Molecular Biochemistry I
F1Fo ATP Synthase
Contents of this page:
H+ flux linked to ATP synthesis or hydrolysis
Composition & roles of major domains of the ATP Synthase
Binding change mechanism
Structure of F1 & central stalk
Evidence for rotation
Fo & peripheral stalk subunits
F1Fo ATP Synthase of mitochondria, chloroplasts,
and bacteria is represented schematically at
right. When the electrochemical H+ gradient is
favorable, F1Fo catalyzes ATP synthesis coupled to
spontaneous H+ flux toward the side of the
membrane where F1 protrudes. E.g., in
mitochondria, the pH and electrical gradients drive
H+ transport from the intermembrane space to the
matrix compartment. The chemiosmotic theory is
discussed elsewhere.
If no membrane potential or pH gradient
exists to drive the forward reaction, the Keq
favors the reverse reaction, ATP hydrolysis
(ATPase activity).
In some bacteria, the reverse reaction has a
physiological role, providing a mechanism for
ATP-dependent creation of a proton gradient
that drives other reactions.
Inhibitors of F1Fo, that block H+ transport
coupled to ATP synthesis or hydrolysis, include:
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oligomycin, an antibiotic
DCCD (dicyclohexylcarbodiimide), a
reagent that reacts with carboxyl groups in
hydrophobic environments, forming a
covalent adduct.
Viewed by electron microscopy with negative
staining, the ATP synthase appeared as
"lollipops" on the inner mitochondrial membrane,
facing the matrix (V & V Fig. 22-36 p. 827).
Higher resolution cryo-electron microscopy later
showed each lollipop to have two stalks. E.g.,
see movie on a website of J. Rubinstein.
Roles of major subunits were determined in
studies of submitochondrial particles (SMP). If
mitochondria are treated with ultrasound, the
inner membrane breaks and reseals as vesicles,
with F1 on the outer surface. Since F1 of intact
mitochondria faces the interior matrix space,
these SMP are said to be inside out.
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F1, the lollipop head, when extracted from SMP, catalyzes ATP hydrolysis (the
spontaneous reaction in the absence of an energy input). Thus F1 contains the
catalytic domain(s).
After removal of F1, the SMP membrane containing Fo is leaky to H+. Adding
back F1 restores the normal low permeability to H+. Thus it was established that
Fo includes a "proton channel."
Either oligomycin or DCCD blocks the H+ leak in membranes depleted of F1.
Thus oligomycin and DCCD inhibit the ATP Synthase by interacting with Fo.
ATP synthase complexes
of bacteria, mitochondria
and chloroplasts are all
very similar, with only
minor differences.
Mitochondria are
believed to have evolved
from symbiotic aerobic
bacteria ingested by an
anaerobic host cell. The
limiting membrane of the
bacterium became the
inner mitochondrial
membrane. Mitochondria
contain a small DNA
chromosome, but genes
that encode most
mitochondrial proteins
are located in the nucleus,
consistent with transfer of
some DNA to the nucleus
during evolution.
The subunit composition of the ATP Synthase was first established for E. coli, which
has an operon that encodes genes for all subunits. Stalk subunits were classified initially
as being part of either F1 or Fo, based on whether they co-purified with extracted F1.
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F1 subunits were named with Greek letters in
order of decreasing molecular weight. They are
present with stoichiometry α3, β 3, γ, δ, ε.
o The α and β subunits (513 and 460 amino
acid residues in E. coli), are homologous
to one another. Looking down at the
membrane, α & β subunits alternate
around a ring. (The γ subunit is discussed
below.)
o There are three nucleotide-binding
catalytic sites, located at αβ interfaces
but predominantly involving residues of
the β subunits.
o Each α subunit contains a tightly bound
ATP, but is inactive in catalysis.
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o Mg binds with the adenine nucleotides
in both α and β subunits.
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Fo subunits were named in Roman letters with decreasing molecular weight.
The stoichiometry of these subunits in the E. coli is a, b2, c10.
Mammalian mitochondrial F1Fo is slightly more complex than the bacterial enzyme,
with a few additional subunits. Also, since names were assigned based on apparent
molecular weights, some subunits were given different names in different organisms.
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The bovine δ subunit turned out to be homologous to the E. coli ε subunit.
The bovine ε subunit is unique.
A bovine subunit called OSCP (oligomycin sensitivity conferral protein) is
homologous to the E. coli δ subunit.
The bovine enzyme has additional subunits d and F6.
