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Abstract

The Na⁺-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c₁₁ rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7 Å resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c11 ring of I. tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F₁F_o holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na⁺. From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5 mM NaCl, respectively. These pK values are identical with those determined for the F₁F_o complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na⁺ from the cytoplasm through access channels within the c-ring itself.