The Photosynthetic Steps
Chloroplast synthesize ATP after generation
of pH gradient
 Proton motive force (∆p) from pH gradient
generates ATP - Peter Mitchell
 Two components: a charge gradient and
a chemical gradient
 In mitochondrion, membrane potential plays a
major role.
 In chloroplast, nearly all ∆p is from pH gradient.
 Because, the thylakoid membrane is quite
permeable to Cl- and Mg2+.
 ex Mg2+ (out) and Cl- (in) upon transfer of H+ into
lumen
 This is in contrast to mitochondria inner membrane
 Electrical neutrality is maintained and no membrane
potential is generated.
ATP is generated in stroma Chloroplast ATP synthase: CF1-CF0 complex
Closely resembles F1-F0 complex of mitochondria.
CF0 conducts protons across the thylakoid membr.
CF1 catalyzes ATP formation.
CF0: embedded in the thylakoid membrane
has four polypeptides: I, II, III and IV with a
ratio of 1:2:12:1.
I and II similar to subunit b of mito F0.
III corresponds to subunit c of mito F0.
IV is similar in sequence to subunit a of F0.
CF1: the site of ATP sythase
has composition α3β3γδε.
β contains the catalytic site, 60% identical in
amino acid sequence with those of human ATP
synthase, despite the passage of 1 billion years
since the separation of the plant and animal.
ATP is generated in matrix
CF1-CF0 membrane orientation is reversed.
However, the functional orientation of two enzymes
is identical: protons flow from the lumen through
the enzyme to the stroma or matrix where ATP is
made.
Comparison of Chemiosomosis in
Chloroplast and Mitochondria
FEATURE
CHLOROPLAST
MITOCHONDRIA
membrame involved
thylakoid
cristae
High H+
thylakoid Space
Intermembrane space
Hydrogen/ Electron
donor
H2O
NADH and Succinate
Final electron/hydrogen
acceptor
NADP to form NADPH
O2 to form H20
Nature of reactions
Light dependent reaction
ETS of aerobic
respiration
Where ATP is formed
stroma
matrix
e travels from Fdred to cyto. bf to Pc
Cyclic photo
phosphorylation
Reduced Pc is reoxidized by P700+
Cyclic electron flow through Photosystem I leads to production of ATP instead of NADPH
When the ratio of NADPH to NADP+ is high, NADP+ is unable to accept electrons from
reduced ferredoxin.
Instead, the electron in reduced ferredoxin is transferred to the cytochrome bf complex.
Then, the e travels to plastocyanin, which can be reoxidized by P700+ to complete a cycle.
The net outcome of the cyclic flow of e is pumping protons by cytochrome bf complex
Cyclic Electron Transport
 The resulting proton gradient from cyclic electron transport drives
ATP synthesis .
 The process is called Cyclic photophosphorylation,
 ATP is generated without NADPH formation.
 H20 is not making Oxygen – PS II is not working
 Leads to the synthesis of a higher amount of ATP
Overall stoichiometry of the light reactions
--Photosystem II needs to absorb 4 photons to generate 1 O2.
--This will release 4 protons into the lumen.
--2Q + 2 H2O
Light
O2 + 2QH2. (Photosystem II reaction)
--2 molecules of Q are reduced by water to 2 molecules of QH2.
-- 2 molecules of QH2 are oxidized by Q cycle of cyto bf to rel.
8 protons into lumen.
2+
2Q + 4Pc (Cu+) + 4H+ thylakoid lumen
-- 2QH2 + 4Pc (Cu )
Light
-- 4Pc (Cu+) + 4Fdox
4Pc (Cu2+) +4Fdred (Photosys I react.)
--E from 4 of reduced Pc travels to Ferredoxin by abs of 4 photons
--Four Fdred (1e carrier) generate 2 NADPH (2e carrier).
--Overall:
2H2O + 2NADP+ +10 H+stroma
O2 +2NADPH +12 H+lumen
Absorption of 8 Photons yields 1 O2, 2 NADPH and 3 ATP
12 protons generated in the lumen will flow through ATP synthase.
There are 12 subunit III in CF0
Thus, 12 protons must pass through CF0 to complete one full rotation of CF1.
