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LECTURE 3:NADPH AND ATP

SYNTHESIS

LECTURE OUTCOMEStudents, after mastering the materials ofthe present lecture, should be able to:1. To explain the conversion mechanism of light energy to

chemical energy (NADPH & ATP)2. To explain electron excitation and flow in the

conversion mechanism of light energy to chemicalenergy

3. To explain the relationship between light energy andwave length

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LECTURE OUTLINE

FluorescencePhosphorescence

LIGHT ABSORBTIONAND TRANSFERTO THE REACTIONCENTERS

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Resonance Energy Transfer - Radiationless

e- e-

e-

e-

e-

e-

e-

e-

hv

Ground state

Excited state

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1. Absorpsi foton mengakibatkan pengaturanelektron intramolekul pada pusat reaksi yangdiikuti dengan tranfer elektron antar molekul

2. Pada mulanya, elektronkhlorofil pada pusat reaksitereksitasi pada orbityang menjauhi inti atomdan molekul denganabsorpsi foton langsungatau lebih mungkinmelalui transfer energifoton dari antena pigmen

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e-

e-

e-

e-

hc

E

hc

E

Excited

ATPmill

Two types ofphotosystems(PS) cooperate inthe light reactions

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Water-splittingphotosystem

NADPH-producingphotosystemPS II PS I

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Fluorescence Emisi cahaya dari molekul yang sedang diiradiasi

sebagai akibat dari penurunan elektron dari orbait 1ke orbit dasar. Proses ini tidak tergantung suhu danberlangsung cepat (lifetime 10-8 detik). Panjanggelombang lebih besar dari panjang gelombangyang diabsorpsi (chlorophyll a mengabsorpsi cahayapada 430 & 630 nm, dan mengemisi cahaya pada668 nm).

Phosphorescence Emisi cahaya dari molekul sebagai akibat penurunan

elektron dari “triplet state” ke orbit dasar. Cahayayang dihasilkan berlangsung relatif perlahan (10-4 –2 detik), dan panjang gelombang relatif sangatpanjang

Excited statee

HeatLight

Photon

Light(fluorescence)

Chlorophyllmolecule

Groundstate

2

Absorption of a photon

Excitation of chlorophyll in a chloroplast

Fluorescence of tonic water (Left),and an acetone solution ofchlorophyll under direct UVirradiation (Right).

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3. Red light absorbed by photosystem II (PSII)produces a strong oxidant and a weakreductant.

4. Far-red light absorbed by photosystem I (PSI)produces a weak oxidant and a strongreductant.

5. The strong oxidant generated by PSIIoxidizes water, while the strongreductant produced by PSI reducesNADP.

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1. H2O2. Z (PSII)3. P680* (PSII reactioncenter chlorophyll)4. Pheo (pheophytin)5. QA and QB(plastoquinoneacceptors)6. Cytochrome b.-fcomplex7. PC (plastocyanin)

8. P700+ (PSI reactioncenter chlorophyll)9. A0 (chlorophyll ?)10. A1 (quinone?)11. FeSx, FeSB, & FeSA(membrane-boundiron-sulfur proteins)12. Fd (soluble ferredoxin)13. Fp (flavoproteinferredoxin-NADPreductase)14. NADP

Primaryelectron acceptor

Primaryelectron acceptor

Photons

PHOTOSYSTEM I

PHOTOSYSTEM II

Energy forsynthesis of

by chemiosmosis

Noncyclic Photophosphorylation Photosystem II regains electrons by splitting water, leavingO2 gas as a by-product

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The O2 liberated by photosynthesis is madefrom the oxygen in water (H+ and e-)

Plants produce O2 gas by splitting H2O

The red X indicates that protonsdo not directly pass through thecytochrome complex.

Protons cross the membrane viaoxidation and reduction of quinones

X

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1. Z is a tyrosine side chain on the reactioncenter protein D1. Electrons are extractedfrom water (H2O) by the oxygen-evolvingcomplex (OEC)and rereduce Z+.

