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

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.