Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein.

We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating Acanthamoeba castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP>ATP>GDP>ADP≫GMP>AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH₂ concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH₂ concentration). The most important finding was that ubiquinol (QH₂) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH₂, the linoleic acid-induced GTP-inhibited H(+) leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH₂) was changed, a competitive influence on the UCP activity was observed. QH₂ decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH₂. These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH₂ in the mitochondria of a unicellular eukaryote.
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