Photosynthesis

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Lectured by Dr. Qin Yongmei (???)Nov. 28, 2007
Photosynthetic Carbohydrate synthesis (Calvin
Cycle)
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The relationship between the light dependent
reactions and the light independent reactions
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Assimilation of CO2 into biomass in plants
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Experiment done by M. Calvin during 1946-1953
Due to the use of isotopes were M. Calvin able to
reveal completely the reactions taking place
during the incorporation of carbon dioxide 14CO2
into carbohydrates. Based on Sensitive methods
two-dimensional paper chromatography autoradiog
raphy Culture the single-celled green alga
Clorella pyrenoidosa Supplied light and an even
stream of air containing 14CO2 .
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At a given time (from t0) was 14CO2 added to
the stream of air for a short time. After 5
seconds, etc, were the experiment stopped and
the newly produced 14Clabeled intermediates
were separated and identified.
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In the absence of CO2,the 3PG concentration
rapidly decreases. Conversely,the RuBP
concentration transiently increases as it is
synthesized from the residual pool of Calvin
cycle intermediates, but in the absence of CO2,
cannot be used for their regeneration.
CO2 withdraw
RuBP
3PG
CO2
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Results

• All the 14C was found to be in the carboxyl group
  of 3-phosphoglycerate (a phenomenon not found
  using animal cells).
• Carboxylation of a C2 compound was immediately
  hypothesized, but never approved.
• Ribulose-1,5-bisphosphate (RuBP) was revealed to
  be the CO2 acceptor by comparing the steady-state
  concentrations of various compounds by CO2
  withdraw.

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In 1950s, Calvin cycle was elucidated by Melvin
Calvin, Andrew Benson and James A. Bassham. CO2
assimilation (CO2 fixation) Green plants contain
their chloroplasts unique enzymatic machinery
that catalyzes the conversion of CO2 to simple
(reduced) organic compounds.
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Three major stages for CO2 assimilation via the
Calvin cycle

• Fixation stage CO2 is condensed to a five-carbon
  
• acceptor (ribulose-1,5-bisphosphate) to form
• 3-phosphoglycerate
• Reduction stage 3-phosphoglycerate is reduced
• to glyceraldehyde-3-phosphate
• Regeneration stage ribulose-1,5-bisphosphate
• is regenerated using glyceraldehyde-3-phosphate.

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• Use NADPH and ATP generated by the light
  reactions to fix CO2 into
• carbohydrates
• - In stroma of the chloroplast

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Stage 1 Fixation of CO2
CO2 is first incorporated into
ribulose-1,5-bisphosphate, catalyzed by
ribulose-1,5-bisphosphate carboxylase (or RuBP
carboxylase/oxygenase, rubisco). The end
product is 3-phosphoglycerate.
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RuBP carboxylase
RuBP
-OO
????
3-phosphoglycerate
3-phosphoglycerate
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RuBPs carboxylase activity
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• RuBP Carboxylase reaction mechanism
• Extraction of a proton from C3 of ribulose-1,5-
• bisphosphate promotes formation of an endiolate
• intermediate.
• Nucleophilic attack on CO2 is proposed to yield a
  
• ?-keto intermediate, that reacts with H2O and
• cleaves to form 2 x 3-phosphoglycerate via
• a carbanion.

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Rubisco has complicated structure, (complex of
L8S8), low efficiency and large quantity.
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Large subunits within Rubisco are arranged as
antiparallel dimers. Each active site is at an
interface between monomers with in an L2 dimers.
Large subunits colored blue and cyan, and small
subunits colored red.
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Properties of RuBP Carboxylase Complex of L8S8
(plants) 8 large subunits (nuclear-encoded,477
aa, catalytic regulatory sites) 8 small
subunits (chloroplast-encoded, 123 aa, unknown
function) Some bacteria contain only the large
subunit, L2(homodimer). Possessing both
carboxylase and oxygenase activities, sharing the
same active site, where O2 competes with CO2
The enzyme has a low efficiency (catalytic
rate only 3s-1), thus with large amounts of
enzyme needed ( 250 mg/ml in the chloroplast
stroma, most abundant enzyme in the biosphere)
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Stage 2 Conversion of 3-phosphoglycerate to
glyceraldehyde 3-phosphate (Reduction)
The reaction catalyzed by the G3Pdehydrogenase is
analogous to G3P dehydrogenase of glycolysis
(reverse) except the dehydrogenase in
chloroplasts uses NADPH as electron donor, while
the cytosolic glycolysis enzyme uses NAD as
electron acceptor.
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The chloroplast stroma lacks phosphoglycerate
mutase, thus no glycolysis will occur there.
Fate I
Fate II
ADP-Glu
Glu-1-P
Three alternative fates of glyceraldehyde-3-phosp
hate
Fate III
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Alternative fates of glyceraldehyde-3-phosphate

