The Photosynthetic Steps

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The Photosynthetic Steps
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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.

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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
12121. 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 a3ß3?de. ß
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. 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.
ATP is generated in stroma
ATP is generated in matrix
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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
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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
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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

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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 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. -- -- 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 Hstroma
O2 2NADPH 12 Hlumen
Light
2QH2 4Pc (Cu2) 2Q 4Pc (Cu) 4H
thylakoid lumen
Light
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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
Hstroma O2 2NADPH 12
Hlumen 3ADP3- 3Pi2- 3H 12 Hlumen
3ATP4- 3H2O 12 Hstroma ______________________
_________________________________ 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

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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

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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.
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Photosynthetic Pigments

• Chlorophylls
• Transmit mainly green light
• Chlorophyll a
• Chlorophyll b
• Carotenoids
• Transmits mainly orange, yellow or red

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Chlorophyll
CHO in chlorophyll b
CH3 in chlorophyll a
H2C
CH
Ring Structure In Head Absorbs Light
H3C
CH2CH3
N
N

• Chlorophyll similar to that of the heme group of
  myoglobin, hemoglobin, and cytochromes
• Mg2 in center
• tetrapyrrole ring
• A long hydrophobic side chain (phytol group)
• 4 isoprenoid units binding to hydrophobic region
  of thylakoid membrane
• chlorophyll a methyl group
• chlorophyll b aldehyde group

Mg
N
N
CH3
H3C
CH2
O
COCH3
CH2
O
C
O
O
CH2
Tail
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Carotenoids

• Accessory Pigment
• Present in
• Light Harvesting Complexes

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Different Pigments Absorb Different Wavelengths
of Light

• Every pigment has a characteristic Absorption
  Spectrum

(Absorbs light between 450 and 500nm)
Chlorophyll a
Chlorophyll b
(responsible for yellow and red colors of plants)
Carotenoids
Amount of light absorbed
400
600
500
700
Wavelength of light (nm)
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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 lt500nm (blue) and
gt600nm (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 lt500nm (blue)
and gt600nm (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.
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Loss of chlorophyll
The carotenoids are responsible for the
brilliance of fall, when the Chlorophyll
molecules are degraded.
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Resonance Energy Transfer
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.
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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
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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
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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.

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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 (O2- and OH )
• Damages membrane lipids double bonds

.
.
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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.
• 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 plants
• ability to perform cyclic photophosphorylatio
  n.
• A No effect.
• Because it blocks photosystem II,
• and cyclic photophosphorylation
• use Photosystem I and the
• cytochrome bf complex.

DCMU
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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)63-. Ferricyanide is reduced to
ferrocyanide Fe2(CN)64- 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
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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.
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Problems-continued

• Function of Cyclic Photophosphorylation.
• When the NADPH / NADP ratio in chloroplasts
  is high, photophosphorylation is
• Predominantly cyclic.
• 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.
• 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.

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Problems-continued

• Effect of DNP
• Predict the effect of an uncoupler such as
  dinitrophenol on production of ATP and
• NADPH in a chloroplast.
• 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

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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 plastocyanin-
• ferredoxin oxidoreductase, plastoquinone-plastocya
  nin oxidoreductase, and water-plasto
• quinone oxidoreductase.
• What are the common names of these enzymes?
• A plastocyanin-ferredoxin oxidoreductase
  Photosystem I
• plastoquinone-plastocyanin oxidoreductase
  Cytochrome bf
• water-plastoquinone oxidoreductase
  Photosystem II
• In what order do they act?

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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 photophosphorylatio
n will occur.
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Problems-continued

• In vitro ATP synthesis.
• 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.