Subunit rotation of ATP synthase embedded in membranes:: a or β subunit rotation relative to the c subunit ring

ATP synthase FoF1 (alpha(3)beta(3)gammadeltaepsilonab(2)c(10-14)) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F-1 beta or membrane sector F-o a subunit when the F-o c subunit ring was immobilized; a biotin-tag was introduced onto the beta or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the beta or alpha subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the alpha or beta subunit. These results established that the gammaepsilonc(10-14) and alpha(3)beta(3)deltaab(2) complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis.