Both rotor and stator subunits are necessary for efficient binding of F-1 to F-0 in functionally assembled Escherichia coli ATP synthase

Abstract

In F1F0-ATP synthase, the subunit b(2)delta complex comprises the peripheral stator bound to subunit a in F-0 and to the alpha(3)beta(3) hexamer of F-1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F-0 subunits to the binding of F-1 within the assembled holoenzyme, the isolated subunit b dimer, ab(2) subcomplex, and fully assembled F-0 complex were specifically labeled with tetramethylrhodamine- 5-maleimide at bCys(64) and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F-1 ( at gamma Cys(106)) and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F-1 dissociation constants of 2.7 nM for the binding of F-0 and 9 - 10 nM for both the ab(2) subcomplex and subunit b dimer. This indicates that both rotor and stator components of F-0 contribute to F-1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F-1 to F-0, whereas subunit a does not contribute significantly.