The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.