The document summarizes the structure and function of F1Fo ATP synthase, a membrane-bound enzyme that catalyzes ATP synthesis using an electrochemical proton gradient. It consists of two main components, F1, which protrudes into the mitochondrial matrix and contains the catalytic sites, and Fo, which forms a proton channel embedded in the membrane. During ATP synthesis, protons flow through the Fo channel, causing the central stalk to rotate and drive conformational changes in the F1 catalytic sites that catalyze ATP formation from ADP and phosphate. The rotation mechanism and roles of key subunits like c and a are described.

1. F1Fo ATP Synthase
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.
Molecular Biochemistry I

2. F1
Fo
ATP Synthase of mitochondria, chloroplasts, bacteria:
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.,
toward the mitochondrial matrix).
ADP + Pi ATP
F1
Fo
3 H+
matrix
intermembrane
space
ATP synthesis with ∆pH & ∆Ψ
− − −
+ + +

3. If no ∆pH or ∆Ψ exists to drive the forward reaction, Keq
favors the reverse, ATP hydrolysis (ATPase).
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.
ADP + Pi ATP
F1
Fo
3 H+
ATPase with H+
gradient dissipated
matrix
intermembrane
space
ADP + Pi ATP
F1
Fo
3 H+
matrix
intermembrane
space
ATP synthesis with ∆pH & ∆Ψ
− − −
+ + +

4. Inhibitors of F1Fo,that block H+
transport coupled to
ATP synthesis or hydrolysis, include:
 oligomycin, an antibiotic
 DCCD (dicyclohexylcarbodiimide), a reagent that
reacts with carboxyl groups in hydrophobic
environments, forming a covalent adduct.

5. Roles of major subunits were determined in studies of
submitochondrial particles (SMP).
In mitochondria treated with ultrasound, inner membrane
breaks & reseals as vesicles, with F1 on the outer surface.
Since F1 of intact mitochondria faces the matrix, these SMP
are said to be inside out.
By EM with negative stain,
ATP Synthase appeared as
"lollipops" on the inner
mitochondrial membrane,
facing the matrix.
Higher resolution cryo-EM
later showed each lollipop
to have 2 stalks. See Movie.
mitochondrion
ultrasound
SMP
F1

6.  After removal of F1, the SMP membrane containing Fo is
leaky to H+
.
Adding back F1 restores normal low permeability to H+
.
Thus it was established that Fo includes a “H+
channel."
 F1, the lollipop head,
when extracted from
SMP, catalyzes ATP
hydrolysis (spontaneous
reaction in the absence
of an energy input).
Thus F1 contains the
catalytic domain(s).
mitochondrion
ultrasound
SMP
F1

7.  Either oligomycin or DCCD blocks the H+
leak in
membranes depleted of F1
.
Thus oligomycin & DCCD inhibit the ATP Synthase by
interacting with Fo
.
ATP synthase complexes of bacteria, mitochondria &
chloroplasts are very similar, with only minor differences.
ADP + Pi ATP
F1
Fo
3 H+
matrix
intermembrane
space
ATP synthesis with ∆pH & ∆Ψ
− − −
+ + +

8. 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 in the nucleus,
consistent with transfer of some DNA during evolution.
aerobic
bacterium
protoeukaryotic cell
lacking aerobic metabolism
DNA
mitochondrial
precursor
nucleus

9. Looking down at the membrane,
α & β subunits alternate around
a ring (γ to be discussed later.)
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 part of F1 or Fo,
based on whether they co-purified with extracted F1.
F1 subunits were named with Greek letters in order of
decreasing MW.
They are present with stoichiometry α3, β3, γ, δ, ε.
The α & β subunits (513 & 460 aa residues in E. coli) are
homologous to one another.
α α
α
β
β β
γ
F1 in cross
section

10. There are three nucleotide-binding catalytic sites,
located at αβ interfaces but predominantly involving
residues of the β subunits.
Each α subunit contains a tightly bound ATP, but is
inactive in catalysis.
Mg++
binds with the adenine nucleotides in both α & β
subunits.
α α
α
β
β β
γ
F1 in cross
section

11. Fo subunits were named in
Roman letters with decreasing
molecular weight.
Stoichiometry of these subunits in E. coli Fo 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.
ADP + Pi ATP
F1
Fo
3 H+
− − −
+ + +

12.  Bovine δ subunit turned out to be homologous to the
E. coli ε subunit.
 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 & F6.
There is evidence that the ATP Synthase (F1Fo) may form a
complex with adenine nucleotide translocase (ADP/ATP
antiporter) & phosphate carrier (Pi/H+
symporter).
This complex has been designated the ATP Synthasome.