• Can be converted to other carbohydrate
• metabolites (F1P and G1P), energy stores (
  sucrose
• or starch), or cell wall constituents
  (cellulose).
• Can be transported out of the chloroplast (using
  
• the Pi-triose phosphate antiporter) and then
  used
• for glycolysis in the cytosol to generate ATP

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Stage 3 Regeneration of ribulose
1,5-Bisphosphate from triose phosphates
A portion of the G3P is converted back to
Ribulose1,5BP, via reactions catalyzed by triose
phosphate isomerase, aldolase, fructose
bisphosphatase, sedoheptulose bisphosphatase, tran
sketolase, epimerase, ribose phosphate isomerase
and phosphoribulokinase. Many of these enzymes
are equivalent to enzymes of the glycolysis,
gluconeogenesis and pentose phosphate pathways,
but are separate gene products resident within
the chloroplast stroma.
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Essentially the reversal of the pentose
phosphate pathway.
transaldolase
C3 C3 C6
TPP
transketolase
C3 C6 C4 C5
transaldolase
C3 C4 C7
TPP
transketolase
C3 C7 C5 C5
3 ATP molecules are consumed.
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C3 C3 ? C6 C3 C6 ?
C5 C4 C3 C4 ? C7 C3
C7 ? C5 C5 overall
5C3 ? 3C5

• One G3P (3C) exists in the pathway as product.
• Five of 3C molecules (total 15C) are recycled
• back into three 5C molecules of Ribulose1,5BP.

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Stoichiometry of the Calvin Cycle
C1
6C1 6C5 12C3 12C3 1C6
6C5 Net 6C1 1C6
C5
C3
C5
C3
C3
C3
Two NADPH and three ATP are consumed for fixing
each CO2
C3
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Summary of Calvin Cycle 3CO2 9ATP
6NADPH ?
Glyceraldehyde-3-P 9 ADP 8Pi 6NADP
Energy cost for converting three molecules of CO2
to one molecule of glyceraldehyde-3-phosphate
requires 9ATP and 6NADPH. The chemical change
that occurs is technically a "reduction
reaction" so this storage of energy is called
carbon reduction. Because this pathway makes
both the product (carbohydrates and the initial
substrates, it is called autocatalytic. That
means it catalyzes, or encourages, itself, it is
a real self-starter.
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Pi-triose phosphate antiport system of the inner
chloroplast membrane
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ATP NADPH used for biosynthesis
ATP NADPH produced in light reaction
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Regulation of Carbohydrate Metabolism in Plants
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Rubisco is both positively (via convalent
modification by CO2) and negatively (via a
transition state analog) regulated
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Activated by the formation of carbamate
carboxylate (binding Mg2) At high CO2 levels,
this occurs nonenzymatically At low CO2 levels,
this reaction is catalyzed by Rubisco activase,
which is ATP-dependent (light- dependent
activation).
positive regulation
Rubisco Activase
Carbamate carboxylate activation
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Rubisco activase overcomes the inhibition by
promoting ATP-dependent release of the ribulose
1,5-bisphosphate, exposing the Lys 201 amino
group to nonenzymatic carbamoylation by CO2.
Rubisco Activase
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• Rubisco Activase
• an ATPase
• causing a conformational
• change in Rubisco from a
• close to an open form
• allowing release of tightly
• bound substrate (RuBP
• leaving the active site),
• promoting carbamate
• formation.

Active Site Rubisco
Negative regulation
(nocturnal inhibitor)
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Certain photosynthetic enzymes are indirectly
activated by light
The high pH and high Mg2 in chloroplast
stroma, resulting from illumination, activate
enzymes like rubisco and fructose-1,6-bisphosphat
ase
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Light-dependent activation of dark reaction
enzymes
High levels of ferredoxin accumulate when NADP
stores are low, and this occurs during light
exposure.
Sedoheptulose-1,7-bisphosphatase,
glyceradehyde-3-phosphate dehydrogenase, and
ribulose-5-phosphate kinase are activated by
reduced ferredoxin.
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1. High pH, CO2, and Mg 2 increase activity
of rubisco. 2. Three enzymes (sedoheptulose-1,7-bi
sphosphatase, glyceradehyde-3-phosphate
dehydrogenase, and ribulose-5-phosphate
kinase) are activated by reduction of
disulfides to sulfhydryls, which is dependent
upon a disulfide exchange reaction promoted by
the protein thioredoxin. 3. In the dark, plants
metabolize some of the stored energy. In general,
the pathways of catabolism glycolysis, the citric
acid cycle, and the pentose phosphate pathway -
are inhibited in the presence of sunlight and
become more active in the dark. (The key
light-inhibited enzymes are phosphofructokinase,
in glycolysis and glucose-6-phosphate
dehydrogenase, in pentose phosphate pathway).

