Abstract

F1FO ATP synthases catalyze the synthesis and hydrolysis of adenosine triphosphate (ATP), the universal biological energy carrier, and are thus integral to life. The membrane-embedded Fo sector utilizes the proton electrochemical gradient to generate rotation of the c ring in in the FO sector, which drives ATP’s chemical synthesis in the F1 sector. Recent structural models of ATP synthase from cryo-electron microscopy have revealed the architecture of the rotor- stator interface. However, despite the higher resolution information, the intricacies of the rotation mechanism are not fully understood. Previous research identified specific residues of the c subunit of E. coli F1FO, including Arg50 on the cytoplasmic end of transmembrane helix 2, that may be necessary for function. Mutation of Arg50 to Cys abolishes ATP-driven H+ pumping and blocks H+ -permeability, but these mutants are still able to grow in succinate minimal medium, suggesting that ATP synthesis activity is unaffected. Since positively-charged amino acids are conserved in this region of subunit c and have been proposed to form a salt bridge with subunit a, we tested the necessity of positive charge and steric bulk at this position using chemical and genetic modifications. Chemically appending side chains including ones containing a positive charge onto Cys50 with methanethiosulfonate did not restore H+ pumping activity. However, mutation of position 50 to nonpolar and carboxyl groups as well as His partially supported ATP- driven H+ pumping as well as ATP synthesis activity. And, mutation to Lys fully supported ATP-driven H+ pumping activity. These results indicate that position 50 of subunit c is tolerant to mutations excluding cysteine, but that a positive change at the end of a four carbon linker is highly favorable.

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