13. The binding change mechanism of energy coupling was
proposed by Paul Boyer.
He shared the Nobel prize for this model that accounts
for the existence of 3 catalytic sites in F1
.
For simplicity, only the catalytic β subunits are shown.
 It is proposed that an irregularly shaped shaft linked
to Fo rotates relative to the ring of 3 β subunits.
 The rotation is driven by flow of H+
through Fo
.
ADP + Pi ATP
ATP
ATP ADP
+ Pi
ATP ADP
+ Pi
ATP
open tight
binding
loose
binding
repeat
Binding Change Mechanism

14. ADP + Pi ATP
ATP
ATP ADP
+ Pi
ATP ADP
+ Pi
ATP
open tight
binding
loose
binding
repeat
Binding Change Mechanism
The conformation of each β subunit changes sequentially
as it interacts with the rotating shaft.
Each β subunit is in a different stage of the catalytic cycle
at any time. E.g, the green subunit sequentially changes to:
 a loose conformation in which the active site can loosely
bind ADP + Pi
 a tight conformation in which substrates are tightly
bound and ATP is formed
 an open conformation that favors ATP release.

15. The γ subunit includes a bent helical loop that constitutes a
"shaft" within the ring of α & β subunits.
Shown is bovine F1 treated with DCCD to yield crystals in
which more of the central stalk is ordered, allowing
structure determination. Colors: α, β, γ, δ, ε.
F1 ATPase PDB 1E79
side view base view
Supporting
evidence:
1. The crystal
structure of F1
with the central
stalk was solved
by J. Walker,
who shared the
Nobel prize.

16. 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 at the base of
the shaft, is equivalent to ε of bacterial F1.
F1 ATPase PDB 1E79
side view base view
Bovine F1
(DCCD-
treated)

17. In crystals of F1 not treated with DCCD, less of the shaft
structure is solved, but ligand binding may be observed
under more natural conditions.
The 3β subunits are found to differ in conformation &
bound ligand.
Two views of F1 (γ subunit red) PDB file 1COW

18.  Bound to one β subunit is a non-hydrolyzable ATP
analog (assumed to be the tight conformation).
 Bound to another β subunit is ADP (loose).
 The third β subunit has an empty active site (open).
This is consistent with the binding change model, which
predicts that each β subunit, being differently affected by
the irregularly shaped rotating shaft, will be in a different
stage of the catalytic cycle.
ADP + Pi ATP
ATP
ATP ADP
+ Pi
ATP ADP
+ Pi
ATP
open tight
binding
loose
binding
repeat
Binding Change Mechanism

19. Additional data are consistent with an intermediate
conformation between each of the 3 states shown.
This intermediate conformation may have nucleotide bound
at all 3 sites. By one model, in the left image above:
ATP synthesis (on green subunit) is associated with
transition to an intermediate conformation that allows
binding of ADP + Pi to the previously empty site (magenta).
A further conformational change then occurs as ATP formed
in the previous step is released (from cyan subunit).
ADP + Pi ATP
ATP
ATP ADP
+ Pi
ATP ADP
+ Pi
ATP
open tight
binding
loose
binding
repeat
Binding Change Mechanism

20. Explore with Chime the structure of bovine F1 with
bound ADP and AMPPNP.
The non-hydrolyzable AMPPNP is used as a substitute
for ATP, which would hydrolyze during crystallization.
AMPPNP (ADPNP) ATP analog
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OPOPNP-O
O
O- O-
O O
O-
H

21. β subunits of F1 were tethered to a glass surface.
A fluorescent-labeled actin filament (yellow) was attached
to the protruding end of the γ subunit (shaft).
Video recordings showed the actin filament rotating like a
propeller. The rotation was ATP-dependent.
α
ββ
γ
Rotation of γ relative to α & β
2. Rotation of the
γ shaft relative to
the ring of α & β
subunits was
demonstrated by
Noji, Yasuda,
Yoshida &
Kinoshita.

22. Some observations indicate that each 120o
step consists of
80-90o
& 30-40o
substeps, with a brief intervening pause.
Such substeps are consistent with evidence for an
intermediate conformation between the major
transitions, discussed above.
α
ββ
γ
Rotation of γ relative to α & β
Studies using
varied techniques
have shown ATP-
induced rotation
to occur in discrete
120o
steps, with
intervening pauses.

