Dark reactions of
Photosynthesis
Andy Howard
Introductory Biochemistry
10 April 2008
10 April 2008
Dark reactions matter!
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Not all of these reactions really take
place in the dark; but some do, and
even the ones that take place in
daylight are not directly dependent
on photon absorption
Dark Reactions
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10 April 2008
What we’ll discuss
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Dark reactions of
Photosynthesis
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RuBisCO
Calvin Cycle
overview
C5 to C3 to C6
Regenerating C5’s
Energy bookkeeping
Dark Reactions
Sucrose & Starch
Other C-fixation
paths
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10 April 2008
Dark reactions
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Series of ordinary chemical reactions
Powered by reducing power in NADPH
Anabolic
Some common features with pentose
phosphate pathway
Dark Reactions
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10 April 2008
Dark reactions: overview
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RuBisCO fixes atmospheric CO2 into
carbon skeletons
Reductions of 3-phosphoglycerate
build up carbohydrate
Pathway is cyclic in that RuBP is
regenerated for additional reactions
Dark Reactions
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10 April 2008
RuBisCO reaction
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Condensation of ribulose
1,5-bisphosphate (RuBP)
with CO2 to produce two
molecules of 3phosphoglycerate
Enzyme is ribulose
1,5-bisphosphate
carboxylase / oxygenase
(RuBisCO)
RuBP
3-phosphoglycerate
Dark Reactions
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The unwanted (?) sidereaction of RuBisCO
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Secondary reaction is
ribulose 1,5-bisphosphate
+ O2 
3-phosphoglycerate +
2-phosphoglycolate
Uses up oxygen rather than
CO2
No net carbon incorporation
into organic molecules
Dark Reactions
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2-phosphoglycolate
10 April 2008
RuBisCO structure
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L8S8 stoichiometry
in higher plants:
Mol.Wt. L=55kDa;
Mol. Wt. S=12 kDa
TIM barrels in both
All (?) catalytic activity in L
(large) subunit
L coded for by chloroplast gene
S by nuclear genome
Does S play a controlling role?
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Dark Reactions
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PDB 1WDD
Octamer of
L8S8 units
L2S2 shown
from rice
(cf. fig. 15.21)
10 April 2008
RuBisCO regulation
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Plant growth closely associated with
carboxylation / oxygenation ratio:
Carboxylation high means fast growth
Easy way to alter that: grow plants in
high CO2
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Difficult to do that without animal toxicity!
Expensive to put your cornfield in a plastic
bubble (but not impossible)
Dark Reactions
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Could you win genetically?
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Attempts to engineer proteins that
don’t do oxygenation
(or even that have improved CO2/O2
activity ratios) have failed
There are some plants whose
RuBisCO has a better SC/O than that
of others
Maybe O2 and CO2 bind in precisely
the same way!
Dark Reactions
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Subsequent dark reactions, I
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Pair of 3-phosphoglycerate
molecules enter reductive pathway
toward bigger sugars
Note that this reaction appears in
glycolysis (in reverse) and in
gluconeogenesis
Phosphoglycerate kinase
activation:
3-P-glycerate + ATP 
1,3-bisP-glycerate + ADP
Dark Reactions
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PDB 1PHP
43 kDa monomer
Bacillus
stearothermophilus
(unfortunately!)
10 April 2008
Subsequent dark reactions,
II (cf. fig. 15.18)
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Three glycolysis / gluconeogenesis rxns:
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GAPDH reaction:
1,3-bisP-glycerate + NADPH + H+
glyceraldehyde-3-phosphate + NADP + Pi
TIM required to convert G3P to DHAP
Aldolase makes fructose 1,6-bisphosphate
Some RuBP is recycled back in to provide
input to subsequent condensations with CO2
Dark Reactions
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10 April 2008
RuBisCO, revisited
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2-phosphoglycolate is the product
of the oxygenation reaction
2-P-glycolate is decarboxylated:
2 2-P-glycolate  CO2 + 3-P-glycerate +Pi
The 3-P-glycerate can re-enter the Calvin
cycle, but at the cost of some carbon
This lossy pathway is known as
photorespiration
Dark Reactions
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10 April 2008
Be careful how you describe
transketolase and transaldolase
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A few days ago we said (in lecture) that
the transketolase reaction was
Kn + Am  Kn-2 + Am+2
That’s wrong: we do donate two carbons
from the ketose to the aldose, but they
swap carbonyl positions when you do, so
the reaction is really
Kn + Am  An-2 + Km+2
The notes have already been corrected!