There is evidence that the ATP Synthase (F1Fo) may form a complex with the adenine
nucleotide translocase (ADP/ATP antiporter) and the phosphate carrier (Pi/H+ symporter).
This complex has been designated the ATP Synthasome.
The binding
change
mechanism
of energy
coupling was
proposed by
Paul Boyer.
He shared the
Nobel prize
for this model,
which
accounts for
the existence
of 3 catalytic
sites in F1.
For
simplicity,
only the
catalytic β
subunits are
shown in the
diagram at
right.
It is proposed that an irregularly shaped "shaft" linked to Fo rotates relative to the 3β
subunits, which are arranged in a ring. The rotation is driven by flow of H+ through Fo.
The conformation of each β subunit changes sequentially, as it interacts with the rotating
shaft. Each of the 3 β subunits is in a different stage of the catalytic cycle at any time. For
example, the green subunit shown above sequentially changes to:
1. a loose conformation in which the active site can loosely bind ADP + Pi
2. a tight conformation in which substrates are tightly bound and ATP is formed
3. an open conformation that favors ATP release.
This model is supported by two major lines of evidence:
1. The crystal structure of F1 with the central stalk was determined by John Walker,
who shared the Nobel prize for that achievement. The γ (gamma) subunit was found to
include a bent helical loop that constitutes a "shaft" within the ring of α and β subunits.
Shown at right is bovine F1, treated
with DCCD to yield crystals in which
more of the central stalk is ordered,
allowing structure determination.
(Structure solved by C. Gibbons, M. G.
Montgomery, A. G. W. Leslie, & J. E. Walker,
2000, PDB 1E79).
Subunit colors: α yellow, β green, γ
red, δ blue, and ε magenta.
Note the wide base of the rotary shaft,
including part of γ as well as δ and ε
subunits.
Recall that the bovine δ subunit, which
is located at the base of the shaft, is
equivalent to the ε subunit of bacterial
F1.
In crystals of F1 not treated with
DCCD (PDB file 1COW), less of the
shaft structure is elucidated, but
ligand binding may be observed
under more natural conditions.
The 3 β subunits are found to
differ in conformation and
bound ligand:
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Bound to one β subunit is a non-hydrolyzable analog of ATP (assumed to be the
tight conformation).
Bound to another β subunit is a molecule of ADP (assumed to be the loose
conformation).
The third β subunit has an empty active site (assumed to be the open
conformation).
These findings are consistent with the binding change mechanism, which predicts that
each of the three β subunits, being differently affected by the irregularly shaped rotating
shaft, will be in a different stage of the catalytic cycle.
Additional data are consistent with there being an intermediate conformation between
the major transitions discussed above. This intermediate conformation may have
nucleotide bound at all three sites. By one model, considering the left-most image in the
diagram above: ATP synthesis (on the green subunit) is associated with transition to an
intermediate conformation that allows binding of ADP + Pi to the adjacent, previously
empty site (magenta subunit). A further conformational change then occurs as ATP
formed in the previous step is released (from the cyan subunit).
See also recent articles, especially the paper by Kagawa et al.
Explore at
right the
structure of
bovine F1 with
bound ADP
and AMPPNP.
The nonhydrolyzable
AMPPNP is
used as a
substitute for
ATP, which
would
hydrolyze
during
crystallization.
2. Rotation of the γ shaft relative to the
ring of α and β subunits was demonstrated
by H. Noji, R. Yasuda, M.Yoshida & K.
Kinoshita.
β subunits of a bacterial F1 were tethered
to a glass surface, as represented at right. A
fluorescent-labeled actin filament
(shown in yellow) was attached to the
protruding end of the γ subunit.
Video recording showed the fluorescent
actin filament rotating like a propeller.
The rotation was found to be ATPdependent.
Studies using varied techniques have
shown ATP-induced rotation to occur in
discrete 120o steps, with intervening
pauses. Some observations indicate that
each 120o step consist of 80-90o and 30-40o
F1 ATPase
substeps, with a brief intervening pause.
Such substeps are consistent with evidence
for an intermediate conformation
between the major transitions, discussed
above.
Although the binding change mechanism is
widely accepted, some details of the
reaction cycle are still debated.
View videos showing F1 rotation, at a website that includes
details of the experimental approach used.
Then view at right an animation based on observed variation
in conformation of F1 subunits attributed to rotation of the γ
shaft.
of rotation in F1
The c subunit of Fo has a hairpin structure, with 2
transmembrane α-helices and a short connecting loop. (Structure
at right determined via NMR by M. E. Girvin, V. K. Rastogi, F. Abildgaard,
J. L. Markley, & R. H. Fillingame, 1998).
The small c subunit (79 amino acid residues in E. coli), is also
referred to as proteolipid, because of its hydrophobicity.
