ATP synthase uses a rotary mechanism to couple transmem-brane proton translocation to ATP synthesis and hydrolysis, which occur at the catalytic sites in the subunits. In the presence of Mg2, the three catalytic sites of ATP synthase have vastly different affinities for nucleotides, and the position of the central subunit determines which site has high, medium, or low affinity. Affinity differences and their changes as rotation progresses underpin the ATP synthase catalytic mechanism. Here, we used a series of variants with up to 45- and 60-residue-long truncations of the N- and C-terminal helices of the subunit, respectively, to identify the segment(s) responsible for the affinity differences of the catalytic sites. We found that each helix carries an affinity-determining segment of 10 residues. Our findings suggest that the affinity regulation by these segments is transmitted to the catalytic sites by the DELSEED loop in the C-terminal domain of the subunits. For the N-terminal truncation variants, presence of the affinity-determining segment and therefore emergence of a high-affinity binding site resulted in WT-like catalytic activity. At the C terminus, additional residues outside of the affinity-determining segment were required for optimal enzymatic activity. Alanine substitutions revealed that the affinity changes of the catalytic sites required no specific interactions between amino acid side chains in the and 33 subunits but were caused by the presence of the helices themselves. Our findings help unravel the molecular basis for the affinity changes of the catalytic sites during ATP synthase rotation.