Dark Reactions
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Calvin cycle:
first reaction
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Begins with ATP-dependent
phosphorylation of 3phosphoglycerate to make
1,3-bisphosphoglycerate via
phophosphoglycerate kinase
Same reaction found in
gluconeogenesis; reverse of
glycolytic step
Enzyme is 3-layer
sandwich
Dark Reactions
PDB 1V6S
86 kDa dimer
Thermus thermophilus
Monomer shown
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2nd Calvin-cycle
reaction: GAPDH
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NADPH-dependent
reduction of 1,3bisphosphoglycerate to
glyceraldehyde 3-phosphate
As in gluconeogenesis,
reverse of glycolytic reaction
GAPDH: typical NAD(P)
dependent oxidoreductase
Dark Reactions
PDB 1RM4
297 kDa octamer
dimer + monomer shown
spinach
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The fates of glyceraldehyde-3phosphate
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The pathway divides three ways at this
metabolite
One equivalent toward fructose 1,6bisphosphate and gluconeogenesis
Two head toward pentose phosphate
pathway, where a second bifurcation
happens
Dark Reactions
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C3 to C6
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TIM converts one molecule of
glyceraldehyde 3-phosphate to
dihydroxyacetone phosphate
Glyc-3-P and DHAP condense to form
fructose 1,6-bisphosphate in standard
aldolase reaction
Fructose 1,6-bisphosphatase removes the
1-phosphate to make fructose 6phosphate
All of this happens in gluconeogenesis
Dark Reactions
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Transketolase
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As we saw in the PPP,
fructose-6-P can react
with glyceraldehyde-3-P
in a transketolase
reaction to form xylulose5-phosphate and
erythrose-4-phosphate
K6 + A3  A4 + K5
Typical TPP binding
structure
Dark Reactions
PDB 1ITZ
297 kDa octamer
dimer+monomer shown
maize
p. 19 of 47
10 April 2008
Fates of DHAP
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Can participate in F-6-P production
Can condense with erythrose-4-P in an
aldolase reaction to form sedoheptulose
1,7-bisphosphate (K3 + A4  K7)
This can be dephosphorylated at the 1position to form sedoheptulose 7-P via
sedoheptulose 1,7-bisphosphatase
Dark Reactions
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10 April 2008
The final Glyc3-P
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It can condense with sedoheptulose 7phosphate in another transketolase
reaction to form xylulose-5-phosphate
and ribose-5-phosphate:
K7 + A3  A5 + K5 (fig. 15.19)
The ribose-5-phosphate is an endpoint
but it can also be isomerized to ribulose5-phosphate
Xylulose-5-phosphate can be epimerized
to form ribulose-5-phosphate too
Dark Reactions
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10 April 2008
Activation of
ribulose-5-phosphate
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Phosphoribulokinase uses
ATP as a phosphate
source to convert
ribulose-5-phosphate to
RuBP
Enzyme is similar to
PDB 1A7J
adenylate kinase
Dark Reactions
32 kDa monomer
Rhodobacter sphaeroides
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What is unique here?
