Atp synthesis

1. ATP SYNTHESIS
CentreforNanoscienceandTechnology
Course:BiologyforNanotechnology.
Code:NST623
Courseinstructor:Dr.S.Kannan.
PRESENTED BY
ROOPAVATH UDAY KIRAN
M.Tech1
st
year

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

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

4. Structure
Purinebase
1’C
5’C
Pentossugar
Three
phosphate
groups

5. •Substrate-level phosphorylation
direct transfer of a phospategroup to ADP

6. In mitochondrion

8. •ChemiosmoticPhosphorylation
Electrochemical gradient + Osmosis
1.Oxidative Phosphorylation
2.Photophosphorylation

9. ATP is synthesized using the same strategy in
oxidative phosphorylationand
photophosphorylation
•OxidativephosphorylationistheprocessinwhichATPis
generatedasaresultofelectronflowfromNADHor
FADH
2toO
2viaaseriesofmembrane-boundelectron
carriers,calledtherespiratorychain(reducingO
2toH
2O
attheend).
•PhotophosphorylationistheprocessinwhichATP(and
NADPH)issynthesizedasaresultofelectronflowfrom
H
2OtoNADP
+
viaaseriesofmembrane-boundelectron
carriers(oxidizingH
2OtoO
2atthebeginning).

10. •Oxidativephosphorylationandphotophosphorylationare
mechanisticallysimilarinthreerespects.
(1)Bothprocessesinvolvetheflowofelectronsthrougha
chainofmembrane-boundcarriers.
(2)Thefreeenergymadeavailablebythis―downhill‖
(exergonic)electronflowiscoupledtothe―uphill‖
transportofprotonsacrossaproton-impermeable
membrane,conservingthefreeenergyoffueloxidation
asa.transmembraneelectrochemicalpotential
(3)Thetransmembraneflowofprotonsdowntheir
concentrationgradientthroughspecificproteinchannels
providesthefreeenergyforsynthesisofATP,
catalyzedbyamembraneproteincomplex(ATP
synthase)thatcouplesprotonflowtophosphorylationof
ADP.

11. ATP Generation
Glycolysis
•Conversion of glucose to pyruvate
•Net synthesis of 2 ATP by substrate level
phosphorylation
Krebs Cycle
•Converts pyruvateto acetyl CoA& carbon dioxide
•10moleculesofcoenzymesNADHand2ofFADH
2are
produced.Resultsinsynthesisof30ATPand4ATPmolecules,
respectivelyintherespiratorychain.
Electron Transport (Respiratory) Chain
•The reduced coenzymes enter into the respiratory
chain of the inner mitochondrial membrane
•Electron transport along the chain generates aproton
electrochemical gradientand this is used to produce ATP

13. Chemiosmotictheory:
•Introduced by Peter Mitchell in 1961
•Transmembranedifferencesinproton
concentrationarethereservoirfortheenergy
extractedfrombiologicaloxidationreactions.
•Itprovidesinsightintotheprocessesof
oxidative phosphorylation and
photophosphorylation,andintosuch
apparentlydisparateenergytransductionsas
activetransportacrossmembranesandthe
motionofbacterialflagella.

14. Proton Gradient Across the Membrane:
“Chemiosmosis”
•ItistheuniversalmechanismofATPproduction
whichinvolvestheproductionofaprotonmotive
force(pmf)basedonaprotongradientacross
themembrane.
•Energytoestablishthiselectrochemicalproton
gradientisprovidedbytheenergyreleasedas
electronsmovetolowerenergylevelsdownthe
electrontransportchainandthecouplingofthis
freeenergytothemovementofprotonsacrossthe
IMMagainsttheprotongradient[frommatrixto
IMS]
•ATPissynthesizedbytheATPsynthaseF
oF
1
complex:protonsmovewiththeprotongradient
throughF
oF
1togenerateATP[fromIMStomatrix]

15. The chemiosmotic
model of Mitchell

16. OXIDATIVE PHOSPHORYLATION
•Thediscoveryin1948byEugeneKennedyand
AlbertLehningerthatmitochondriaarethesiteof
oxidativephosphorylationineukaryotesmarkedthe
beginningofthemodernphaseofstudiesin
biologicalenergytransductions.
•Oxidativephosphorylationbeginswiththeentryof
electronsintotherespiratorychain.
•Mostoftheseelectronsarisefromtheactionof
dehydrogenasesthatcollectelectronsfrom
catabolicpathwaysandfunnelthemintouniversal
electronacceptors—nicotinamidenucleotides
(NAD+orNADP+)orflavinnucleotides(FMNor
FAD).

