Competition of fatty acids (FA) and alkylsulfonates with 5-DOXYL-stearic acid (5-SASL) binding to isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy. A distinct peak of the bound 5-SASL (h+1I) decreased with increasing concentration of competitors. Since alkylsulfonates are UcP substrates, it suggests that the FA binding site is located in the anion channel. Moreover, with increasing ATP the h+1I peak decreased and was smoothed with the ‘micellar’ peak into a single wider peak. A pH of 8.5 reversed this effect. It could reflect an allosteric release of 5-SASL from the ATP binding site which mimics the ATP gating mechanism.