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Not much
Last reaction is specific to Calvin cycle
Others are found in gluconeogenesis or
pentose phosphate pathway or both
In this direction these reactions require
the NADPH and ATP derived from the
light reactions of photosynthesis
Dark Reactions
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10 April 2008
Bookkeeping for dark
reactions
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Numbers given on fig.15.19 presuppose 3
input RuBP molecules per run of the cycle
This makes it easy to divide up the
Glyceraldehyde 3-P later
Net reaction is:
3 CO2 + 9ATP + 6 NADPH + 5 H2O 
glyceraldehyde 3-P + 9ADP +
8 Pi + 6 NADP+
Dark Reactions
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10 April 2008
Cost of making Acetyl CoA
• We get back 2 NADH, 2 ATP when we
convert glyceraldehyde 3-P to acetyl CoA
• Therefore acetyl CoA costs 9-2 = 7 ATP
and 6-2=4 NAD(P)H
• At 2.5 ATP per NAD, that total is 7 + 2.5 * 4
= 17 ATP required per acetyl CoA
• When we oxidize acetyl CoA we get 10
ATP (see TCA-cycle lecture),
so we’re 10/17 = 59% efficient
Dark Reactions
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10 April 2008
Carbohydrate storage in plants
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Glyc3P is converted to glucose-6-P or
glucose by gluconeogenesis
Glycogen is storage polysaccharide in
bacteria, algae, some plants
Other plants make starch (amylose or
amylopectin) from glucose-6-P
Pathway begins with conversion of
glucose-6-P to glucose-1-P, catalyzed by
phosphoglucomutase
Dark Reactions
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10 April 2008
Starch synthesis
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Glucose 1-P activated with ATP, not UDP
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-D-glucose 1-P + ATP  ADP-glucose + PPi
Reaction driven to the right by hydrolysis of PPi
ADP glucose is added to growing starch
molecule with release of ADP:
ADP-glucose + (Starch)n 
ADP + (Starch)n+1
Branching in amylopectin accomplished as
in glycogen
(Yao et al (2004) Plant Physiol. 136:3515)
Dark Reactions
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10 April 2008
Diurnal variations
in starch
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Starch synthesis in daylight:
ATP is readily available
Starch degradation at night
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Starch phosphorylase cleaves
starch to produce glucose-1phosphate;
glucose-1-P to triose
phosphates by glycolysis
Enzyme is similar to glycogen
phosphorylase
PLP-dependent
Dark Reactions
p. 28 of 47
PDB 2C4M
350 kDa tetramer
Corynebacterium
callunae
10 April 2008
Alternative path for
night-time starch
degradation
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Starch to dextrins via amylase
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Dextrins are oligosaccharides
beginning with a -1,6 link
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Dextrins eventually degraded
to glucose
Glucose is phosphorylated by
hexokinase
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PDB 1HT6
45 kDa monomer
barley
Enzyme:
 sheet domain + TIM barrel
Dark Reactions
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10 April 2008
Sucrose: mobile
carbohydrate
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Synthesized in chloroplastcontaining cells; exported to
vascular system so other
plant parts can use it
Two fructose 6-phosphate
molecules are starting points
(fig 15.25)
One is converted to Glucose-1-P (via glucose 6-P) and thence
to UDP-glucose
That condenses with the other Fructose-6-P with the help of
sucrose 6-P synthase to form sucrose 6-P
That gets dephosphorylated to make sucrose
Dark Reactions
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10 April 2008
Enzymes in sucrose synthesis
Enzyme
 Glucose 6-phosphate
isomerase
 Phosphoglucomutase
 UDP-glucose
pyrophosphorylase
 Sucrose 6-phosphate
synthase
 Sucrose phosphate
phosphatase
Dark Reactions
Reactant Product
F-6-P
G-6-P
G-6-P
G-1-P
G-1-P + UDP-glucose
UTP
+ PPi
F-6-P +
Sucrose-6-P
UDP-glucose
Suc-6-P Sucrose
+ H2O
+ Pi
p. 31 of 47
10 April 2008
UDP-glucose
pyrophosphorylase
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Catalyzes
glucose-1-P + UTP 
UDP-glucose + PPi
Dark Reactions
PDB 2ICY
103 kDa dimer
Arabidopsis
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10 April 2008
Sucrose 6phosphate
phosphatase
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Contains “tongs” that
release free sucrose
into the cell:
Fieulaine et al, Plant
Cell 17: 2049-2058
Rossmann fold + 
complex
Dark Reactions
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PDB 1TJ5
27 kDa monomer
Synechocystis
10 April 2008
How sucrose is used
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Sucrose taken up by non-photosynthetic
cells
Broken down to glucose and fructose