A single rotation generates 3 ATP
2H2O + 2NADP+ +10 H+stroma
O2 +2NADPH +12 H+lumen
3ADP3- + 3Pi2- +3H+ +12 H+lumen
3ATP4- + 3H2O + 12 H+stroma
_______________________________________________________
2NADP+ +3ADP3- +3Pi2- +H+
O2 +2NADPH + 3ATP4- + H2O
Thus, absorption of 8 Photons yields 1 O2, 2 NADPH and 3 ATP
2.7 photons needed for one ATP
Cyclic Photophosphorylation
 In cyclic electron transport drives cyclic photophosphorylation
 Photosystem I transfers electrons to plastoquinone (PQ).
 4 photons by photosystem I = 8 protons rel into lumen by ETC--- flow of 8 protons
through ATP synthase generates 2 ATPs --- Thus 2 photons/ATP – higher yield of
ATP production.
 8 photons = 12 protons and 3 ATPs = 2.7 photons/ATP- for PSII & I
4e–
Ferredoxin
PQ
Cytochrome
complex
Photon
Pc
ATP
produced via proton
motive force
P700
Photosystem I
Accessory pigments
A light harvesting system only relies on the chlorophyll a
molecules of the special pair would be inefficient.
Two reasons:
1. Chlorophyll a only absorbs light at specific
wavelengths.
A large gap between 450 and 650nm
The gap region is the peak of the solar spectrum
So, failure to absorb the gap region is a huge waste
2. Chlorophyll a density is not very great in the reaction
center.
Thus, many photos that do not fall in the gap pass
through without being absorbed.
3. Thus, accessory pigments, both additional chlorophyll
and other pigment (Carotenoid), are closely associated
with reaction centers.
Photosynthetic Pigments
Chlorophylls:
Transmit mainly green light
Chlorophyll a
Chlorophyll b
Carotenoids:
Transmits mainly orange, yellow or red
Chlorophyll
 Chlorophyll: similar to that
of the heme group of
myoglobin, hemoglobin,
and cytochromes

in center
 tetrapyrrole ring
 A long hydrophobic side
chain (phytol group)
Mg2+
 4 isoprenoid units binding
to hydrophobic region of
thylakoid membrane
 chlorophyll a: methyl
group
 chlorophyll b: aldehyde
group
CHO in chlorophyll b
H2C
Ring
Structure
In Head
Absorbs
Light
CH3 in chlorophyll a
CH
H3C
N
N
CH2CH3
Mg
N
N
CH3
H3C
CH2
CH2
C
COCH3 O
OO
O
CH2
Tail
Carotenoids
CH3
CH3
 Accessory Pigment
 Present in
Light Harvesting Complexes
H3C
CH
-carotene
H3C
CH
CH3
CH3
HO
Different Pigments Absorb Different
Wavelengths of Light
Every pigment has a characteristic Absorption Spectrum
Amount of light absorbed
Chlorophyll a
(Absorbs light between 450 and 500nm)
Chlorophyll b
(responsible for yellow and red colors of plants)
Carotenoids
400
500
600
Wavelength of light (nm)
700
Properties of Light absorption
When a pigment absorbs a wavelength of light, you do not see that color!
Chlorophyll a and b absorb light with wavelengths <500nm (blue) and
>600nm (orange or red). Thus, we do not see these colors.
Chlorophyll a and b do not absorb light with wavelength between
500-600nm, which is green region. Thus, we mainly see green color.
Carotenoids do not absorb light with wavelength longer than 600nm (orange and red).
In summer, Chlorophyll a and b are domainant, absorbing light with wavelength
<500nm (blue) and >600nm (orange or red). Thus, we do not see these colors.
We see green color (500-600nm), instead.
In fall, Chlorophylls are degraded. Carotenoids are dominant. Since they do not absorb
light with wavelength longer than 600nm, plants are orange or red.
Loss of chlorophyll
The carotenoids are responsible for the brilliance of fall, when the
Chlorophyll molecules are degraded.
Resonance Energy Transfer
Energy transfer
1st donor
chromophore
1st acceptor
chromophore
Energy transfer
2nd donor
chromophore
2nd acceptor
chromophore
The absorp of a photo does not always lead to e excitation and transfer.
More commonly, excitation energy is transferred from one mol. to a nearby mol. through
electromagnetic interactions through space. (Resonance Energy Transfer)
RET depends strongly on the distance between energy donor and acceptor.
An increase of 2 results in a decrease in the energy transfer rate by a factor of 26 = 64.
RET must be from a donor in the excited state to an acceptor of equal or lower energy.
Energy transfer from accessory pigments to reaction centers
Carotenoid
Accessory chlorophyll
Light energy absorbed by accessory
chlorophyll or other pigments can be
transferred to reaction centers, where it
drives photoinduced charge separation.
