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The membrane F_o factor of ATP synthase is highly sensitive to mutations in the proton half-channel leading to the functional blocking of the entire protein. To identify functionally important amino acids for the proton transport, we performed molecular dynamic simulations on the selected mutants of the membrane part of the bacterial F_oF₁-ATP synthase embedded in a native lipid bilayer: there were nine different mutations of <i>a</i>-subunit residues (<i>a</i>E219, <i>a</i>H245, <i>a</i>N214, <i>a</i>Q252) in the inlet half-channel. The structure proved to be stable to these mutations, although some of them (<i>a</i>H245Y and <i>a</i>Q252L) resulted in minor conformational changes. <i>a</i>H245 and <i>a</i>N214 were crucial for proton transport as they directly facilitated H⁺ transfer. The substitutions with nonpolar amino acids disrupted the transfer chain and water molecules or neighboring polar side chains could not replace them effectively. <i>a</i>E219 and <i>a</i>Q252 appeared not to be determinative for proton translocation, since an alternative pathway involving a chain of water molecules could compensate the ability of H⁺ transmembrane movement when they were substituted. Thus, mutations of conserved polar residues significantly affected hydration levels, leading to drastic changes in the occupancy and capacity of the structural water molecule clusters (W1–W3), up to their complete disappearance and consequently to the proton transfer chain disruption.