18. •Themitochondrialrespiratorychainconsistsofaseriesof
sequentiallyactingelectroncarriers,mostofwhichare
integralproteinswithprostheticgroupscapableof
acceptinganddonatingeitheroneortwoelectrons.
•Threetypesofelectrontransfersoccurinoxidative
phosphorylation:
(1)Directtransferofelectrons,asinthereductionofFe+3
toFe+2;
(2)Transferasahydrogenatom(H++e);and
(3)Transferasahydrideion(:H),whichbearstwo
electrons.
•Thetermreducingequivalentisusedtodesignatea
singleelectronequivalenttransferredinanoxidation-
reductionreaction.

19. Electrons collected in NADH and FADH
2are
released and transported to O
2viathe respiratory
chain
•Thechainislocatedontheconvolutedinner
membrane(cristae)ofmitochondriain
eukaryoticcells(revealedbyEugene
KennedyandAlbertLehningerin1948)or
ontheplasmamembraneinprokaryoticcells.
•A1.14-voltpotentialdifference(E`
0
)
betweenNADH(-0.320V)andO
2(0.816V)
driveselectronflowthroughthechain.

20. •Therespiratorychainconsistsoffourlargemulti-
proteincomplexes(I,II,III,andIV;threebeing
protonpumps)andtwomobileelectroncarriers,
ubiquinone(QorcoenzymeQ,andcytochromec.
•Prostheticgroupsactingintheproteinsof
respiratorychainincludeflavins(FMN,FAD),
hemes(hemeA,ironprotoporphyrinIX,hemeC),
iron-sulfurclusters(2Fe-2S,4Fe-4S),andcopper.

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

22. •Ubiquinone(alsocalledcoenzymeQ,orsimply
Q)isalipid-solublebenzoquinonewithalong
isoprenoidsidechain
•Becauseubiquinoneisbothsmalland
hydrophobic,itisfreelydiffusiblewithinthelipid
bilayeroftheinnermitochondrialmembraneand
canshuttlereducingequivalentsbetweenother,
lessmobileelectroncarriersinthemembrane.And
becauseitcarriesbothelectronsandprotons,it
playsacentralroleincouplingelectronflowto
protonmovement.

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

24. Heme groups of
cytochrome
proteins
Heme groups
Of cytochromes

25. Different types of
iron-sulfur centers
•IronatomscyclebetweenFe
2+
(reduced)andFe
3+
(oxidized).
•AtleasteightFe-Sproteins
actintherespiratorychain.
4Fe-4S2Fe-2S
A ferredoxin

26. NADH:Ubiquinone
Oxidoreductase
a.k.a. Complex I
•Oneofthelargestmacro-
molecularassembliesinthe
mammaliancell
•Over40differentpolypeptide
chains,encodedbybothnuclear
andmitochondrialgenes
•NADHbindingsiteinthematrix
side
•Non-covalentlyboundflavin
mononucleotide(FMN)accepts
twoelectronsfromNADH
•Severaliron-sulfurcenterspass
oneelectronatthetimetoward
theubiquinonebindingsite

27. NADH:UbiquinoneOxidoreducaseis a
Proton Pump
•TransferoftwoelectronsfromNADHtoubiquinoneis
accompaniedbyatransferofprotonsfromthematrix(N)
totheinter-membranespace(P)
•Experimentssuggestthataboutfourprotonsare
transportedperoneNADH
NADH+Q+5H
+
N=NAD
+
+QH
2+4H
+
P
•ReducedcoenzymeQpicksuptwoprotons
•Despite50yearsofstudy,itisstillunknownhowthefour
otherprotonsaretransportedacrossthemembrane

