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LECTURE 3:
NADPH AND ATP
SYNTHESIS
LECTURE OUTCOME
Students, after mastering the materials of
the present lecture, should be able to:
1.
2.
3.
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To explain the conversion mechanism of light energy to
chemical energy (NADPH & ATP)
To explain electron excitation and flow in the
conversion mechanism of light energy to chemical
energy
To explain the relationship between light energy and
wave length
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LECTURE OUTLINE
Fluorescence
Phosphorescence
LIGHT ABSORBTION
AND TRANSFER
TO THE REACTION
CENTERS
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Resonance Energy Transfer - Radiationless
Excited state
e-
e-
e-
e-
hv
e-
e-
e-
e-
Ground state
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1.
Absorpsi foton mengakibatkan pengaturan
elektron intramolekul pada pusat reaksi yang
diikuti dengan tranfer elektron antar molekul
2.
Pada mulanya, elektron
khlorofil pada pusat reaksi
tereksitasi pada orbit
yang menjauhi inti atom
dan molekul dengan
absorpsi foton langsung
atau lebih mungkin
melalui transfer energi
foton dari antena pigmen
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E
hc

ee-
Excited
E
hc

ee-

Two types of
photosystems
(PS) cooperate in
the light reactions
ATP
mill
Water-splitting
photosystem
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PS II
NADPH-producing
photosystem
PS I
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
Fluorescence


Emisi cahaya dari molekul yang sedang diiradiasi
sebagai akibat dari penurunan elektron dari orbait 1
ke orbit dasar. Proses ini tidak tergantung suhu dan
berlangsung cepat (lifetime 10-8 detik). Panjang
gelombang lebih besar dari panjang gelombang
yang diabsorpsi (chlorophyll a mengabsorpsi cahaya
pada 430 & 630 nm, dan mengemisi cahaya pada
668 nm).
Phosphorescence

Emisi cahaya dari molekul sebagai akibat penurunan
elektron dari “triplet state” ke orbit dasar. Cahaya
yang dihasilkan berlangsung relatif perlahan (10-4 –
2 detik), dan panjang gelombang relatif sangat
panjang
Excitation of chlorophyll in a chloroplast
e
2
Excited state
Heat
Light
Light
(fluorescence)
Photon
Ground
state
Chlorophyll
molecule
Absorption of a photon
Fluorescence of tonic water (Left),
and an acetone solution of
chlorophyll under direct UV
irradiation (Right).
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3.
4.
5.
Red light absorbed by photosystem II (PSII)
produces a strong oxidant and a weak
reductant.
Far-red light absorbed by photosystem I (PSI)
produces a weak oxidant and a strong
reductant.
The strong oxidant generated by PSII
oxidizes water, while the strong
reductant produced by PSI reduces
NADP.
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1.
2.
3.
4.
5.
6.
7.
H2O
Z (PSII)
P680* (PSII reaction
center chlorophyll)
Pheo (pheophytin)
QA and QB
(plastoquinone
acceptors)
Cytochrome b.-f
complex
PC (plastocyanin)
8.
9.
10.
11.
12.
13.
14.
P700+ (PSI reaction
center chlorophyll)
A0 (chlorophyll ?)
A1 (quinone?)
FeSx, FeSB, & FeSA
(membrane-bound
iron-sulfur proteins)
Fd (soluble ferredoxin)
Fp (flavoprotein
ferredoxin-NADP
reductase)
NADP
Noncyclic Photophosphorylation

Photosystem II regains electrons by splitting water, leaving
O2 gas as a by-product
Primary
electron acceptor
Primary
electron acceptor
Photons
Energy for
synthesis of
PHOTOSYSTEM I
PHOTOSYSTEM II
by chemiosmosis
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Plants produce O2 gas by splitting H2O

