Amino Acid Substitutions in theaSubunit Affect the ϵ Subunit of F1F0ATP Synthase fromEscherichia coli

Amino acid substitutions at many positions in theasubunit of F1F0ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in theasubunit affect the ϵ subunit. In mutant F1F0ATP synthases, the ϵ subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0ATP synthase, the ϵ subunit in enzymes carrying either theaarg-210→ileoragly-218→aspsubstitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the ϵ subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying theaala-217→argsubstitution, the trypsin digestion of the ϵ subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0ATP synthase, theaala-217→argsubstitution resulted in reduced efficiency of crosslinking between the ϵ and β subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in theasubunit are propagated to the ϵ subunit and are specific to the individual substitutions.