Resolving stepping rotation in Thermus thermophilus H(+)-ATPase/synthase with an essentially drag-free probe

Vacuole-type ATPases (V(o)V(1)) and F(o)F(1) ATP synthases couple ATP hydrolysis/synthesis in the soluble V(1) or F(1) portion with proton (or Na(+)) flow in the membrane-embedded V(o) or F(o) portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V(1) and the whole V(o)V(1) from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V(1) made 120° steps, commensurate with the presence of three catalytic sites. Dwells between the steps involved at least two events other than ATP binding, one likely to be ATP hydrolysis. V(o)V(1) exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the V(o) rotor in T. thermophilus. Unlike F(1) that undergoes 80°–40° substepping, chemo-mechanical checkpoints in isolated V(1) are all at the ATP-waiting position, and V(o) adds further bumps through stator–rotor interactions outside and remote from V(1).