Electron partitioning between the two branching quinol-oxidizing pathways in Acanthamoeba castellanii mitochondria during steady-state state 3 respiration

Amoeba mitochondria possess a respiratory chain with two quinol-oxidizing pathways: the cytochrome pathway and the cyanide-resistant alternative oxidase pathway. The ADP/O method, based on the non-phosphorylating property of alternative oxidase, was used to determine contributions of both pathways in overall state 3 respiration in the presence of GMP (an activator of the alternative oxidase in amoeba) and succinate as oxidizable substrate. This method involves pair measurements of ADP/O ratios plus and minus benzohydroxamate (an inhibitor of the alternative oxidase). The requirements of the method are listed and verified. when overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a non-penetrating inhibitor of succinate uptake), the quinone reduction level declined. At the same time, the alternative pathway contribution decreased sharply and became negligible when quinone redox state was lower than 50%, whereas the cytochrome pathway contribution first increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This study is the first attempt to examine the steady-state kinetics of the two quinol-oxidizing pathways when both are active and to describe electron partitioning between them when the steady-state rate of the quinone-reducing pathway is varied.