In contrast to most bacteria, the mycobacterial F₁ F_O -ATP synthase (α₃ :β₃ :γ:δ:ε:a:b:b':c₉ ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F₁ -ATPase component α₃ :β₃ :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F₁ -ATPase (MsF₁ -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF₁ αβγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF₁ αβγε_Δ2-5 , revealed similar ATP hydrolysis as MsF₁ αβγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF₁ αβγε_Δ121 and MsF₁ αβγε_Δ103-121 with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α₃ β₃ -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.

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