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Regulation of sucrose synthesis F2,6BP

• In the light (phtosynthesis is active),
  photosynthetic 3-carbon products inhibit FPK-2,
  F2,6BP?, thus increases the activity of FBPase-2,
  F6P? and synthesis of sucrose is favored, ?
  gluconeogenesis
• The high level of Pi generated in the dark
  stimulates FPK-2, ? F2,6BP, thus increases the
  activity of PFK-1,
• ? glycolysis.

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Biosynthesis of starch and sucrose
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ADP-glucose is the substrate for starch
synthesis in plants
Starchn G1P ATP ? Starchn1 ADP 2Pi
G
-50 kJ/mol
Starch is an ?-glucan (D-glucose in ?1?4
linkage). It can be synthesized either in
chloroplasts or in amyloplasts (non- photosyntheti
c tissues).

• Catalyzed by starch synthase
• Essentially similar to the mechanism of glycogen
  synthesis in animals
• Two pathways
• Amyloplast pathway (glucose 1-phosphate)
• ADP-glucose is the preferred glucose
  precursor
• Chloroplast pathway (fructose-6-phosphate)

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rate limiting step of starch biosynthesis
ADP-glucose pyrophosphorylase
ADP-glucose
starch synthase (chloroplast stroma)
Starch synthesis
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Starch synthesis proceeds by a two-site
insertion mechanism
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UDP-Glucose is the substrate for sucrose
synthesis in plants

• From UDP-glucose and fructose-6-phosphate
• Catalyzed by sucrose-6-phosphate synthase and
  sucrose-6-phosphate phosphatase
• Sucrose (a nonreducing sugar) is a readily
  transportable and mobilizable sugar that is
  stored in many plants.

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Regulation of starch synthesis ADP-glucose
pyrophosphorylase
When sucrose synthesis slows, 3-phosphoglycerate
formed by CO2 fixation accumulates, activating
this enzyme and stimulating the synthesis of
starch.
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Regulation of sucrose synthesis sucrose
6-phosphate synthase
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Photorepiration results from rubiscos oxygenase
activity
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Under conditions of high O2 and low CO2, rubisco
has oxygenase activity Ribulose-1,5-bisphosphat
e is lost from the Calvin cycle, resulting in
no carbon fixation. Phosphoglycolate is produced.
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Glycolate pathway (salvage pathway)
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Photorespiration

• It is converted to 3-phosphoglycerate
• via a long pathway involving three cellular
• compartments
• The whole process is called photorespiration
• because O2 is consumed and CO2 is released ,
• and ATP is expended without apparent benefit
• The oxygenase activity increases more rapidly
• with high temperature
• So, photorespiration is wasteful for plants.

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C4 plants (maize, sugarcane and sorghum) have
evolved a mechanism to minimize photorespiration
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The C4 Cycle (HATCH-SLACK cycle)
Mesophyll Cell
C4 pathway
PEP carboxylase
pyruvate phosphate dikinase
C3 pathway
malic enzyme
The processes of photosynthesis in these two
types of cells are spatially separated.
Bundle sheath cell
To the Calvin cycle
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• Photosynthesis of C4 plants
• CO2 is bound to PEP in mesophyll cells, and
• the product is oxaloacetate (C4)
• Oxaloacetate is then converted to malate,
• which moves to the bundle-sheath cells
• via the plasmodesmata linkage
• In bundle sheath cell, CO2 split off the
• malate and fed into the Calvin cycle
• (CO2 ?, O2?)
• Pyruvate is transported back into mesophyll
• cells (active transport).

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Why do C4 plants outgrow C3 plants in the summer?
C4 plants consume five ATP to fix one CO2, (C3
plants consume only three) When temperature
increases to about 28oC to 30oC, the gain in
efficiency from the elimination of
photorespiration in C4 plants more than
compensates for this higher energy cost.
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• Summary of Plant Photosynthesis
• Photosynthesis in plants takes place in
  chloroplast
• 2. In Calvin cycle, ATP and NADPH are used to fix
  and
• reduce CO2 to form carbohydrate. Enzymes and
• intermediates involved are located in the
  chloroplast
• stroma
• 3. Properties of Rubisco structure, reaction
  mechanism
• and its regulation
• 4. Pi-triose phosphate antiport system in the
  inner chloroplast
• membrane transport ATP and NADH/NADPH from
• chloroplast to cytosol.
• 5. Regulation of carbohydrate metabolism in
  plants
• 6. Photorespiration and the C4 cycle.
• 7. UDP-Glc is used for glycogen and sucrose
  syntheses
• ADP-Glc is used for starch synthesis.