Photosynthesis 2

Calvin Cycle
C4 and CAM Pathways

Lecture 32

Key Concepts

• Three stages of the Calvin Cycle

– Stage 1: Fixation of CO2 to form 3-phosphoglycerate
– Stage 2: Reduction of 3-phosphoglycerate to form hexose
sugars
– Stage 3: Regeneration of ribulose-1,5-bisphosphate

• Regulation of the Calvin Cycle

• The C4 and CAM pathways reduce photorespiration in

hot climates
 Three stages of the Calvin Cycle:

Fixation, Reduction and Regeneration

Plants store light energy in the form of carbohydrate, primarily starch

and sucrose. The carbon and oxygen for this process comes from CO2,
and the energy for carbon fixation is derived from the ATP and NADPH
made during photosynthesis.

The conversion of CO2 to carbohydrate is called the Calvin Cycle and is

named after Melvin Calvin who discovered it. The Calvin Cycle requires
the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase
commonly called rubisco. The Calvin cycle generates the triose
phosphates 3-phosphoglycerate (3-PGA), glyceraldehyde-3P (GAP)
and dihydroxyacetone phosphate (DHAP), all of which are used to
synthesize the hexose phosphates fructose-1,6-bisphosphate and
fructose 6-phosphate.

Hexose phosphates produced by the Calvin Cycle are

converted to:

1) sucrose for transport to other plant tissues

2) starch for energy stores within the cell
3) cellulose for cell wall synthesis
4) pentose phosphates for metabolic intermediates
 Basic scheme of the Calvin Cycle
Three turns of the
cycle results in the
fixation of three
molecules of CO2 .
Stage 1 is catalyzed
by rubisco enzyme
which combines three
molecules of
ribulose-1,5-
bisphosphate
(RUBP), a C5
compound, with three
molecules of CO2 to
form six molecules of
3-PGA, a C3
compound. Five 3-
phosphoglycerate
molecules are used
to reform RUBP for
another round and
one is used for sugar.

Basic scheme of the Calvin Cycle

The Calvin Cycle is sometimes called the Dark Reactions, but do not be
fooled by this name - the Calvin Cycle is the most active during the daylight
hours when ATP and NADPH are plentiful.

The net reaction of three turns of the Calvin cycle can be written as:

3 CO2 + 3 RuBP + 6 NADPH + 9 ATP + 6 H2O →

1 GAP + 3 RuBP + 6 NADP+ + 9 ADP + 9 Pi

If we just look at the fate of the carbons coming from CO2 (C1) in this
reaction, we see that one net C3 compound (GAP) is formed and three C5
molecules (RuBP) are regenerated:

3 C1 + 3 C5 → 1 C3 + 3 C5

Why was the Calvin Cycle originally called the Dark Reactions?
 Stage 1: Fixation of CO2 to form 3-phosphoglycerate

To identify the metabolic intermediates in this process, Calvin and his

colleagues used radioactive labeling with 14CO2 to follow carbon fixation in
photosynthetic algae cells grown in culture.

They found that within a few seconds of

adding 14CO2 to the culture, the cells
accumulated 14C-labeled 3-PGA, suggesting
that this was the first product of the
carboxylation reaction. 3-PGA is converted to
GAP by two enzymes in the Calvin Cycle.

Within a minute of adding 14CO2 to the culture,

they found numerous compounds were labeled
with 14C, many of which were later identified as
Calvin Cycle intermediates.

Rubisco reaction can be broken down into four basic steps

1) formation of an enediolate intermediate of ribulose-1,5-bisphosphate

2) carboxylation by nucleophilic attack on the CO2

3) hydration of 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate

4) aldol cleavage to form two molecules of 3-PGA

(3-PGA)

The rubisco reaction is very exergonic (ΔG°' = -35.1 kJ/mol), with the aldol
cleavage step being a major contributor to the favorable change in free energy
Rubisco is a multisubunit enzyme consisting of eight identical catalytic
subunits at the core surrounded by eight smaller subunits that function to
stabilize the complex and presumably enhance enzyme activity.

Considering that rubisco plays a central role in all photosynthetic autotrophic

organisms on earth, of which ~85% are photosynthetic plants and microorganisms
that inhabit the oceans, rubisco is the most abundant protein on planet earth.

Stage 2: Reduction of 3-phosphoglycerate to form hexose sugars

3-phosphoglycerate (3-
PGA) is converted to 1,3-
bipsphosphoglycerate
which is reduced to
glyceraldehyde-3-
phosphate (GAP) These
two reactions require the
ATP and NADPH made
during the light reactions.
Some of the GAP is
isomerized to
dihydroxyacetone
phosphate (DHAP).

Remember that for every 3

CO2 that are fixed by
carboxylation of 3 RuBP
molecules, six moles of
3PGA are generated by
aldol cleavage.