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

29. SuccinateDehydrogenase
a.k.a. Complex II
•FAD acceptstwo
electronsfromsuccinate
•Electronsarepassed,one
atatime,viairon-sulfur
centerstoubiquinone
thatbecomesreduced
QH
2

30. •Thecytochromesareproteinswith
characteristicstrongabsorptionofvisiblelight,
duetotheiriron-containinghemeprosthetic
groups.Mitochondriacontainthreeclassesof
cytochromes,designateda,b,andc,whichare
distinguishedbydifferencesintheirlight-
absorptionspectra.
•Eachtypeofcytochromeinitsreduced(Fe2)
statehasthreeabsorptionbandsinthevisible
range

31. Cytochromebc
1Complex a.k.a. Complex III
•Uses two electrons from QH
2to reduce two
molecules of cytochromec

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

33. Cytochromec
•Cytochromecis a soluble
heme-containing protein
in the intermembrane
space
•Hemeiron can be either
ferrous(Fe
3+
, oxidized) or
ferric(Fe
2+
, reduced)
•Cytochromeccarries a
single electron from the
cytochromebc
1complex
to cytochromeoxidase

34. CytochromecAbsorbs Visible
Light
•Intense Soretband near
400 nm absorbs blue light
and gives cytochromec
an intense red color
•Cytochromesare
sometimes named by the
position of their longest-
wavelength peak

35. CytochromeOxidase
a.k.a. Complex IV
•Mammalian cytochromeoxidaseis a membrane
protein with 13 subunits
•Contains two hemegroups
•Contains copper ions
–Two ions (Cu
A) form a binuclear center
–Another ion (Cu
B) bonded to hemeforms Fe-Cu
center

36. CytochromeC Oxidase(complex IV) Transport

37. Structure of the CytochromeC OxidaseMonomer
•The hemegroups 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

38. Aproposedreactioncycleforthefour-electron
reductionofO
2bycytochromeoxidase(atthe
Hemea
3-Cu
Bcenter)

39. Structure of Beef Heart CytochromeOxidase
The protein is a dimerof two 13 monomers
3 dimensional structure of beef heart cytochrome
oxidaseat 2.8 angstrom resolution

40. The order of the many electron carriers on the
respiratory chain have been elucidated via various
studies
•Measurementofthestandardreductionpotential
(E`
0)
):ElectronstendtotransferfromlowE`
0
carrierstohighE`
0
carriers(butmaydeviatefrom
thisinrealcells).
•Oxidationkineticsstudies:Fullreductionfollowed
bysuddenO
2introduction;earlieroxidation,closer
totheendoftherespiratorychain;usingrapidand
sensitivespectrophotometrictechniquestofollow
theoxidationofthecytochromes,whichhave
differentwavelengthofmaximalabsorption).

41. Electron carriers may have an order of increasing E`
0

42. •thestandardreductionpotentialsofthe
individualelectroncarriershavebeen
determinedexperimentally.Wewouldexpect
thecarrierstofunctioninorderofincreasing
reductionpotential,becauseelectronstendto
flowspontaneouslyfromcarriersoflowerE
tocarriersofhigherE.
•Theorderofcarriersdeducedbythismethodis
NADH →Q→cytochromeb→
cytochromec1→cytochromec→
cytochromea→cytochromea3→O2.

43. •Effectsofvariousspecificinhibitors:those
beforetheblockedstepshouldbereducedand
thoseafterbeoxidized.
•Isolationandcharacterizationofeachofthe
multiproteincomplexes:specificelectron
donorsandacceptorscanbedeterminedfor
portionsofthechain.

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

45. Oxidative
Phosphorylation
(0n inner membrane
of mitochondria)

46. Electron transfer to O
2was found to be coupled to
ATP synthesis from ADP + P
iin isolated mitochondria
•ATPwouldnotbesynthesizedwhenonlyADP
andP
iareaddedinisolatedmitochondria
suspensions.
•O
2consumption,anindicationofelectronflow,
wasdetectedwhenareductant(e.g.,succinate)is
added,accompaniedbyanincreaseofATP
synthesis.
•BothO
2consumptionandATPsynthesiswere
suppressedwheninhibitorsofrespiratorychain
(e.g.,cyanide,CO,orantimycinA)wasadded.
•ATPsynthesisdependsontheoccurrenceof
electronflowinmitochondria.

