F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits

F-1- ATPase, the catalytic part of FoF1- ATP synthase, rotates the central c subunit within the alpha(3)beta(3) cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F-1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at similar to 200 degrees and carries out a third catalytic event at similar to 320 degrees. This demonstrates the concerted nature of the F-1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the c subunit with a 120 degrees phase difference, a process we describe as a 'sequential three- site mechanism'.