2. On the oxidizing side of PSII (to the left ofthe arrow joining P680 with P680*), P680+is rereduced by Z, the immediate donor toPSII.https://en.wikipedia.org/wiki/File:Photosystem-II_2AXT.PNG

3. The excited PSII reaction center chlorophyll,(P680*) transfers an electron to pheophytin(Pheo).4. On the reducing side of PSII (to the right ofthe arrow joining P680 with P680*), thepheophytin transfers electrons to theplastoquinone acceptors QA and QB.5. The cytochrome b.-f complex transferselectrons to plastocyanin (PC), which in turnreduces P700+.6. The b,-f complex contains a Rieske iron-sulfurprotein (FeSR), two b-type cytochromes (cytb), and cytochrome f (cyt f).7. The acceptor of electrons from P700* (A0) isthought to be a chlorophyll, and the nextacceptor (A1) may be a quinone.

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8. A series of membrane-bound iron-sulfurproteins (FeSx, FeSB, and FeSA) transferelectrons to soluble ferredoxin (Fd).9. The flavoprotein ferredoxin-NADP reductase(Fp) serves to reduce NADP, which is used inthe Calvin cycle to reduce CO2.10. The dashed line indicates cyclic electron flowaround PSI.11. PSII produces electrons that reduce thecytochrome b.-f complex, while PSI producesan oxidant that oxidizes the cytochrome b.-fcomplex.P680 and P700 refer to the wavelengths ofmaximum absorption of the reaction centerchlorophylls in PSII and PSI

12. The electron of PSI is then excited uponabsorption of radiation energy and transfer toA0 (chlorophyll)13. The transfer of electron occurs from A0 to A1(quinone), FeSx, FeSB, & FeSA(membrane-bound iron-sulfur proteins), Fd(soluble ferredoxin ), Fp (flavoproteinferredoxin-NADP reductase) and finally toNADP+14. It was found antagonistic effects of light oncytochrome oxidation.

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H2O Z (PSII)P680* (PSII reaction centerchlorophyll) Pheo (pheophytin)QA andQB (plastoquinone acceptors)Cytochromeb.-f complex PC (plastocyanin)P700+(PSI reaction center chlorophyll) A0(chlorophyll ?)A1 (quinone?) FeSx,FeSB, & FeSA (membrane-bound iron-sulfurproteins) Fd (soluble ferredoxin )Fp(flavoprotein ferredoxin-NADPreductase)NADP

1. Eksitasi 1 mol e pada setiap pusat reaksi (PSII &PSI) membutuhkan 1 kuanta cahaya. Reduksi 1mol NADP 1 mol NADPH membutuhkan 2 mole2. Berapa kuanta cahaya dibutuhkan untukpembentukan 1 mol NADPH ?3. Berapa NADPH dihasil dari hasil fotolisis air ?4. Tingkat Cahaya di Malang sekitar 1 mmol.s-1,berapa NADPH yang dihasilkan dengan tingkatcahaya demikian ?

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1. Where does the light reaction happen ?2. What is the function of water inphotosynthesis ?3. What is the event to happen after the lightinterception by pigments4. What is the first molecule receivingelectrons from the pigments (chlorophyll)excited at PS I5. What is the first molecule receivingelectrons from the pigments (chlorophyll)excited at PS II

Compiled crystal structure of chloroplast F1F0 ATP synthase

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A portion of the photon’s energy is capturedas the high-energy phosphate bond in ATP

Electron flow must occur for the formation ofATP (photophosphorylation), althoughunder some conditions electron flow maynot be accompanied by phosphorylation.

It is now widely accepted thatphotophosphorylation works via thechemiosmotic mechanism, first proposed inthe 1960s by Peter Mitchell

Outer membraneInner membrane

Thylakoid membraneThylakoid space

StromaGrana

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1. The basic principle of chemiosmosis is that ionconcentration differences and electrical potentialdifferences across membranes are Chemiosmosiseenergy that can be utilized by the cell.

2. As described by the second law of thermodynamics,any nonuniform distribution of matter or energyrepresents a source of energy.

3. Chemical potential differences of any molecularspecies whose concentrations are not the same onopposite sides of a membrane provide such a source ofenergy.

4. Proton flow from one side of the membrane to the otheraccompanies electron flow in addition to theasymmetric nature of the photosynthetic membrane

5. The direction of proton translocation is such thatthe STROMA BECAMES MORE ALKALINE and theLUMEN BECAMES MORE ACIDIC when electrontransport occurs.

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The synthesis of ATP in chloroplasts occurs when theenergy of a proton gradient is coupled to phosphorylateADP to ATP by an F-type ATPase Protons are accumulated on the inside of the thylakoidlumen and depleted in the stroma compartment

pH of chloroplast compartmentThylakoid lumen Stroma

Dark 7.0 7.0Light 5.2 8.2

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Some of the early evidence in support of achemiosmotic mechanism.