The excited state of special pair of
chlorophyll mol. is lower in energy than
that of single chlorophyll mol., allowing
reaction centers to trap the energy
transferred from other mole.
Protein
Bacterial Light-harvesting complex
The accessory pigments are arranged in many
light-harvesting complexes surround the reaction
center.
Eight polypeptides
Each polypeptide binds
3 chlorophyll mol.
1 carotenoid mol.
Chlorophyll
Reaction center
Carotenoid
Localization of photosynthesis components
 Stacking (appressed) increases surface area
 Unstack (non-appressed) regions make direct contact with stroma
 PS I and ATP synthase locate in unstacked regions
PS I is given direct access to the stroma for the reduction of NADP+.
ATP synthase is provided enough space for its large CF1 globule and access to ADP
 PS II mainly in stack regions.
The tight quarters of these region pose no problem for PS II, which interacts with a
small polar e donar (H2O) and a lipid soluable e carrier (plastoquinone).
 Cytochrome bf in both regions
 Plastocyanin and plastoquinone mobile carriers
 A common internal lumen enables protons released by PS II in stacked region to be
used by ATPase located in unstacked region.
Inhibitors
 Kill weeds by inhibiting PS II or PS I
 Diuron (urea derivative)
 Bind QB site of PS II
 Prevent formation of plastoquinol QH2.
 Paraqat
 Targets PS I
 Accept electrons from PS I
 Becomes a radical
 Reacts with O2 to produce ROS (O2and OH )
 Damages membrane lipids double
bonds
.
.
Problems
Weed killer DCMU. DCMU (dichlorophenyldimethylurea), a herbicide, interferes with
Photophosphorylation and O2 evolution. However, it does not block O2 evolution in the
Presence of an artificial e acceptor such as ferricyanide.
a) Propose a site for the inhibitory action of DCMU.
A: --- DCMU inhibits e transfer in the link between photosystem II and I.
--- O2 can evolve in the presence of DCMU if an artificial e acceptor such as
ferricyanide can accept electrons from Q.
b) Predict the effect of DCMU on a plant’s
ability to perform cyclic photophosphorylation.
A: No effect.
Because it blocks photosystem II,
and cyclic photophosphorylation
use Photosystem I and the
cytochrome bf complex.
DCMU
Problems-continued
Hill reaction. In 1939, Robert Hill discovered that chloroplasts evolve O2 when they are
Illuminated in the presence of an artificial electron acceptor such as ferricyanide
[Fe3+(CN)6]3-. Ferricyanide is reduced to ferrocyanide [Fe2+(CN)6]4- in this process.
No NADPH or reduced plastocyanin is produced. Propose a mechanism for the Hill
reaction .
A: The electrons flow through photosystem II directly to ferricyanide.
No other steps are required.
Close approach. Suppose that energy transfer
between two chlorophyll a molecules separated
by 10A takes place in 10picoseconds. Suppose
that this distance is increased to 20A with all
other factors remaining the same. How long
would energy transfer take?
A: The distance doubles, and so the rate should
decrease by a factor of 64 to 640ps.
Ferricyanide can accept e here
Problems-continued
Photochemical Efficiency of Light at Different Wavelengths.
The rate of photosynthesis, measured by O2 production, is higher when a green plant is
Illuminated with light of wavelength 680nm than with light of 700nm. However,
Illumination by a combination of light at 680nm and 700nm gives a higher rate of
photosynthesis than light of either wavelength alone. Explain.
A: For the maximum photosynthetic rate, photosystem I (which absorbs light of 700nm)
and photosystem II (which absorbs light of 680nm) must be operating simultaneously.
Effect of Monochromatic Light on Electron Flow.
The extent to which an e carrier is oxidized or reduced during photosynthetic e transfer
can sometimes be observed directly with a spectrophotometer. When chloroplasts are
illuminated with 700nm light, cytochrome f, plastocyanin, and plastoquinone are
oxidized. When chloroplasts are illuminated with 680nm light, however, these e carriers
are reduced. Explain.
A: Light of 700nm excites photosystem I but not photosystem II. E flow from P700 to
NADP+, but no e flow from P680 to replace them.
When light of 680nm excites photosystem II, e tend to flow to photosystem I, but the e
carriers between the two photosystems quickly become completely reduced.
Problems-continued
Function of Cyclic Photophosphorylation.
When the [NADPH] / [NADP+] ratio in chloroplasts is high, photophosphorylation is
Predominantly cyclic.
a) Is O2 evolved during cyclic photophosphorylation? Explain.
