Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation

F-1-ATPase is a rotary molecular motor in which unidirectional rotation of the central subunit is powered by ATP hydrolysis in three catalytic sites arranged 120degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an similar to80degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further 40degrees rotation.