Chemistry Reference
In-Depth Information
predictions of the Boyer mechanism.
13
The structure supports a catalytic mechanism in intact ATP synthase in
which the three catalytic subunits are in different states of the catalytic cycle at any instant. As indicated in the
insert, the front of the three
subunits is in the O (here E for empty) form, that to the left has dinucleotide bound
(L) and that on the right is in the T form. This convincingly demonstrates that ATP synthase functions by
rotational catalysis.
What drives the interconversion of the three states is the rotation of the
b
g
subunit. As the proton flux causes
subunit,sayby120
,thethree
rotation of the
subunits will change position and conformational state. So, the
subunit which had ATP tightly bound will adopt the open conformation, and ATP will be released. The loosely
bound ADP and P
i
will find itself in the tight conformation, and its high affinity for ATP will drive ATP
synthesis. Finally, the subunit previously in the open form, will adopt the L form, and bind ADP and P
i
.Themost
elegant proof that the ATP synthase is a rotary molecular motor comes from studies in which the
g
b
a
3
b
3
hexamer
was fixed to a Ni- surface (using a short sequence of His residues attached to the end of the protein chain
e
aHis
tag) with the
g
subunit pointing upward and attached to a fluorescently labelled actin filament (
Figure 5.22
)
.
FIGURE 5.22
Direct observation of ATP-driven rotation in ATP synthase. The
a
3
b
3
hexamer is fixed to a surface with the
g
subunit pointing
upward and linked to a fluorescently labeled actin filament. Addition of ATP results in rotation of the
g
subunit, which can be observed with
a fluorescence microscope. (From
Berg et al., 2002
: pp. 974.)
Addition of ATP (to stimulate the reverse reaction of ATP synthesis) resulted in a rotation of 120
for each
equivalent of ATP added.
One final conceptual question remains. How does the flow of protons through F
0
drive the rotation of the
g
subunit? It is suggested that the c subunit (
Figure 5.19
), which has an aspartate residue (Asp 61) in the middle of
a pair of helices which traverse the membrane, plays a key role. There are channels in the a subunit, which
surrounds the central ring of c subunits, but which do not cross the membrane, but rather go more or less half way
across from each side of the membrane (
Figure 5.23
)
. Suppose further that two residues of Asp 61 of two subunits
c are in contact with the two half-channels of the a subunit. One has picked up a proton from the high concen-
tration of protons on the cytosolic (intermembrane space) of the mitochondria, and will be in its protonated
(neutral) form. The other, coming from the half-channel on the matrix side, which is proton deficient, will be in its
charged, nonprotonated form. Rotation of the c ring by 360
will now align the protonated Asp 61 with the matrix
half-channel, whereas the unprotonated Asp 61 of the second will be confronted by the proton-rich cytosolic
half-channel (25 times higher [H
þ
] concentration than in the matrix). The net result is vectorial proton migration
across the inner mitochondrial membrane as a consequence of rotation of the c ring.
13. Boyer and Walker received the 1997 Nobel prize for Chemistry together with Jens Skou, who discovered the Na
þ
/K
þ
ATPase (of which
more in Chapter 9).