23. Although the binding change mechanism is widely
accepted, some details of the reaction cycle are still
debated.
View an animation of ATP synthesis based on
observed variation in conformation of F1 subunits
attributed to rotation of the γ shaft.

24. The c subunit of Fo has a hairpin
structure with 2 transmembrane
α-helices & a short connecting loop.
The small c subunit (79 aa in E.
coli) is also called proteolipid,
because of its hydrophobicity.
One α-helix includes an Asp or Glu
residue whose carboxyl reacts with
DCCD (Asp61 in E coli).
Mutation studies have shown that
this DCCD-reactive carboxyl, in the
middle of the bilayer, is essential
for H+
transport through Fo.
Asp61
Fo subunit c
PDB 1A91

25. At right: a low resolution
partial structure of yeast F1
with central stalk & attached Fo
c subunits.
View this file by Chime.
 Count the number of Fo c
subunits, arranged in a ring.
 Look for the Asp near the
middle of one transmembrane
segment of each c subunit.
Partial
structures
of F1, Fo
PDB 1Q01

26. 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.
Partial
structures
of F1, Fo
PDB 1Q01

27. Mitochondrial ATP Synthase E. coli ATP Synthase
These images depicting models of ATP Synthase subunit
structure were provided by John Walker. Some equivalent
subunits from different organisms have different names.

28. Mitochondrial F1Fo E. coli F1Fo
In some bacteria a portion of ε has an added role inhibiting
the reverse rotation that accompanies ATP hydrolysis.
A separate inhibitory peptide in mitochondria prevents
F1Fo from hydrolyzing ATP when there is no H+
gradient to
drive ATP synthesis, e.g., under anoxic conditions.
Proposed "rotor": the
ring of 10 c subunits,
plus the central stalk
• γ & ε in E. coli
• γ, δ, & ε in
mitochondria.
E. coli ε (mito. δ) helps
attach γ to the rotating
ring of c subunits.

29. Mitochondrial F1Fo E. coli F1Fo
The proposed "stator" consists of the 3α & 3β F1 subunits,
a subunit of Fo, & a peripheral stalk that connects these.
The peripheral stalk consists of 2b & δ in E. coli or
subunits b, d, F6, & OSCP in bovine mitochondria.

30. Mitochondrial F1Fo E. coli F1Fo
OSCP, homologous to E. coli δ, interacts with the end of
the b subunit & with the distal end of an F1 α subunit.
This linkage, plus interactions of b with residues on F1,
are postulated to hold back the ring of α & β subunits,
keeping it from rotating along with the central stalk.
The b subunit includes
a membrane anchor,
1 transmembrane
α-helix in E. coli; 2 in
mammalian F1Fo, that
interacts with the intra-
membrane a subunit.
A polar α-helical
domain of b extends out
from the membrane.

31. The a subunit of Fo (271 amino
acid residues in E. coli) is
predicted from hydropathy plots,
to include several trans-membrane
α-helices.
It has been proposed that the
intramembrane a subunit contains
2 half-channels or proton wires
(each a series of protonatable
groups or embedded waters), that
allow passage of protons between
the two membrane surfaces & the
bilayer interior.
a
subunit
ring of
c subunits
H+
H+

32. 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.
Asp61
Fo subunit c
PDB 1A91
An essential arginine residue on one of the trans-
membrane a-subunit α-helices has been identified as the
group that accepts a proton from Asp61 and passes it to
the exit channel.

33. As the ring of 10 c subunits
rotates, the c-subunit carboxyls
relay protons between the
2 a-subunit half-channels.
This allows H+
gradient-driven
H+
flux across the membrane
to drive the rotation.
a
subunit
ring of
c subunits
H+
H+

34. Rotation of the ring of c subunits may result from
concerted swiveling movements of the c-subunit helix
that includes Asp61, & transmembrane a-subunit helices
with residues that transfer H+
to or from Asp61, as protons
are passed from or to each half-channel.
a
subunit
ring of
c subunits
H+
H+
Asp61
Fo subunit c
PDB 1A91
Proposed
mechanism:

35. • A webpage with animations
relevant to this mechanism.
• A webpage with a diagram
of E. coli F1Fo, based on a
composite of solved structures,
with cartoons representing
parts of the complex whose
structure has not yet been
determined.
a
subunit
ring of
c subunits
H+
H+
• A website with movies depicting conformational
changes in F1 during rotation and catalysis.