ATP SYNTHESIS
Centre for Nano science and Technology
Course: Biology for Nanotechnology.
Code: NST 623
Course instructor: Dr. S.Kannan.
PRESENTED BY
ROOPAVATH UDAY KIRAN
M.Tech 1st year

Outline
• Introduction
• Electron-Transfer Reactions in Mitochondria
• ATP Synthesis
• Regulation of Oxidative Phosphorylation
• General Features of Photophosphorylation
• Light Absorption
• The Central Photochemical Event: Light-
Driven Electron Flow
• ATP Synthesis by Photophosphorylation

Adenosine Triphosphate
Energy source
photosynthesis and cellular
respiration
Signal
transduction
second messenger cAMP
DNA replication
AMP

Structure
Purine base
1’C
5’C
Pentos sugar
Three
phosphate
groups

• Substrate-level phosphorylation
direct transfer of a phospate group to ADP

In mitochondrion

• Chemiosmotic Phosphorylation
Electrochemical gradient + Osmosis
1.Oxidative Phosphorylation
2. Photophosphorylation

ATP is synthesized using the same strategy in
oxidative phosphorylation and
photophosphorylation
• Oxidative phosphorylation is the process in which ATP is
generated as a result of electron flow from NADH or
FADH2 to O2 via a series of membrane-bound electron
carriers, called the respiratory chain (reducing O2 to H2O
at the end).
• Photophosphorylation is the process in which ATP (and
NADPH) is synthesized as a result of electron flow from
H2O to NADP+ via a series of membrane-bound electron
carriers (oxidizing H2O to O2 at the beginning).

• Oxidative phosphorylation and photophosphorylation are
mechanistically similar in three respects.
(1) Both processes involve the flow of electrons through a
chain of membrane-bound carriers.
(2) The free energy made available by this ―downhill‖
(exergonic) electron flow is coupled to the ―uphill‖
transport of protons across a proton-impermeable
membrane, conserving the free energy of fuel oxidation
as a. transmembrane electrochemical potential
(3) The transmembrane flow of protons down their
concentration gradient through specific protein channels
provides the free energy for synthesis of ATP,
catalyzed by a membrane protein complex (ATP
synthase) that couples proton flow to phosphorylation of
ADP.

ATP GenerationGlycolysis
• Conversion of glucose to pyruvate
• Net synthesis of 2 ATP by substrate level
phosphorylation
Krebs Cycle
• Converts pyruvate to acetyl CoA & carbon dioxide
• 10 molecules of coenzymes NADH and 2 of FADH2 are
produced. Results in synthesis of 30 ATP and 4 ATP molecules,
respectively in the respiratory chain.
Electron Transport (Respiratory) Chain
• The reduced coenzymes enter into the respiratory
chain of the inner mitochondrial membrane
• Electron transport along the chain generates a proton
electrochemical gradient and this is used to produce ATP

Chemiosmotic theory:
• Introduced by Peter Mitchell in 1961
• Transmembrane differences in proton
concentration are the reservoir for the energy
extracted from biological oxidation reactions.
• It provides insight into the processes of
oxidative phosphorylation and
photophosphorylation, and into such
apparently disparate energy transductions as
active transport across membranes and the
motion of bacterial flagella.

Proton Gradient Across the Membrane:
“Chemiosmosis”
• It is the universal mechanism of ATP production
which involves the production of a proton motive
force (pmf) based on a proton gradient across
the membrane.
• Energy to establish this electrochemical proton
gradient is provided by the energy released as
electrons move to lower energy levels down the
electron transport chain and the coupling of this
free energy to the movement of protons across the
IMM against the proton gradient [from matrix to
IMS]
• ATP is synthesized by the ATP synthase FoF1
complex : protons move with the proton gradient
through FoF1 to generate ATP [from IMS to matrix]

The chemiosmotic
model of Mitchell

OXIDATIVE PHOSPHORYLATION
• The discovery in 1948 by Eugene Kennedy and
Albert Lehninger that mitochondria are the site of
oxidative phosphorylation in eukaryotes marked the
beginning of the modern phase of studies in
biological energy transductions.
• Oxidative phosphorylation begins with the entry of
electrons into the respiratory chain.
• Most of these electrons arise from the action of
dehydrogenases that collect electrons from
catabolic pathways and funnel them into universal
electron acceptors—nicotinamide nucleotides
(NAD+ or NADP+) or flavin nucleotides (FMN or
FAD).

