Evidence for Structural Integrity in the Undecameric c-Rings Isolated from Sodium ATP Synthases

Abstract

The Na +-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c 11 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 c 11 ring of I. tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F 1F 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 p K of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5 mM NaCl, respectively. These p K values are identical with those determined for the F 1F 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.