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supplies energy by glycolysis and TCA
Glucose and fructose can be built back up to
starch in storage tissues:
Amyloplasts (modified chloroplasts with no
photosynthetic mechanisms) in root cells do this
Dark Reactions
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Other carbon-fixation pathways
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Purpose: increase local [CO2] / [O2] to
improve performance of RuBisCO
C4 pathway (high temp, lots of light)
Crassulacean acid metabolism (high
temp, limited water)
Dark Reactions
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C4 pathways
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Common in maize, sorghum, sugarcane,
weeds
Needed at high temp because
rate(oxidation)/rate(carboxylation) increases
with temperature
External CO2 acceptor is PEP via PEP
carboxylase; product is oxaloacetate
This occurs in mesophylls; bundle sheath cells
continue to do ordinary RuBisCO-based
carbon fixation using CO2 released from
metabolites
Dark Reactions
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10 April 2008
PEP Carboxylase
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PEP + HCO3- 
oxaloacetate + Pi
Occurs outside C4
metabolism too
One TIM barrel per
monomer
Dark Reactions
PDB 1JQO
427 kDa tetramer
maize
p. 37 of 47
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C4 interplay
Diagram courtesy
MIT: ESG Biology program
Dark Reactions
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Crassulacean acid
metabolism
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Leaf cells open to CO2 uptake lose a
lot of water during the day
(high evaporation rate)
Solution: assimilate carbon at night
Reactions are as in C4 pathway;
cellular specialization and enzyme
regulation are different
Dark Reactions
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Stomata and vacuoles
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Stomata (spaces between cells that
can open to allow access for
respiration) near mesophylls open only
at night, enabling PEP carboxylation to
oxalacetate and then reduction to
malate
Malate stored in central vacuole, then
released during the day when the
stomata are closed
Dark Reactions
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CAM: day and night
University of Newcastle, Plant Physiology program
Dark Reactions
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10 April 2008
iClicker quiz question 1
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Oxidation of a 2ncarbon fatty acid yields
(n-1) QH2,
(n-1) NADH, and n
acetyl CoA. Initiating
the process costs 2
ATPs. Assume we can
get 10 ATP per acetyl
CoA. How much ATP
can we get from
oxidizing palmitate?
Dark Reactions
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(a) 104 ATP
(b) 106 ATP
(c ) 108 ATP
(d) 112 ATP
(e) Undeterminable
given the data
supplied
p. 42 of 47
10 April 2008
Answer to 1st question
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Palmitate is a C16 carboxylic acid.
Therefore in the conditions of the
problem, 2n = 16, n = 8, n-1 = 7.
Thus we get 7 QH2, 7 NADH,
8 acetyl CoA produced by its oxidation
Thus we get 7*2.5 + 7 * 1.5 + 8 * 10 =
17.5 + 10.5 + 80 = 108 ATP produced
Starting the process costs 2 ATP, so the
net result is 106 ATP gained
Dark Reactions
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10 April 2008
iClicker quiz question 2
Why would you not expect to find crassulacean
acid metabolism in tropical plants?
 (a) Tropical plants do not photosynthesize.
 (b) Tropical plants cannot develop the stomata
that close off the chloroplast-containing cavities
 (c) Water conservation is less critical in areas of
high rainfall
 (d) The waxy coating required to close off the
leaves’ access to O2 would dissolve in the high
humidity and high temperature of the tropics
 (e) None of the above
Dark Reactions
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Answer: (c)
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The primary significance of CAM is
conservation of water in regions of low
humidity, where evaporation rates are
high and water is scarce. Neither of these
conditions pertains in the tropics.
Dark Reactions
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Control of CAM
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PEP carboxylase inhibited by
malate and low pH
That prevents activity during
daylight, which would lead to futile
cycling and competition for CO2
between PEP carboxylase and
RuBisCO
Dark Reactions
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Compartmentation in bacteria
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In photosynthetic bacteria,
RuBisCO is concentrated in protein
microcompartment called a
carboxysome
Active carbonic anhydrase there:
catalyzes HCO3-  OH- + CO2
That tends to keep the CO2 / O2
ratio high
Dark Reactions
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10 April 2008