Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the KMox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KKMox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (KMox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in KMox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the KMox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.