Oxidative Phosphorylation III

ATP synthase: Structure and

Mechanism of ATP synthesis
ATP Synthase
❖ Mitochondrial ATP synthase, also known as Complex V is a F-type
ATPase.
❖ ATP synthase is a remarkable molecular machine. It is an enzyme, a
proton pump, and a rotating molecular motor. Nearly all the ATP
that fuels our cellular processes is made by this ATP synthase.
❖ It resemble in structure and mechanism to the ATP synthases of
chloroplasts and bacteria.
❖ Located in inner mitochondrial membrane catalyzes the formation
of ATP from ADP and Pi, accompanied by the flow of protons from
the P (intermembrane space) to the N (matrix) side of the
membrane.
❖ It consist of two multi-protein components:
✓ F1, a peripheral membrane protein
✓ Fo (o denoting oligomycin-sensitive), an integral multi-
transmembrane protein complex is a proton channel.
ATP Synthase structure

ATP synthase, also called

Complex V, has two distinct
components:
F1, a peripheral membrane
protein complex (discovered
by Efraim Racker and his
colleagues in the early 1960s).
Fo (o denoting oligomycin-
sensitive), which is integral to
the membrane.

Figure is from Lehninger Principles of Biochemistry by Nelson & Cox,

5th Edition 2008, W.H. Freeman and Company, New-York
F1 subunit
❖ F1 binds to the ADP and Pi and form ATP. It is catalytic unit of
ATP synthase. It is present in the matrix region of Mitochondria.
❖ Mitochondrial F1 has nine subunits of five different types, with
the composition α3β3γδε.
❖ 3α3β form the hexameric ring structure in the alternate
arrangement. Both α and β can bind to ATP but only β unit
possessed the catalytic activities.
❖ γ and ε combined to form central stalk is an elongated structure
which runs along the inner cavity of the hexameric α3β3 ring
structure. It is also connects the Fo structure.
❖ Function of γε central stalk is to connect Fo and F1 subunits of
ATP synthase and as central stalk rotates it stimulate catalysis of
ADP and Pi to synthesis and release of ATP.
❖ δ subunit hold hexameric α3β3 ring structure in place and will
keep it from rotating.
Fo subunit
❖ Fo (o denoting oligomycin-sensitive) is proton channel that
allows downhill movement of proton according to the
electrochemical gradient.
❖ Fo is highly hydrophobic and consist of 10-14 “c” subunit (su)
organized into a ring structure that acts as hydrogen ion
channel. It allows H+ ion to flow from intermembrane space
to matrix according to electrochemical gradient.
❖ Subunit “a” bind to the outside of c-ring and help connect Fo
to F1 unit and play a role in proton transport.
❖ Fo and F1 connected at two points
1) Through the γε central stalk
2) through the arm formed by “a” subunit,“2b” subunit and
“δ” subunit.
Mechanism of ATP synthase