• The mitochondrial respiratory chain consists of a series of
sequentially acting electron carriers, most of which are
integral proteins with prosthetic groups capable of
accepting and donating either one or two electrons.
• Three types of electron transfers occur in oxidative
phosphorylation:
(1) Direct transfer of electrons, as in the reduction of Fe+3
to Fe+2;
(2) Transfer as a hydrogen atom (H+ +e); and
(3) Transfer as a hydride ion (:H), which bears two
electrons.
• The term reducing equivalent is used to designate a
single electron equivalent transferred in an oxidation-
reduction reaction.

Electrons collected in NADH and FADH2 are
released and transported to O2 via the respiratory
chain
• The chain is located on the convoluted inner
membrane (cristae) of mitochondria in
eukaryotic cells (revealed by Eugene
Kennedy and Albert Lehninger in 1948) or
on the plasma membrane in prokaryotic cells.
• A 1.14-volt potential difference (E`0)
between NADH (-0.320 V) and O2 (0.816 V)
drives electron flow through the chain.

• The respiratory chain consists of four large multi-
protein complexes (I, II, III, and IV; three being
proton pumps) and two mobile electron carriers,
ubiquinone (Q or coenzyme Q, and cytochrome c.
• Prosthetic groups acting in the proteins of
respiratory chain include flavins (FMN, FAD),
hemes (heme A, iron protoporphyrin IX, heme C),
iron-sulfur clusters (2Fe-2S, 4Fe-4S), and copper.

Four multi-protein
Complexes (I, II,
III, and IV)
Two mobile
Electron carriers
I
II
III
IV

• Ubiquinone (also called coenzyme Q, or simply
Q) is a lipid-soluble benzoquinone with a long
isoprenoid side chain
• Because ubiquinone is both small and
hydrophobic, it is freely diffusible within the lipid
bilayer of the inner mitochondrial membrane and
can shuttle reducing equivalents between other,
less mobile electron carriers in the membrane. And
because it carries both electrons and protons, it
plays a central role in coupling electron flow to
proton movement.

Complete reduction
of ubiquinone
requires two
electrons and two
protons, and occurs
in two steps through
the semiquinone
radical
intermediate.

Heme groups of
cytochrome
proteins
Heme groups
Of cytochromes

Different types of
iron-sulfur centers
•Iron atoms cycle between Fe2+
(reduced) and Fe3+(oxidized).
•At least eight Fe-S proteins
act in the respiratory chain.
4Fe-4S2Fe-2S
A ferredoxin

NADH:Ubiquinone
Oxidoreductase
a.k.a. Complex I
• One of the largest macro-
molecular assemblies in the
mammalian cell
• Over 40 different polypeptide
chains, encoded by both nuclear
and mitochondrial genes
• NADH binding site in the matrix
side
• Non-covalently bound flavin
mononucleotide (FMN) accepts
two electrons from NADH
• Several iron-sulfur centers pass
one electron at the time toward
the ubiquinone binding site

NADH:Ubiquinone Oxidoreducase is a
Proton Pump
• Transfer of two electrons from NADH to ubiquinone is
accompanied by a transfer of protons from the matrix (N)
to the inter-membrane space (P)
• Experiments suggest that about four protons are
transported per one NADH
NADH + Q + 5H+
N = NAD+ + QH2 + 4 H+
P
• Reduced coenzyme Q picks up two protons
• Despite 50 years of study, it is still unknown how the four
other protons are transported across the membrane

Iron-Sulfur Centers
• Found in several proteins of
electron transport chain,
including NADH:ubiquinone
oxidoreductase
• Transfers one electron at a
time

Succinate Dehydrogenase
a.k.a. Complex II
• FAD accepts two
electrons from succinate
• Electrons are passed, one
at a time, via iron-sulfur
centers to ubiquinone
that becomes reduced
QH2

• The cytochromes are proteins with
characteristic strong absorption of visible light,
due to their iron-containing heme prosthetic
groups. Mitochondria contain three classes of
cytochromes, designated a, b, and c, which are
distinguished by differences in their light-
absorption spectra.
• Each type of cytochrome in its reduced (Fe2)
state has three absorption bands in the visible
range

Cytochrome bc1 Complex a.k.a. Complex III
• Uses two electrons from QH2 to reduce two
molecules of cytochrome c

