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The enormous progress in the area of ATP synthesis during the 1990s has not yet been communicated to students
through biochemistry textbooks. In this article, we summarize the breakthroughs in the elucidation of the structure of
F0F1-ATPase/synthase, and relate this information to previous and new kinetic mechanisms and bioenergetic
considerations. The emerging picture of ATP synthase is nothing less than fantastic: a rotary engine of high e$ciency.
During synthesis, the rotor is driven by the electrochemical protonmotive force; the energy of the rotation is converted to
elastic-strain energy within the stator subunits, followed by conformational changes a!ecting the release of ATP.
Rotational catalysis was predicted by kinetic models, and the rotation itself was subsequently demonstrated by video
kinematography. The structures of F 1-ATPases of mitochondria from beef heart and rat liver and of bacteria were
obtained by X-ray di!raction analysis, while the structure of the F 0 subunit was deduced from NMR and other physical-
chemical methods. The complexity and perfection of this molecular machine will generate fascination and appreciation
among students of both science and engineering. ( 1999 IUBMB. Published by Elsevier Science Ltd. All rights

1. Introduction
Although the signi"cance of ATP synthesis has been
given due recognition in all textbooks of biochemistry,
perhaps no other subject has remained so confusing
for so long. Only in the last couple of years have we
seen the long-awaited advances at the detailed
molecular level, advances that o!er some degree of
satisfaction to the student or educator who is weary of
the black boxes of contentious hypotheses.

The purpose of this article is to summarize the recent
developments in the "eld of ATP synthesis and to "t the
new material into the fabric of previous knowledge. We
hope that this account, with its emphasis on molecular
details, will make a useful adjunct material to the
teaching of oxidative phosphorylation based on the
existing textbooks. Our order of emphasis represents
the combined views of the authors, one of whom (JB)
is a student of biochemistry, and the other has been
teaching biochemistry for 35 years.
The current understanding of ATP synthesis is the
result of several approaches which unfolded over the
period of almost 50 yr. Of paramount signi"cance was
the discovery of the enzyme complex F0F1
ATPase/synthase, which is widely distributed in
animals, plants, fungi

and bacteria. The culmination of structural work was
the elucidation of the F1-ATPase structure, via X-ray
diffraction analysis, of beef heart mitochondria [1], of
rat liver mitochondria [2] and of bacteria [3,4]. Other
important issues concerning the driving force and the
mechanism of
ATP synthesis were clari"ed by the ingenious
theoretical considerations of Peter Mitchell and by
kinetic studies of Paul Boyer. In fact, their predictions
have inspired much of the recent and still ongoing

2. The bioenergetic and kinetic problems of ATP
synthesis In order to synthesize ATP from ADP and P*,
the two molecules must be brought in close vicinity for
the condensation reaction, in which 1 molecule of
water must be produced from the displacement of an
OH group of the P* molecule. The main problem is the
coulombic repulsions that arise on bringing the
negatively charged reactants, presumably the dianion
of ADP and monoanion of P*, together in close vicinity.
The standard free energy change of ATP hydrolysis,!31
kJ/mol, seems a forbiddingly high value to overcome. It
has been strangely ignored, however, that all this
impressive `high energya is due to the di!erence
between *G0 and *G0@, and that the relevant value of
hydrolysis at pH 0, *G0, may be slightly positive.

Based on the above argument, the simplest way
to negate the high-activation energy caused by
the repulsive forces between two phosphoanions
is their neutralization by protons, which also
catalyze the condensation reaction. This is a well-
established fact: just think of the formation of
pyrophosphoric acid in concentrated phosphoric
acid solution. No wonder Peter Mitchell looked
for an acidic compartment to facilitate the
synthesis of ATP. He predicted the existence of
the proton gradient because it o!ered the
simplest chemical solution to the problem [5]. 3.
The merits of the chemiosmotic theory The
chemiosmotic theory is aptly presented in all
modern textbooks, and we only summarize its
conclusions. f Protons are transferred vectorially
from the mitochondrial matrix into the
periplasmic space by redox loops, in which the
purely organic electron carriers (FMN and Co-Q)
interact with iron-containing redox systems. f
Also important is the resulting membrane
potential, which together with the pH di!erence
makes up the protonmotive force. f Protons
return through the ATP synthase, thus enabling
the synthesis of ATP in the active site.
In spite of some inaccuracies, history may well
judge Mitchell's theory among the few most
in#uential ones in modern biochemistry.
Students "nd it encouraging that this work was
not a product of large research grants, and its
author had to overcome much initial resistance.
The great bene"t of the chemiosmotic theory
was that it o!ered the best rationale for the
observed P/O ratio, which is the number of
molecules of ATP synthesized per pair of
electrons carried through electron transport. The
chemiosmotic theory postulates the transfer of 6
protons per NADH oxidized, which seemed
su$cient to Mitchell for the synthesis of three
ATPs. Recent textbooks report experimental
"ndings of 10}12 protons pumped across the
inner membrane. The details of this process are
yet clear, but they are not the focus of our
here. Su$ce it to say, there are more than
enough protons
available to support ATP synthesis. Where
proved to be eventually wrong was his
assumption that
seemed very logical to many of us: the creation
a strongly acidic environment in the catalytic
site. The
direct involvement of protons in catalysis was
shared by
all textbook authors who depicted ATP synthase
a channel in the middle, leading to the active
site. This
view was proven wrong only by the recent
X-ray di!raction images of the F1 complex, but
were previously raised by the kinetic studies of
Boyer in the 1970s.
4. ADP#Pi/ATP equilibrium at neutral pH
Paul Boyer's quest to elucidate the mechanism of
synthesis began in the 1950s but his "rst major
was achieved only in the early 1970s. It was the
demonstration that ADP#P* can be converted to
bound ATP by the F1-ATPase enzyme at pH 7. His
elegant kinetic experiment describing the
of 18O into P* is in most textbooks, and we do
not have to
describe it in detail. However, one can hardly
the impact of this discovery once it was "nally
which required a lag period of several years.
The "nding of the kinetic studies, i.e., that the
constant for the inter-conversion of ADP and ATP
is near unity, suggests that the ATP synthase
active site
can su$ciently neutralize the negative charges of
and P* monoanion and bring them close enough
for the
required in-line displacement reaction. The
attack on the
phosphate dianion, as shown in Fig. 1, is still
Boyer's explanation was that energy input is not
for the synthesis of ATP but for the release
He believed that the release of ATP is achieved
a conformational change of the active site, which
is energized
by the proton gradient. Before the arrival of the
recent X-ray di!raction images, Mitchell and
others continued
to assume that the protons arriving at the active
site were directly involved in the release process.
view has become obsolete.
5. The structure of F0F1-ATPase/synthase
The chronological presentation would have some
pedagogical merits, but it is more expedient to
o!er here
the current information on F0F1-
The early work of Racker showed that the
consists of the membrane embedded Fo portion
and the
F1 knob which can be dissociated. There are only