Paul Boyer proposed a simple catalytic scheme commonly

known as the binding change mechanism, which predicted
that F-ATPase implements a rotational mechanism in the
catalysis of ATP
The F1 subunit of ATP synthase responsible for:
a) Binding to ADP and Pi molecule Do not req H+
b) Catalyzing the synthesis of ATP Channel
c) Releasing the ATP molecule Required H+ channel
❖ Although α subunit contain ATP, they do not release it and
they do not actively participate in the reaction.
❖ The each β subunit can bind to ADT and Pi, synthesize the
ATP and releases them into matrix. At any given time the
each of three β subunit can exist in one of the three forms:
a) Tense state: the ADP and Pi are brought close and
they are combined to form ATP
b) Open state: The formed ATP is released and new
ADP and Pi set can bind
c) The loose state: The ADP and Pi are trapped and can
not leave
❖ These three states i.e., T, O, L are interchangeable and at
given time all three β su will be existed in three different
states.
Binding change mechanism of F1 subunit of ATP synthase
❖ The change in the state (T, O, L) of β su is brought by
sequential rotation of γ su. This occurs in following steps:
a) A rotation of γ su of 120o in the counterclockwise direction
causes conformational change in the β su which convert
the tense state of β su into open state. This allows open β
su to release ATP.
b) Once the ATP is release, the sets of ADP and Pi enters in to
open β su. Another rotation of γ su to further 120o in the
counterclockwise direction locks the open β su and convert
into loose state.
c) Another 120o rotation of γ su caused loose β su to convert
into T state and ADP and Pi are brought closure and ATP
is formed
❖ The cycle is repeated. This mechanism is called binding change
mechanism. The overall 360o rotation of γ su leads to synthesis
of 3 ATP as there are 3 β su.
How downhill movement of proton across inner
mitochondrial membrane site causes ATP production?
❖ Fo of the ATP synthase is responsible for the movement of
proton across inner mitochondrial membrane from
intermembrane space to the matrix.
❖ Fo consist of “a” and “c” subunits. “a” contains two
hydrophilic half channels that do not cross the entire
membrane. “c” su is very small (8kD) and highly hydrophobic.
❖ One of the half channel opens in the inner membrane space
while the other half channels open in the matrix site.
Interaction of this two half channels and “c” su of ring
causes proton movements
❖ “a” subunit is positioned to interacts with “c” subunit. The
center of each “c” subunit contains an aspartate residue that
can readily bind to H+ under acidic condition near the ‘a’ half
channel.
Proton movement through
aspartic acid in every “c”
su of Fo subunit
❖ The mechanism of proton movement is as follows.
a) A H+ will enter the half-channel of the “a”su facing the
inner membrane space and it will bind to the aspartate
residue of a nearby “c” subunit.
b) The entire “c” ring then rotate clockwise due to the
movement of aspartic acid (which become more
hydrophobic after protonation) towards the
hydrophobic region of the membrane until a “c”
subunit with protonated aspartic acid enter the other
half channel that faces the proton poor environment of
the matrix.
c) The H+ ion is released into the matrix of mitochondria
due to slight alkaline environment of the matrix.
❖ Therefore, the movement of the H+ through the half
channel powers the rotation of the “c” ring. Since the “c”
ring is directly connected to γε central stalk, it causes
central stalk to rotate as well as.
❖ This intern stimulates the synthesis of ATP in the α3β3 via
the binding change mechanism.
How many H+ ions movement leads to generation of an ATP ?
(360o of rotation of γε/3 ATP)= 10-14 of H+/360o rotation)
= 3.33-4.67 H+ /ATP
3 to 4 H+ are needed to cross the membrane to generate 1 ATP.
What is the P/O ratio of oxidative phosphorylation
in mitochondria?
❖ The P/O ratio is the number of molecules of ATP formed in
oxidative phosphorylation per two electrons flowing through a
defined segment of the electron transport chain to reduced one
atom of oxygen.
❖ For 2e- from NADH + H+ to the oxygen →10 H+ to P site

❖ For 2e- from FADH to the oxygen →6 H+ to P site

The complete oxidation of glucose yields about 30
molecules of ATP
Reaction sequence ATP yield per
glucose molecule
Glycolysis 2
TCA cycle 2
Oxidative Phosphorylation
2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP 3
(assuming transport of NADH by the glycerol 3-phosphate shuttle)
2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each 5
yields 2.5 molecules of ATP
2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules 3
of ATP
6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules 15
of ATP
NET YIELD PER MOLECULE OF GLUCOSE 30
The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier one of 36
molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport
should be regarded as estimates. About two more molecules of ATP are formed per molecule of glucose
oxidized when the malate-aspartate shuttle rather than the glycerol 3- phosphate shuttle is used.
References
❖Nelson DL and Cox MM, Lehninger Principles of
Biochemistry, 5th Edition 2008, W.H. Freeman and
Company, New-York​​
❖Berg, Tymoczko and Stryer, Biochemistry, 5
E W.H. Freeman and Company, New-York​​
❖Garrett and Grisham - Biochemistry (4E) – 2010,
Brooks/Cole, Cengage Learning )