ATP SYNTHESIS

Centre for Nano science and TechnologyCourse: Biology for Nanotechnology.Code: NST 623Course instructor: Dr. S.Kannan.

PRESENTED BY

ROOPAVATH UDAY KIRANM.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 sourcephotosynthesis and cellular

respiration

Signal

transductionsecond messenger cAMP

DNA replicationAMP

Structure

Purine base

1’C5’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 isgenerated as a result of electron flow from NADH orFADH2 to O2 via a series of membrane-bound electroncarriers, called the respiratory chain (reducing O2 to H2Oat the end).

• Photophosphorylation is the process in which ATP (andNADPH) is synthesized as a result of electron flow fromH2O to NADP+ via a series of membrane-bound electroncarriers (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 productionwhich involves the production of a proton motiveforce (pmf) based on a proton gradient acrossthe membrane.

• Energy to establish this electrochemical protongradient is provided by the energy released aselectrons move to lower energy levels down theelectron transport chain and the coupling of thisfree energy to the movement of protons across theIMM against the proton gradient [from matrix toIMS]

• ATP is synthesized by the ATP synthase FoF1

complex : protons move with the proton gradientthrough FoF1 to generate ATP [from IMS to matrix]

The chemiosmoticmodel 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 mobileElectron carriers

III

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 groupsOf cytochromes

Different types of iron-sulfur centers•Iron atoms cycle between Fe2+

(reduced) and Fe3+(oxidized).

•At least eight Fe-S proteinsact 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-electronreduction of O2 by cytochrome oxidase (at theHeme 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 theindividual electron carriers have beendetermined experimentally . We would expectthe carriers to function in order of increasingreduction potential, because electrons tend toflow spontaneously from carriers of lower Eto carriers of higher E.

• The order of carriers deduced by this method isNADH → 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 ADPand Pi are added in isolated mitochondriasuspensions.

• O2 consumption, an indication of electron flow,was detected when a reductant (e.g., succinate) isadded, accompanied by an increase of ATPsynthesis.

• Both O2 consumption and ATP synthesis weresuppressed when inhibitors of respiratory chain(e.g., cyanide, CO, or antimycin A) was added.

• ATP synthesis depends on the occurrence ofelectron flow in mitochondria.

• O2 consumption (thus electron flow) wasneither observed if ADP was not added tothe suspension, although a reductant isprovided!

• The O2 consumption was also not observed in thepresence 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, allowingc 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

matrixhalf-channel

cytosolichalf-channel

asp

subunit ac subunit

cannot rotate ineither 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 tightlybound 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 TO 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 thatrotates

the C ring structure and the attached subunit.

• -subunits contain the catalytic sites of ATP synthesis. 120degree

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-ATPaseHIROYUKI 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 movesin oppositedirection

ADP-ATPexchange

Active Transport of ATP, ADP & Pi

• Adenine Nucleotide Translocase

– Antiporter

– (ATP4-matrixADP3-

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 thylakoidmembrane 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.