The Q Cycle
• 4 H+ / 2 e-
that reach
CytC
• 2 H+ from
QH2
• 2 H+ from
the matrix

Cytochrome c
• Cytochrome c is a soluble
heme-containing protein
in the intermembrane
space
• Heme iron can be either
ferrous(Fe3+, oxidized) or
ferric(Fe2+, reduced)
• Cytochrome c carries a
single electron from the
cytochrome bc1 complex
to cytochrome oxidase

Cytochrome c Absorbs Visible
Light
• Intense Soret band near
400 nm absorbs blue light
and gives cytochrome c
an intense red color
• Cytochromes are
sometimes named by the
position of their longest-
wavelength peak

Cytochrome Oxidase
a.k.a. Complex IV
• Mammalian cytochrome oxidase is a membrane
protein with 13 subunits
• Contains two heme groups
• Contains copper ions
– Two ions (CuA) form a binuclear center
– Another ion (CuB) bonded to heme forms Fe-Cu
center

Cytochrome C Oxidase (complex IV) Transport

Structure of the Cytochrome C Oxidase Monomer
• The heme groups are
shown in blue and red
and copper sites in
green
• The catalytic core
consists of I yellow, II
blue, III pink
• The entire complex
consists of 13 subunits

A proposed reaction cycle for the four-electron
reduction of O2 by cytochrome oxidase (at the
Heme a3-CuB center)

Structure of Beef Heart Cytochrome Oxidase
The protein is a dimer of two 13 monomers
3 dimensional structure of beef heart cytochrome
oxidase at 2.8 angstrom resolution

The order of the many electron carriers on the
respiratory chain have been elucidated via various
studies
• Measurement of the standard reduction potential
(E`0)): Electrons tend to transfer from low E`0
carriers to high E`0 carriers (but may deviate from
this in real cells).
• Oxidation kinetics studies: Full reduction followed
by sudden O2 introduction; earlier oxidation, closer
to the end of the respiratory chain; using rapid and
sensitive spectrophotometric techniques to follow
the oxidation of the cytochromes, which have
different wavelength of maximal absorption).

Electron carriers may have an order of increasing E`0

• the standard reduction potentials of the
individual electron carriers have been
determined experimentally . We would expect
the carriers to function in order of increasing
reduction potential, because electrons tend to
flow spontaneously from carriers of lower E
to carriers of higher E.
• The order of carriers deduced by this method is
NADH → Q → cytochrome b →
cytochrome c1 → cytochrome c →
cytochrome a → cytochrome a3 → O2.

• Effects of various specific inhibitors: those
before the blocked step should be reduced and
those after be oxidized.
• Isolation and characterization of each of the
multiprotein complexes: specific electron
donors and acceptors can be determined for
portions of the chain.

Various inhibitors generate various patterns of
reduced/oxidized carriers
Reduced Oxidized
Reduced Oxidized
Reduced

Oxidative
Phosphorylation
(0n inner membrane
of mitochondria)

Electron transfer to O2 was found to be coupled to
ATP synthesis from ADP + Pi in isolated mitochondria
• ATP would not be synthesized when only ADP
and Pi are added in isolated mitochondria
suspensions.
• O2 consumption, an indication of electron flow,
was detected when a reductant (e.g., succinate) is
added, accompanied by an increase of ATP
synthesis.
• Both O2 consumption and ATP synthesis were
suppressed when inhibitors of respiratory chain
(e.g., cyanide, CO, or antimycin A) was added.
• ATP synthesis depends on the occurrence of
electron flow in mitochondria.

• O2 consumption (thus electron flow) was
neither observed if ADP was not added to
the suspension, although a reductant is
provided!
• The O2 consumption was also not observed in the
presence of inhibitors of ATP synthase (e.g.,
oligomycin or venturicidin).
• Electron flow also depends on ATP synthesis!

Electron transfer was found to be obligatorily
coupled to ATP Synthesis in isolated
mitochondria suspensions:
neither occurs without the other.

3 D Model of ATP Synthase:
An Electrical Mechano-Chemical
Molecular Complex
• The Fo portion is composed of
integral transmembranous
proteins a, b and 9-14 copies of c
which forms a ring-like structure
in the plane of the membrane.
• The F1 head piece is composed of
a hexagonal array of alternating 
and  subunits, a central  protein
with a helical coil that associates
with  and  proteins and extends
into the c protein ring in the Fo.

