Structure and function of the F_o complex of the ATP synthase from <i>Escherichia coli</i>

Abstract

The membrane-bound ATP synthase (F1Fo) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions. In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F-1 and F-o. The water-soluble F-1 part carries the catalytic sites for ATP synthesis and hydrolysis, It is associated with the membrane-embedded F-o complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12. Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer. Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F-1 binding, thereby demonstrating that subunit b had recovered its native conformation. Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of alpha-helical conformation of 80%. After addition of a His(6)-tag to the N terminus of subunit a, a stable ab(2) subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits, After addition of subunit c and reconstitution into phospholipid vesicles, an F-o complex was obtained exhibiting rates of proton translocation and F-1 binding comparable with those of wild-type F-o. The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31-Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides. Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F-1. Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits gamma and epsilon Of the F-1 part, the mAb molecules have no effect on the function of F-o, either in proton translocation or in F-1 binding, However, the F-1 part and the mAb molecule(s) are hound simultaneously to the F-o complex, suggesting that not all c subunits are involved in the interaction with F-1.