*2.8. High-Resolution Respirometry in Different Respiratory States*

Oxygen consumption of intact epimastigotes (5 × 107 parasites/chamber) was measured using an O2k-system high-resolution oxygraph (Oxygraph-2K; Oroboros Instruments, Innsbruck, Austria) at 28 ◦C with continuous stirring. The cells were suspended in a 2 mL respiration solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris–HCl (pH 7.2), and 50 μM digitonin was added to permeabilize the parasites. Following that, 10 mM succinate and 200 μM ADP were added. Uncoupled respiration was induced with 3 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and then blocked with 2.5 μg/mL antimycin A to assess residual O2 consumption [29]. Oxygen concentrations and O2 consumption were recorded using DatLab software coupled to Oxygraph-2K.

## *2.9. Succinate-Cytochrome C Oxidoreductase Activity*

The activity of succinate-cytochrome c oxidoreductase (complex II/III) was determined by the increase in absorbance due to the reduction of ferricytochrome c at 550 nm [40,41]. Frozen-thawed parasite homogenates (50 μg) were incubated with 25 mM potassium phosphate (pH 7.4), 10 mM succinate, 1 mM KCN, and 5 mM MgCl2 for 20 min to allow for complete activation of succinate dehydrogenase, after which the reaction was initiated with 50 μM horse heart cytochrome c and monitored for 2 min. Protein concentrations were determined by the Bradford method, using bovine serum albumin as a standard [42].