Atomic Force Microscopy of C-subunit Ring Structures
Isolated from Chloroplast ATP Synthase and Inserted
Into Liposomes

c ring & a subunit structure
•each c subunit has 2 membrane-
spanning
a helices
– midway along 1 helix: asp
– COOH↔COO–
•a subunit has 2 half-channels
H+ path
•H+ from cytosol diffuses via half-
channel
to asp on c ring subunit (c1)
•this subunit can now move to
interface membrane, allowing
c ring to rotate
•c9 now interfaces matrix half-channel,
allowing H+ to diffuse into matrix
c ring
subunit a
H+ path through membrane
c1 c9
matrix
half-
channel
cytosolic
half-channel
asp
subunit ac subunit

cannot rotate in
either direction
can rotate
clockwise
matrix
H+ flow drives rotation of c ring

Binding-change mechanism of ATP synthesis
• Rotation of gama subunit drives release of tightly
bound ATP
• 3 active sites cycle through 3 structural states:
O, open; L, loose-binding; T, tight-binding
• At T site, ADP + Pi  ATP, but ATP can’t dissociate
• G rotation causes T  O, L  T, O  L
• As a result of the TO structural change,
ATP can now dissociate from what is now an O site.
T O
ATP
ADP + Pi
ATP
120° rotation of 
(counterclockwise)
T
TO
O
L
L
1 1
2 2
3 3

Synthesis of ATP: Rotary Catalysis
• ATP is synthesized by coupling the energy liberated during
proton translocation through the FoF1 to a motive force that
rotates
the C ring structure and the attached  subunit.
• -subunits contain the catalytic sites of ATP synthesis. 120
degree
units of rotation of the  protein around the stationary /
hexagonal array results in altered associations of the  protein
with the  protein forming the L, T and O states for the 3 β-
subunits.
ATP is produced in the T state where the ∆G = ~ 0.
• Each rotation of 360 degrees of the γ subunit results in 3 ATP,
one
for each β-subunit.
The model shows the rotation as arbitrarily clockwise.
∆G = ~ 0

Nature 386, 299 - 302 (20 March 1997); doi:10.1038/386299a0
Direct observation of the rotation of F1-ATPase
HIROYUKI NOJI*, RYOHEI YASUDA†, MASASUKE YOSHIDA* & KAZUHIKO KINOSITA JR†
†Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1,
Kohoku-ku, Yokohama 223, Japan

Transport across inner mitochondrial membrane
• p also drives flow of substances across inner membrane
• Transported by specific carrier proteins
• Cotransport: coupled transport of 2 substances
–Symport:
both move
in same
direction
CH3CCOO–
+ H+
O
HPO4=
+ H+
–Antiport:
each moves
in opposite
direction
ADP-ATP
exchange

Active Transport of ATP, ADP & Pi
• Adenine Nucleotide Translocase
– Antiporter
– (ATP4-
matrix  ADP3-
inter membrane)
• Phosphate trans locase
– Symporter
– {Pi- , H+} inter membrane => {Pi- , H+} matrix

Summary of ATP synthesis & translocation of ATP,ADP & Pi

Energy of Light is Used to
Synthesize ATP in
Photosynthetic Organisms
• Light causes charge separation between
a pair chlorophyll molecules
• Energy of the oxidized and reduced
chlorophyll molecules is used drive
synthesis of ATP
• Water is the source of electrons that are
passed via a chain of transporters to the
ultimate electron acceptor, NADP+
• Oxygen is the byproduct of water
oxidation

Various Pigments Harvest the
Light Energy
The energy is transferred to the photosynthetic reaction
center

Light-Induced Redox Reactions and Electron
Transfer Cause Acidification of Lumen
The proton-motive
force across the
thylakoid
membrane drives
the synthesis of
ATP

Flow of Protons: Mitochondria,
Chloroplasts, Bacteria
• Mitochondria and chloroplasts arose endosymbionts - entrapped
bacteria
• Bacterial cytosol became mitochondrial matrix and chloroplast stroma

Photophosphorylation
(on thylakoid of chloroplasts)

References
• Lehninger Principles of Biochemistry, 5th
Edition- © 2008 W.H Freeman and company.
• Fundamentals of Biochemistry- a text book ,
H.P. Gajera, S.V. Patel, B.A. Golakiya.
• Fundamentals of Biochemistry- J.L. Jain,
Sunjay Jain, Nitin Jain.