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

Abstract

Vacuole-type ATPases (V(o)V₁) and F(o)F₁ ATP synthases couple ATP hydrolysis/synthesis in the soluble V(1) or F₁ 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 V₁ and the whole V(o)V₁ from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V₁ 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)V₁ exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the V(o) rotor in T. thermophilus. Unlike F₁ that undergoes 80°-40° substepping, chemo-mechanical checkpoints in isolated V₁ are all at the ATP-waiting position, and V(o) adds further bumps through stator-rotor interactions outside and remote from V₁.