As the primary producer of adenosine triphosphate (ATP), the energy currency of the cell, F1Fo ATP synthase is essential for life. F1Fo is composed of two coupled rotary motors, membrane-embedded Fo and soluble F1. Using the H+ gradient across a membrane, the Fo complex generates torque on the F1 complex, which synthesizes ATP. During the synthesis process, protons enter Fo through a half channel within subunit a and exit through a half channel at the interface of subunit a (stator) and the c10 ring (rotor). In the reverse process, driven by ATP hydrolysis, protons are pumped in the reverse direction. While much is known about the E. coli complex, which serves as a model for ATP synthase structure and function, aspects of Fo functionality, including the role of some interactions between subunits a and c, remain unresolved. Using site-directed mutagenesis and biochemical assays, we have investigated residues involved in a putative salt bridge across this interface. When Arg50 in subunit c is replaced with Cys, H+ movement through Fo ceases; however, the Cys mutant is still able to grow in succinate minimal medium, suggesting that ATP synthesis activity is unaffected. To better understand the necessary chemical properties of residues at these positions, we report the results of H+ permeability, H+ pumping, and ATP synthesis experiments showing the remarkable tolerance of these regions to mutation (with the notable exception of Cys) and the effects of mutations on H+ movement in the ATP-hydrolysis vs. ATP-synthesis directions.

Mutational analysis of a positive charge at the rotor-stator interface of E. coli ATP synthase

ACS Spring Meeting 2021

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