A: No.
The excited electron from P700 returns to refill the electron “hole” created by
illumination.
Photosystem II is not needed to supply electrons, and no O2 is evolved from water.
b) Can the chloroplast produce NADPH in this way?
A: No NADPH is formed, because the excited electrons returns to P700.
c) What is the main function of cyclic photophosphorylation?
A: Cyclic photophosphorylation occurs when the plant cell is
already amply supplied with reducing power in the form of
NADPH but requires additional ATP for other metabolic needs.
Thus, a plant can adjust the ratio of NADPH and ATP to match
the needs of these products in energy-requiring processes.
Problems-continued
Effect of DNP
Predict the effect of an uncoupler such as dinitrophenol on production of ATP and
NADPH in a chloroplast.
a) ATP
A: An uncoupler dissipates the transmembrane proton gradient by providing a route for
proton translocation other than ATP synthase. Thus, chloroplast ATP production would
decrease.
b) NADPH
A: The uncoupler would not affect NADP+ reduction since light-driven electron transfer
reactions would continue regardless of the state of the proton gradient.
More photon absorption.
Why is it possible for chloroplasts to absorb more than 8 photons per O2 evolved?
A: ---When cyclic electron flow occurs, photoactivation of PSI drives electron transport
independently of the flow of electrons derived from water.
---Thus, the oxidation of water by PSII is not linked to the no of photons consumed
byPSI.
Problems-continued
Radioactive labelling
H218O is added to a suspension of chloroplasts capable of photosynthesis. Where does
the label appear when the suspension is exposed to light? Why
A: ---The label appears as 18O2.
----2 H218O + 2NADP+----2NADPH + 2H+ + 18O2
Reaction order
The three e-transport complexes of the thylakoid membrane can be called plastocyaninferredoxin oxidoreductase, plastoquinone-plastocyanin oxidoreductase, and water-plasto
quinone oxidoreductase.
a) What are the common names of these enzymes?
A: plastocyanin-ferredoxin oxidoreductase: Photosystem I
plastoquinone-plastocyanin oxidoreductase: Cytochrome bf
water-plastoquinone oxidoreductase: Photosystem II
a)
In what order do they act?
Water-plastoquinone oxidoreductase---- plastoquinone-plastocyanin oxidoreductase
----- plastocyanin-ferredoxin oxidoreductase,
Problems-continued
Red tide. The “red tide” is a massive proliferation of certain algal species that causes
Seawater to become visibly red. Describe the spectral characteristics of the dominant
Photosynthetic pigments in the algae.
A: The color of the seawater indicates that the photosynthetic pigments of the algae
absorb colors of visible light other than red.
Carotenoids, rather than Chlorophylls, are the likely pigments.
More DCMU DCMU blocks photosynthetic e transport from PSII to the cytochrome bf.
a). When DCMU is added to isolated chloroplasts, will both O2 evolution and
photophosphorylation cease?
A: YES. ---- When DCMU locks e flow, PSII in the P680* state will not be reoxidized
to the P680+ state, which is required as an acceptor of electrons from water.
---If water is not oxidized by P680+, then no O2 will be produced.
---In the absence of e flow through cytochrome bf, no protons will be translocated.
---Without a proton gradient, ATP will not generated.
b). If an external e acceptor that reoxidizes P680* is added, how will this affect O2
Productiion and photophosphorylatiion?
A: External e acceptor for PSII will permit P680* to be oxidized to P680+, thus restore
O2 evolution. No e will flow through the cytochrome bf, however, so that
no photophosphorylation will occur.
Problems-continued
In vitro ATP synthesis.
a) The luminal pH of chloroplasts suspended in a solution of pH 4.0 reaches pH 4.0
within a few minutes. Explain why there is a burst of ATP synthesis when the pH of the
external solution is quickly raised to pH 8.0 and ADP and Pi are added.
A: -When the external pH rises to 8.0, the stromal pH also rises quickly, but the luminal
pH remains low because the thylakoid membrane is relatively impermeable to protons.
--The pH gradient across the thylakoid membrane drives the production of ATP via
proton translocation through chloroplast ATP synthase.
b) If ample ADP and Pi are present, why does ATP synthesis cease after a few seconds?
A: ----Protons are transferred from the lumen to the stroma by ATP synthase,
driving ATP synthesis.
----The pH gradient across the membrane decreases until it is insufficient to drive
the phosphorylatiion of ADP, and ATP synthesis stops.