subunits are responsible for the synthesis of ATP or sometimes, as in the case of bacteria,

ATP hydrolysis. All three functions must be tightly integrated for the production of ATP.

Proton Translocation: Driving Rotation

 The demonstration that the electrochemical gradient of protons drives the rotation

of bacterial flagella (245) in combination with Peter Mitchell's chemiosmotic theory (2)

began the search for evidence of rotation in F1Fo ATP synthesis. At the same time, a

model for proton transport was suggested by Cox et al. (212) and Boyer developed his

ideas for the binding change mechanism (discussed below) (246). But an indication of

rotational catalysis was not evident until the high-resolution crystal structure of F1

became available (20). This was followed a few years later by the first direct observation

of rotation when Noji et al. fixed the top of F1 to a glass coverslip and attached a

fluorescently labeled actin filament to the y subunit (15). Upon addition of ATP, rotation

of the actin filament was observed under an optical microscope at 0.2-10 revolutions per

second. At low concentrations of ATP (<600 nM), the actin filaments were observed to

rotate in a step-wise manner at 1200 intervals, which reflects the three catalytic sites in

the F1 003 3 hexamer (247). Experiments, in which two phenylalanine residues in the

nucleotide binding pocket were mutated to reduce the binding affinity of ATP, indicated

that the binding and hydrolysis of ATP is initially accompanied by a 900 substep

followed by a 300 substep attributed to product release (248, 249). These observations

were consistent with the two proposed "catches" observed between the P and y subunits

in the high resolution structure (see "The y subunit:" above) (20). The observance of the

rotation of the s and c subunits at the same speed and direction, indicating that these three

subunits rotate in synchrony, forming the central rotary machinery of the enzyme