One α-helix of the c subunit includes an aspartate or glutamate
residue whose carboxyl group reacts with DCCD (Asp61 in E.
coli). Mutation studies have shown this DCCD-reactive
carboxyl group, which is located in the middle of the bilayer, to
be essential for H+ transport through Fo.
View at right a low resolution, partial structure of yeast F1 with the central stalk and
attached Fo c subunits (D. Stock, A. Leslie, & J. Walker, 1999, PDB file 1Q01).
Display as backbone and color chain.
Question: How many c subunits, are in the Fo c-ring?
Visualize the aspartate residue near the middle of one transmembrane segment of each
c subunit.
An atomic resolution structure of the complete ATP Synthase, including F1 and Fo with
peripheral as well as central stalks, has not yet been achieved. However partial or
complete structures of individual protein constituents, mutational studies, and evidence
for inter-subunit interactions, have defined the roles of most subunits.
The image at right,
depicting models of
mitochondrial and
bacterial ATP Synthase
subunit structure, was
provided by Dr. John
Walker. Keep in mind
that some equivalent
subunits from different
organisms are assigned
different names.
The proposed "rotor"
consists of the ring 10 c
subunits, plus the
central stalk (subunits γ,
δ, & ε in the
mitochondrial enzyme; or
γ & ε in E. coli).
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The E. coli ε
subunit
(mitochondrial δ)
helps to attach γ
to the rotating
ring of c subunits.
In some bacteria a
portion of the ε
subunit has an
additional role in
inhibiting the
reverse rotation
that accompanies
ATP hydrolysis.
A separate
inhibitory
peptide in
mitochondria
prevents F1Fo
from hydrolyzing
ATP when there
is no
electrochemical
H+ gradient to
Mitochondrial ATP Synthase
E. coli ATP Synthase
drive ATP
synthesis, e.g.,
under anoxic
conditions.
The proposed "stator"
consists of the 3α
α and 3β
β
F1 subunits, the a subunit
of Fo, and a peripheral
stalk that connects these.
The peripheral stalk
consists of 2b, and δ in E.
coli, or subunits b, d, F6,
and OSCP in bovine
mitochondria..
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The b subunit includes a membrane anchor, one transmembrane α-helix in E.
coli and two in mammalian F1Fo, that interacts with the intramembrane a subunit.
A polar, α-helical domain of b extends out from the membrane.
OSCP, which is homologous to the E. coli δ subunit, interacts with the
protruding end of the b subunit and with the distal end of an F1 α subunit. This
linkage, along with interactions of the b subunit with residues on the surface of
F1, are postulated to hold back the ring of α and β subunits, keeping it from
rotating along with the central stalk.
The a subunit of Fo (271 amino acid residues in E. coli) is predicted, e.g., from
hydropathy plots, to include several transmembrane α-helices.
It has been proposed that the intramembrane a subunit contains two half-channels or
proton wires (each a series of protonatable groups or embedded water molecules), that
allow passage of protons between the two membrane surfaces and the interior of the
bilayer.
Protons may be relayed from one half-channel or proton wire to the other only via the
DCCD-sensitive carboxyl group of a c-subunit. Recall that the essential carboxyl group
of each c-subunit (Asp61 in E. coli) is located half way through the membrane (see
above). An essential arginine residue on one of the transmembrane a-subunit α-helices
has been identified as the group that accepts a proton from Asp61 and passes it to the
exit channel.
As the ring of 10 c subunits rotates, the c-subunit
carboxyls relay protons between the 2 a-subunit halfchannels. This allows H+ gradient-driven H+ flux across the
membrane to drive the rotation.
It has been proposed that rotation of the ring of c subunits
may result from concerted swiveling movements of the csubunit helix that includes the DCCD-sensitive Asp61 and
transmembrane a-subunit helices having the residues that
transfer H+ to or from Asp61, as protons are passed from or
to each half-channel. See also Fig. 22-43 p. 832.
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A webpage of Dr. Mark Girvin provides links to
animations relevant to this mechanism.
A webpage of Dr. Joachim Weber includes a
diagram of the E. coli F1Fo complex, based on a
composite of solved X-ray, NMR and modeling
structures, with cartoons representing parts of the
complex whose structure has not yet been
determined.
A website of Nobel laureate John Walker contains
movies depicting conformational changes in F1
during rotation and catalysis.
Copyright © 1998-2007 by Joyce J. Diwan. All rights reserved.