Characterization of the First Cytoplasmic Loop of Subunit a of the Escherichia coli ATP Synthase by Surface Labeling, Cross-linking, and Mutagenesis

Abstract The first cytoplasmic loop of subunita of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit acontains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunitb, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40–64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75–90) is probably near the cytoplasmic surface, perhaps in contact with bsubunits.