Andre Jagendorf and co-workers suspendedchloroplasts in a pH 4 buffer, which permeatedthe membrane and caused the interior, as wellas the exterior, of the thylakoid to equilibrate atthis acidic pH. They then rapidly injected thesuspension into a pH 8 buffer solution therebycreating a pH difference of 4 units across thethylakoid membrane , with the inside acidicrelative to the outside.

They found that large amounts of ATP wereformed from ADP and Pi by this process,without any light input or electron transport.

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The ATP is synthesized by a large (400-kDa)enzyme complex known as the coupling factor,ATPase, ATP synthase or CF0-CF1.

This enzyme consists of two parts: ahydrophobic membrane bound portion calledCF0 and a portion that sticks out into the stromacalled CF1.

CF0 appears to be achannel across themembrane through whichprotons can pass, whileCF1 is the portion of thecomplex that actuallysynthesizes ATP.

Subunit composition ofchloroplast F1F0 ATPsynthase. This enzymeconsists of a largemultisubunit complex,CF1 and CF0. CF1consists of five differentpolypepticles, withstoichiometry of 3, 3,, .

CF0 contains probably four different polypeptides, with astoichiometry of a, b, b', C14. Protons from the lumen aretransported by the rotating c polypepticle and ejected onthe stroma side. (Figure courtesy of W. Frasch in Taiz & Zeiger, 2010)

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Total energy available for ATP synthesis, protonmotive force (p), is the sum of a protonchemical potential and a transmembraneelectrical potential and These two components of the proton motiveforce from the outside of the membrane to theinside are given by

p = Em – 59(pHi – pH0) (mV) Em (or ) is the transmenbrane potentialand pH1- pH0 (or pH) is the pH differenceacross the membrane.

Total energy available for ATP synthesis, protonmotive force (p), is the sum of a proton chemicalpotential and a transmembrane electricalpotential and These two components of the proton motive forcefrom the outside of the membrane to the insideare given by

p = Em – 59(pHi – pH0) (mV) Em (or ) is the transmenbrane potential andpH1- pH0 (or pH) is the pH difference across themembrane.

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PHOTOSYSTEM I

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+ H+H+ H+H+ H+

H+ H+

H+

H+

H+

H+

H+

H+H+ H+H+

H+ H+

H+

H+ADP+ Pi

ATP

4.67 H+

Thylakoid Lumen – compartment of low pH

Chloroplast stroma – region of high pH

PHOTOPHOSPHORYLATION

ATP Synthase(F-type ATPase)

Based on the equation, a transmembranepH difference of one unit is equivalent to amembrane potential of 59 mV. Under conditions of steady state electrontransport in chloroplasts, the membraneelectrical potential is quite small, so p isdue almost entirely to pH The stoichiometry of protons translocatedto ATP synthesis has been to be(14/3)H+/ATP , before 3H+/ATP.

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Herbicides are xenobiotics (foreign chemicals toplants) that are designed to kill plants but are notsupposed to harm humans and animals. Ideally, herbicides will only kill weeds and variousnoxious plants, leaving crops unharmed. Major types of herbicides Hormone mimics: e.g. 2,4-D mimics the planthormone auxin. (trade-name of Weed-B-Gone) Inhibitors of biosynthetic pathways unique to plants:e.g. glyphosphate (Round-up) Inhibitors of photosynthetic electron transport: e.g.methyl viologen (Paraquat) and atrazine

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Process for ATPgenerationassociated withsomePhotosyntheticBacteria

• Electron in Photosystem I is excited and transferred toferredoxin that shuttles the electron to the cytochromecomplex.• The electron then travels down the electron chain andre-enters photosystem I

Cyclic photophosphorylation

In sulfur bacteria, only one photosystem isused

Generates ATP via electron transport Anoxygenic photosynthesis Excited electron passed to electron

transport chain Generates a proton gradient for ATP

synthesis

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1. How many is H+ transported across membranefrom lumen to stroma for the transfer of each1e- to NADP+

2. How many is ATP produced for each formationof 1 NADPH

3. How much is the light quanta required for eachformation of ATP

4. What does it mean by proton motive force5. What is the enzyme catalyzing the synthesis of

ATP from ADP

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