The O2 liberated by photosynthesis is made
from the oxygen in water (H+ and e-)
The red X indicates that protons
do not directly pass through the
cytochrome complex.
X
Protons cross the membrane via
oxidation and reduction of quinones
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1.
Z is a tyrosine side chain on the reaction
center protein D1. Electrons are extracted
from water (H2O) by the oxygen-evolving
complex (OEC)and rereduce Z+.
https://en.wikipedia.org/wiki/File:Photosystem-II_2AXT.PNG
2.
On the oxidizing side of PSII (to the left of
the arrow joining P680 with P680*), P680+
is rereduced by Z, the immediate donor to
PSII.
3.
The excited PSII reaction center chlorophyll,
(P680*) transfers an electron to pheophytin
(Pheo).
On the reducing side of PSII (to the right of
the arrow joining P680 with P680*), the
pheophytin transfers electrons to the
plastoquinone acceptors QA and QB.
The cytochrome b.-f complex transfers
4.
5.
6.
7.
electrons to plastocyanin (PC), which in turn
reduces P700+.
The b,-f complex contains a Rieske iron-sulfur
protein (FeSR), two b-type cytochromes (cyt
b), and cytochrome f (cyt f).
The acceptor of electrons from P700* (A0) is
thought to be a chlorophyll, and the next
acceptor (A1) may be a quinone.
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A series of membrane-bound iron-sulfur
proteins (FeSx, FeSB, and FeSA) transfer
electrons to soluble ferredoxin (Fd).
9. The flavoprotein ferredoxin-NADP reductase
(Fp) serves to reduce NADP, which is used in
the Calvin cycle to reduce CO2.
10. The dashed line indicates cyclic electron flow
around PSI.
11. PSII produces electrons that reduce the
cytochrome b.-f complex, while PSI produces
an oxidant that oxidizes the cytochrome b.-f
complex.
8.
P680 and P700 refer to the wavelengths of
maximum absorption of the reaction center
chlorophylls in PSII and PSI
The electron of PSI is then excited upon
absorption of radiation energy and transfer to
A0 (chlorophyll)
13. The transfer of electron occurs from A0 to A1
(quinone), FeSx, FeSB, & FeSA
(membrane-bound iron-sulfur proteins), Fd
(soluble ferredoxin ), Fp (flavoprotein
ferredoxin-NADP reductase) and finally to
NADP+
14. It was found antagonistic effects of light on
cytochrome oxidation.
12.
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H2O Z (PSII)P680* (PSII reaction center
chlorophyll) Pheo (pheophytin)QA and
QB (plastoquinone acceptors)Cytochrome
b.-f complex PC (plastocyanin)P700+
(PSI reaction center chlorophyll) A0
(chlorophyll ?)A1 (quinone?) FeSx,
FeSB, & FeSA (membrane-bound iron-sulfur
proteins)  Fd (soluble ferredoxin )Fp
(flavoprotein ferredoxin-NADP
reductase)NADP
1.
2.
3.
4.
Eksitasi 1 mol e pada setiap pusat reaksi (PSII &
PSI) membutuhkan 1 kuanta cahaya. Reduksi 1
mol NADP  1 mol NADPH membutuhkan 2 mol
e
Berapa kuanta cahaya dibutuhkan untuk
pembentukan 1 mol NADPH ?
Berapa NADPH dihasil dari hasil fotolisis air ?
Tingkat Cahaya di Malang sekitar 1 mmol.s-1,
berapa NADPH yang dihasilkan dengan tingkat
cahaya demikian ?
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1.
2.
3.
4.
5.
Where does the light reaction happen ?
What is the function of water in
photosynthesis ?
What is the event to happen after the light
interception by pigments
What is the first molecule receiving
electrons from the pigments (chlorophyll)
excited at PS I
What is the first molecule receiving
electrons from the pigments (chlorophyll)
excited at PS II
Compiled crystal structure of chloroplast F 1F0 ATP synthase
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
A portion of the photon’s energy is captured
as the high-energy phosphate bond in ATP

Electron flow must occur for the formation of
ATP (photophosphorylation), although
under some conditions electron flow may
not be accompanied by phosphorylation.

It is now widely accepted that
photophosphorylation works via the
chemiosmotic mechanism, first proposed in
the 1960s by Peter Mitchell
Thylakoid membrane
Outer membrane
Thylakoid space
Inner membrane
Stroma
Grana
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1.
2.
3.
4.
5.
The basic principle of chemiosmosis is that ion
concentration differences and electrical potential
differences across membranes are Chemiosmosise
energy that can be utilized by the cell.
As described by the second law of thermodynamics,
any nonuniform distribution of matter or energy
represents a source of energy.
Chemical potential differences of any molecular
species whose concentrations are not the same on
opposite sides of a membrane provide such a source of
energy.
Proton flow from one side of the membrane to the other
accompanies electron flow in addition to the
asymmetric nature of the photosynthetic membrane
The direction of proton translocation is such that
the STROMA BECAMES MORE ALKALINE and the
LUMEN BECAMES MORE ACIDIC when electron
transport occurs.
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

