EPR spectroscopy of 5‐DOXYL‐stearic acid bound to the mitochondrial uncoupling protein reveals its competitive displacement by alkylsulfonates in the channel and allosteric displacement by ATP

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Abstract
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.