Chemistry Reference
In-Depth Information
property may also be required for the ef
cient ATP synthesis catalyzed by F
o
F
1
in the
physiological composition of ATP (
1mM), ADP (
0.1mM), and Pi (
1mM). If the
af
nity of ATP and ADP is dependent on the angle of the
subunit and reversed
during the rotation, F
1
would be able to bind ADP ef
ciently even in the presence
of high [ATP], when forced to rotate by F
o
. This also facilitates the dissociation of
synthesized ATP from the catalytic sites.
g
10.2.2.2 Highly Coupled ATP Synthesis by F
1
Forced to Rotate in the Reverse Direction
The physiological function of F
1
is ATP synthesis and not hydrolysis. As described
above, when F
1
hydrolyzes ATP, the rotation of the
subunit is tightly coupled to the
chemical reaction, and three ATP molecules are hydrolyzed per turn (3 ATP/turn).
Then, how ef
ciently does F
1
synthesize ATP when the rotation is reversed? ATP
synthesis by the reverse rotation of F
1
was proved in 2004 [34]. In this experiment,
many F
1
molecules enclosed in an observation chamber were forced to rotate in the
reverse direction by using magnetic tweezers, and the synthesized ATP was detected
by a conventional bioluminescence method using the luciferin
-
luciferase system.
However, quantitative estimation of the ratio of mechanochemical coupling was
dif
cult because the number of active F
1
molecules in the chamber was unknown.
The coupling ratio can be directly estimated if the number of ATP molecules
synthesized by a single F
1
molecule during the forced rotation can be measured.
Usingmagnetic tweezers, forced rotation of F
1
could be easily achieved. However, the
quantitative measurement of ATP generated by a single F
1
molecule was not easy
using conventional methods. For example, even if we assume 100%coupling (3 ATP/
turn), the rotation of F
1
at 10Hz for 1min would result in the generation of only 1800
ATP molecules (3.0
g
10
21
mole). To overcome this problem, we developed a very
small reaction chamber having a volume of 1
(
m]
3
) by using conventional
microfabricationmethods (Figure 10.8A) [35]. If 1800 ATPmolecules are enclosed in
this chamber, [ATP] would reach 3
ΒΌ
[1
m
M, which is suf
ciently high to be detected by
conventional methods. In the actual experiment, we did not use the bioluminescence
assay; instead, we used an enzymatic property of F
1
for the estimation of the number
of ATPmolecules synthesized after forced rotation usingmagnetic tweezers. In other
words, we estimated [ATP] in a femtoliter chamber from the rotational speed of an
enclosed F
1
molecule since the speed (or ATP hydrolysis activity) is proportional to
the [ATP] below micromolar level.
After the forced rotation in the reverse direction in the femtoliter chamber, the
rotational speed of F
1
driven by ATPhydrolysis actually increased (Figure 10.8B). This
result indicates that ATP was actually synthesized by reverse rotation (Figure 10.8B);
however, the ratio of coupling catalyzed by the
m
subcomplex was unexpectedly
very low and only 10% (0.3 ATP/turn) of that of ATP hydrolysis (3 ATP/turn)
(Figure 10.8C) [36]. However, when the
a
3
b
3
g
a
3
b
3
g
subcomplex, the ratio signi
cantly increased. Some molecules exhibited an almost
100% coupling ratio, and the average value was 77% (2.3 ATP/turn) (Figure 10.8D).
The difference in the coupling ratio between each
e
subunit was reconstituted with the
subcomplex was relatively
large; this can be attributed to the difference in the volume of chamber and the error
in the estimation of the rotational speed.
a
3
b
3
ge