The synthesis of ATP in chloroplasts occurs when the
energy of a proton gradient is coupled to phosphorylate
ADP to ATP by an F-type ATPase
Protons are accumulated on the inside of the thylakoid
lumen and depleted in the stroma compartment
pH of chloroplast compartment
Dark
Light
Thylakoid lumen
7.0
5.2
Stroma
7.0
8.2
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


Some of the early evidence in support of a
chemiosmotic mechanism.
Andre Jagendorf and co-workers suspended
chloroplasts in a pH 4 buffer, which permeated
the membrane and caused the interior, as well
as the exterior, of the thylakoid to equilibrate at
this acidic pH. They then rapidly injected the
suspension into a pH 8 buffer solution thereby
creating a pH difference of 4 units across the
thylakoid membrane , with the inside acidic
relative to the outside.
They found that large amounts of ATP were
formed 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: a
hydrophobic membrane bound portion called
CF0 and a portion that sticks out into the stroma
called CF1.
CF0 appears to be a
channel across the
membrane through which
protons can pass, while
CF1 is the portion of the
complex that actually
synthesizes ATP.

Subunit composition of
chloroplast F1F0 ATP
synthase. This enzyme
consists of a large
multisubunit complex,
CF1 and CF0. CF1
consists of five different
polypepticles, with
stoichiometry of 3, 3,
, .
CF0 contains probably four different polypeptides, with a
stoichiometry of a, b, b', C14. Protons from the lumen are
transported by the rotating c polypepticle and ejected on
the stroma side. (Figure courtesy of W. Frasch in Taiz & Zeiger, 2010)
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





Total energy available for ATP synthesis, proton
motive force (p), is the sum of a proton
chemical potential and a transmembrane
electrical potential and
These two components of the proton motive
force from the outside of the membrane to the
inside are given by
p = Em – 59(pHi – pH0)
(mV)
Em (or ) is the transmenbrane potential
and pH1- pH0 (or pH) is the pH difference
across the membrane.
Total energy available for ATP synthesis, proton
motive force (p), is the sum of a proton chemical
potential and a transmembrane electrical
potential and
These two components of the proton motive force
from the outside of the membrane to the inside
are given by
p = Em – 59(pHi – pH0)
(mV)
Em (or ) is the transmenbrane potential and
pH1- pH0 (or pH) is the pH difference across the
membrane.
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PHOTOPHOSPHORYLATION
Chloroplast stroma – region of high pH
ATP Synthase
(F-type ATPase)
H+
H+
H+
H+
H+
ADP
+ Pi
ATP
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+
4.67 H
H+
H+
H+
H+
H+
H+
H+
H+
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+
Thylakoid Lumen – compartment of low pH
Based on the equation, a transmembrane
pH difference of one unit is equivalent to a
membrane potential of 59 mV.
 Under conditions of steady state electron
transport in chloroplasts, the membrane
electrical potential is quite small, so p is
due almost entirely to pH
 The stoichiometry of protons translocated
to ATP synthesis has been to be
(14/3)H+/ATP , before 3H+/ATP.

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


Herbicides are xenobiotics (foreign chemicals to
plants) that are designed to kill plants but are not
supposed to harm humans and animals.
Ideally, herbicides will only kill weeds and various
noxious plants, leaving crops unharmed.
Major types of herbicides



Hormone mimics: e.g. 2,4-D mimics the plant
hormone 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 ATP
generation
associated with
some
Photosynthetic
Bacteria
• Electron in Photosystem I is excited and transferred to
ferredoxin that shuttles the electron to the cytochrome
complex.
• The electron then travels down the electron chain and
re-enters photosystem I
Cyclic photophosphorylation
In sulfur bacteria, only one photosystem is
used
 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.
2.
3.
4.
5.
How many is H+ transported across membrane
from lumen to stroma for the transfer of each
1e- to NADP+
How many is ATP produced for each formation
of 1 NADPH
How much is the light quanta required for each
formation of ATP
What does it mean by proton motive force
What is the enzyme catalyzing the synthesis of
ATP from ADP
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