Essential Cell Biology 5th edition

Molecular Mechanisms of Electron Transport and Proton Pumping
477
membranes of the mitochondria in these cells contain a
carrier protein that allows protons to move down their
electrochemical gradient, circumventing ATP synthase.
As a result, the cells oxidize their fat stores at a rapid
rate and produce much more heat than ATP. Tissues
containing brown fat serve as biological heating pads,
helping to revive hibernating animals and to protect
sensitive areas of newborn human babies (such as the
backs of their necks) from the cold.
Artificial ATP generation
If disrupting the electrochemical proton gradient across
the mitochondrial inner membrane terminates ATP synthesis,
then, conversely, generating an artificial proton
gradient should stimulate ATP synthesis. Again, this
is exactly what happens. When a proton gradient is
imposed artificially by lowering the pH on the outside of
the mitochondrial inner membrane, out pours ATP.
How does the electrochemical proton gradient drive
ATP production? This is where the ATP synthase comes
in. In 1974, Efraim Racker and Walther Stoeckenius
demonstrated that they could assemble an artificial
ATP-generating system by combining an ATP synthase
isolated from the mitochondria of cow heart muscle with
a proton pump purified from the purple membrane of
the archaean Halobacterium halobium. As discussed in
Chapter 11, the plasma membrane of this prokaryote is
packed with bacteriorhodopsin, a protein that pumps H +
out of the cell in response to sunlight (see Figure 11−28).
When bacteriorhodopsin alone was reconstituted
into artificial lipid vesicles (liposomes), Racker and
Stoeckenius showed that, in the presence of light, the
protein pumps H + into the vesicles, generating a proton
gradient. (The orientation of the protein is reversed in
these membranes, so that protons are transported into
the vesicles; in the organism, protons are pumped out.)
When the bovine ATP synthase was then incorporated
into these vesicles, much to the amazement of many biochemists,
the system catalyzed the synthesis of ATP from
ADP and inorganic phosphate in response to light. This
ATP formation showed an absolute dependence on an
intact proton gradient, as either eliminating bacteriorhodopsin
from the system or adding uncoupling agents
such as DNP abolished ATP synthesis (Figure 14–27).
This remarkable experiment demonstrated without a
doubt that a proton gradient can cause ATP synthase
to make ATP. Thus, although biochemists had initially
hoped to discover a high-energy intermediate involved
in oxidative phosphorylation, the experimental evidence
eventually convinced them that their search was in vain
and that the chemiosmotic hypothesis was correct.
Mitchell was awarded a Nobel Prize in 1978.
LIGHT
H +
bacteriorhodopsin
LIGHT
H +
sealed vesicle
(liposome)
H +
H +
H + H +
H +
H + H +
ADP + P
ATP synthase
ATP
(A)
(C)
NO ATP GENERATED
LIGHT
ATP synthase
NO ATP GENERATED
(B)
(D)
LIGHT
ATP GENERATED
uncoupling
H +
agent
H +
H +
NO ATP GENERATED
Figure 14–27 Experiments in which
bacteriorhodopsin and bovine
mitochondrial ATP synthase were
introduced into liposomes provided
direct evidence that proton gradients
can power ATP production. (A) When
bacteriorhodopsin is added to artificial lipid
vesicles (liposomes), the protein generates
a proton gradient in response to light.
(B) In artificial vesicles containing both
bacteriorhodopsin and an ATP synthase,
a light-generated proton gradient drives
the formation of ATP from ADP and P i.
(C ) Artificial vesicles containing only ATP
synthase do not on their own produce
ATP in response to light. (D) In vesicles
containing both bacteriorhodopsin and ATP
synthase, uncoupling agents that abolish
the proton gradient eliminate light-induced
ATP synthesis.