Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes

Kathrin Forster, Paola Turina, Friedel Drepper, Wolfgang Haehnel, Susanne Fischer, Peter Graber, Jan Petersen

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The H(+)/ATP synthase from yeast mitochondria, MFF, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MFF liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s(1) at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MFF, was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species HPO.
Original languageEnglish
Pages (from-to)1828 - 1837
Number of pages10
JournalBiochimica et Biophysica Acta - Bioenergetics
Issue number11
Publication statusPublished - 2010

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