47. •O
2consumption(thuselectronflow)was
neitherobservedifADPwasnotaddedto
thesuspension,althoughareductantis
provided!
•TheO
2consumptionwasalsonotobservedinthe
presenceofinhibitorsofATPsynthase(e.g.,
oligomycinorventuricidin).
•ElectronflowalsodependsonATPsynthesis!

48. Electrontransferwasfoundtobeobligatorily
coupledtoATPSynthesisinisolated
mitochondriasuspensions:
neitheroccurswithouttheother.

49. 3 D Model of ATP Synthase:
An Electrical Mechano-Chemical
Molecular Complex
•The F
oportion 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 F
1 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 F
o.

50. Atomic Force Microscopy of C-subunit Ring Structures
Isolated from Chloroplast ATP Synthaseand Inserted
Into Liposomes

51. 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 cytosoldiffuses via half-
channel
to aspon 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
cring
subunit a
H
+
path through membrane
c1
c9
matrix
half-
channel
cytosolic
half-channel
asp
subunit acsubunit

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

53. Binding-change mechanism of ATP synthesis
•Rotationofgamasubunitdrivesreleaseoftightly
boundATP
•3activesitescyclethrough3structuralstates:
O,open;L,loose-binding;T,tight-binding
•AtTsite,ADP+PiATP,butATPcan’tdissociate
•GrotationcausesTO,LT,OL
•AsaresultoftheTOstructuralchange,
ATPcannowdissociatefromwhatisnowanOsite.
T
O
ATP
ADP + Pi
ATP
120°rotation of 
(counterclockwise)
T
T
O
O
L
L
1 1
2 2
3 3

54. Synthesis of ATP: Rotary Catalysis
•ATPissynthesizedbycouplingtheenergyliberatedduring
protontranslocationthroughtheF
oF
1toamotiveforcethat
rotates
theCringstructureandtheattachedsubunit.
•-subunitscontainthecatalyticsitesofATPsynthesis.120
degree
unitsofrotationoftheproteinaroundthestationary/
hexagonalarrayresultsinalteredassociationsoftheprotein
withtheproteinformingtheL,TandOstatesforthe3β-
subunits.
ATPisproducedintheTstatewherethe∆G=~0.
•Eachrotationof360degreesoftheγsubunitresultsin3ATP,
one
foreachβ-subunit.
The model shows the rotation as arbitrarily clockwise.
∆G = ~ 0

55. 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, Hiyoshi3-14-1,
Kohoku-ku, Yokohama 223, Japan

56. 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

57. Active Transport of ATP, ADP & Pi
•Adenine Nucleotide Translocase
–Antiporter
–(ATP
4-
matrixADP
3-
inter membrane)
•Phosphate trans locase
–Symporter
–{Pi
-
, H
+
}
inter membrane => {Pi
-
, H
+
}
matrix

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

59. 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 moleculesis used drive
synthesis of ATP
•Water is the source of electronsthat are
passed via a chain of transporters to the
ultimate electron acceptor, NADP
+
•Oxygen is the byproduct of water
oxidation

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

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

67. Flow of Protons: Mitochondria,
Chloroplasts, Bacteria
•Mitochondria and chloroplasts arose endosymbionts-entrapped
bacteria
•Bacterial cytosolbecame mitochondrial matrixand chloroplast stroma

68. Photophosphorylation
(on thylakoidof chloroplasts)

69. References
•LehningerPrinciplesofBiochemistry,5
th
Edition-©2008W.HFreemanandcompany.
•FundamentalsofBiochemistry-atextbook,
H.P.Gajera,S.V.Patel,B.A.Golakiya.
•FundamentalsofBiochemistry-J.L.Jain,
SunjayJain,NitinJain.