Importance of Hydrophobic Bulk at the Rotor‐Stator Interface of E. coli ATP Synthase

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As the primary producer of adenosine triphosphate (ATP), the chemical energy currency of cells, F1Fo ATP synthase is essential for life. Using the H+ electrochemical gradient, the membrane‐embedded Fo complex generates torque on the cytoplasmic F1 complex, which synthesizes ATP from ADP and phosphate. While much is known about the mechanism of ATP synthesis, aspects of Fo functionality, including the roles of interactions between subunit a (stator) and the c10 ring (rotor), remain unresolved. In E. coli, Phe54 and Ile55 on subunit c have been found in a previous study to be important for H+ movement through Fo during ATP‐driven H+ pumping. We show here that cF54C and cI55C mutations cannot pump or passively translocate protons through Fo. ATP‐driven H+ pumping activity was partially restored by systematically adding steric bulk to Cys54 through chemical modifications with methanethiosulfonate derivatives of increasing size; however, similar restoration did not occur for Cys55. Additionally, we used site‐directed mutagenesis to replace Phe54 or Ile55 with hydrophobic amino acids of various size, all of which supported H+ pumping activity. While the chemical modification of Cys54 suggested that hydrophobic bulk at this position is important for H+ pumping, the chemical properties required at these positions remain unclear.Support or Funding InformationSupported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.