Orientation of the γ Shaft and Catalytic β Subunit in F 1-ATPase in the Intermediate State Revealed at the Single-Molecule Level

Abstract

The enzyme FoF1-ATP synthase catalyzes the synthesis of ATP from ADP and inorganic phosphate (Pi) using proton-motive force (pmf) across a membrane. The F1 sector containing α3β3γδε subunits solely hydrolyzes ATP when isolated, is thus called F1-ATPase. Now it is well established that both Fo and F1-ATPase are rotary molecular motors sharing the common shaft: α3β3 cylinder in F1 rotates the γ shaft when ATP is cooperatively hydrolyzed in three catalytic sites located at α-β interfaces (Nishizaka et al., Nat. Struct. Mol. Biol., 2004), whereas reverse rotation of the shaft by pmf through Fo synthesizes ATP from ADP and Pi. The central question still remained unsolved is how these two different reaction, pmf and hydrolysis/synthesis, are coupled through the shaft from the structural point of view regarding the γ subunit. Because of asymmetric coiled-coil structure of the γ subunit, rotation of the γ subunit is expected to accompany the additional motion against the rotation axis during rotation. Here we scrutinize the rotation radius in an isolated α3β3γ subcomplex under optical microscope at the single-molecule level, and report the change of radius, which notably suggest the tilting motion of the shaft, estimated to be ∼4°, between two chemical states. In contrast, we have already reported that cooperative three-step motions in catalytic subunits of F1 correlate with 80° and 40° substep rotations, and thus revealed a previously undescribed set of β conformations, open, closed and partially closed, in the ATP-waiting dwells (Masaike et al., Nat. Struct. Mol. Biol., 2008). The conformation set presumably correlates the tilting of the shaft, and effectively transforms to the Fo rotation which couples to pmf.