL^–1 alamethicin, and initiated using 200 µM NADH. NADH oxidation was monitored as described above.