(Figure 6.15; Kinosita et al. 2004;Junge & Nelson 2005). The F 1 domain
was attached to a glass slide with a nickel coating through histidine residues
that were on extensions of the protein. A long, thin actin filament with
a fluorescent label was attached through a streptavidin/avidin connector
to the csubunits. When ATP was added, the enzyme ran in reverse and
the filament was found to spin rapidly. In single-molecule experiments
(Chapter 14) the filament was found to be
positioned only along certain discrete angles.
Based upon a long series of experiments, the
binding-change mechanism for ATP synthesis has
been elucidated. The mechanism involves the
presence of three sites, one of which binds ATP
tightly, the second of which binds weakly, and
the third of which is empty (Figure 6.16). Energy
is required to release ATP, not to form it. The
position of the three sites in the subunits is not
fixed, but varies as the enzyme rotates, with
the γsubunit acting like a camshaft and altern-
ately distorting the βsubunits, which can cause
cycling of the three sites. Interactions between
the a subunit and the cring provide a ratchet
that couples proton transfer with a ring rotation
in a counterclockwise direction only.
Although many aspects of the mechanism
of ATP synthesis have been determined, the130 PARTI THERMODYNAMICS AND KINETICS
γδα
βαAttachment to nickle
surface through
histidine residuesRotation is coupled
with ATP conversionRotation of actin
filament visible
in microscpeAvidinActin filamenta
cbATPADP ATPATPATPATPATPATPADP
PiADP
PiADP
Pi3 HN
3 HPααααααα βββββββββαα3 HN3 HN3 HP3 HPFigure 6.15An experimental demonstration of the
rotation of the ATP synthase by use of fluorescently
labeled actin filament. Based upon Kinosita et al.
(2004).Figure 6.16The
binding-change mode
for ATP synthesis.
Modified from Boyer
(2000).
βββγ
αααF 1ATP
b 2a
cH H ADP PiF 0Figure 6.14The structure of the F 0 F 1
complex and a model of how rotation of
the csubunits in the cell membrane relative
to the F 1 domain and aand bsubunits can
couple ATP synthesis to proton transport.
Based upon Murata et al. (2005).