Abstract

In the cell, energy is required to carry out all necessary functions to keep the cell alive. The carrier for this vital energy is known as adenosine triphosphate, ATP. The ATP synthase complex, where a majority of ATP is synthesized, is composed of eight unique protein subunits in a stoichiometry of ab2c10?3?3???, making up two units, F1 and F0. The F0 sector moves protons, driven by the proton gradient, across the cell membrane to drive ATP production via a rotary mechanism. The main pathway by which protons are translocated through F0 is through the a-c interface. In this pathway, we are investigating several conserved residues, including arginine (R) 50 in subunit c and glutamate (E) 196 and aspartate (D) 92 in subunit a, to determine what makes them essential to function. Previous work has suggested that the R50 could be a part of a functionally important salt bridge with E196 or D92 on subunit a, or involved in a water wire within the proton channel. To clarify the role of these residues, mutations were generated and their functionality examined through biochemical assays. In general, mutations made at aD92 significantly impacted functionality and were not well tolerated. We observed substantial functional defects in the aD92A/cR50A mutant, where both positions are replaced with alanine (A), and the aD92R/cR50D mutant, where the positions are swapped. While more investigation is needed, this sensitivity may be due to the disruption of an electrostatic interaction between rotor and stator. The mutations made at the aE196 position showed a mild effect on ATP-driven proton pumping, while moderately inhibiting ATP synthesis. These results indicate that aE196 is not involved in a salt bridge with cR50 but do suggest that aE196 may have a greater role in the synthesis direction.

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