Therefore, 6 ATP and 6 NADPH are required

for every 3 CO2 that are converted to one net
3PGA. An additional 3 ATP are used in stage
3 to regenerate these 3 RuBP molecules.
NADPH and ATP are produced by the Light Reactions of Photosynthesis

Stage 3: Regeneration of ribulose-1,5-bisphosphate

In this final stage of the Calvin cycle, a series of enzyme reactions

convert five C3 molecules (GAP or DHAP) into three C5 molecules
(RuBP) to replenish supplies of this CO2 acceptor molecule which is
required in the rubisco reaction.

This requires an additional 3 ATP.

Two of the primary enzymes in this carbon shuffle are transketolase

and transaldolase which are involved in interconverting C3, C4, C6 and
C7 molecules:
 “Carbon shuffle” reactions to replenish RUBP

5 C3 ---> 3 C5

2X

Summary of the Calvin Cycle reactions

Fix 6 CO2 into one glucose molecule using 12 ATP and 12 NADPH
and then regenerate 6 RuBP from using 6 ATP.

Glucose synthesis
6 CO2 + 6 RuBP + 12 NADPH + 12 ATP + 10 H2O →
6 GAP + 4 DHAP + Glucose + 12 NADP+ + 12 ADP + 16 Pi

Regeneration of RuBP
6 GAP + 4 DHAP + 6 ATP + 2 H2O →
6 RuBP + 6 ADP + 2 Pi

Net reaction from six turns of the Calvin cycle

6 CO2 + 12 NADPH + 18 ATP + 12 H2O →
Glucose + 12 NADP+ + 18 ADP + 18 Pi
 Regulation of the Calvin Cycle

Why must the Calvin Cycle be regulated?

At night, plant cells rely on glycolysis and mitochondrial aerobic

respiration to generate ATP for cellular processes.

Since photophosphorylation and NADPH production by the

photosynthetic electron transport system is shut down in the dark,
it is crucial that the Calvin cycle only be active in the light.

Otherwise, if glycolysis, the pentose phosphate pathway and the

Calvin cycle were all active at night, then simultaneous starch
degradation and carbohydrate biosynthesis would quickly deplete
the ATP and NADPH pools in the stroma.

This is why “Dark Reactions” is a misnomer for the Calvin Cycle; if it

were only active at night, then the plant would die from energy loss!

Light stimulation of Calvin Cycle enzymes by two mechanisms:

1. Several enzymes including Rubisco are activated by

elevated pH and by increased Mg2+ concentrations in
the stroma.

2. Other calvin cycle enzymes are activated thioredoxin-

mediated reduction of disulfide bridges.

Why is it important that light stimulate the Calvin Cycle?

 Activation by elevated pH and by increased Mg2+
concentrations in stroma

Light activation of the

photosynthetic electron transport
system causes stromal pH to
increase from pH 7 to pH 8 as a
result of proton pumping into the
thylakoid lumen.

This influx of H+ into the lumen

causes an efflux of Mg2+ to the
stroma to balance the charge.

Rubisco activity is maximal under

conditions of pH 8 and high Mg2+.

Activation by thioredoxin-mediated reduction of

disulfide bridges
Thioredoxin is a small protein of 12 kDa that is found throughout nature and
functions as a redox protein that can interconvert disulfide bridges and sulfhydrals
in cysteine residues of target proteins.
As long as reduced thioredoxin is present in the stroma, these Calvin Cycle
enzymes are maintained in the active state.
However, when the sun goes down, spontaneous oxidation leads to their
inactivation.
 The C4 and CAM pathways
reduce photorespiration in hot climates

What is photorespiration?
A wasteful oxygenase reaction catalyzed by the enzyme
Rubisco that results in the loss of 2 carbons as CO2.

How do plants limit photorespiration?

By separating CO2 uptake from CO2 fixation, thereby
limiting exposure of Rubisco to O2. This can be done
spatially (C4 pathway) or temporally (CAM pathway).

Photorespiration and Rubisco

Rubisco also catalyzes a oxygenase reaction that combines RuBP

with O2 to generate one molecule of 3-PGA (C3) and one molecule of
2-phosphoglycolate (C2).

It is thought that this wasteful reaction (2-phosphoglycolate cannot be

used for sugar synthesis) belies the ancient history of the rubisco
enzyme which has been around since before O2 levels in the
atmosphere were as high as they are today.

In order to salvage the carbon in 2-phosphoglycolate, it must first be

converted to glycolate, which is exported to peroxisomes to make
glyoxylate and glycine which is then exported to mitochondria where
two molecules of glycine are converted to one molecule of serine.

Oxygenation of RuBP, and metabolism of 2-phosphoglycolate by the

C2 glycolate pathway, is collectively called photorespiration
because O2 is consumed and CO2 is released.
 Rubisco is a carboxylase and an oxygenase

C2 Glycine
Sugar glycolate
pathway
Serine
Buchanon et al., Fig. 14.45

C2 Glycolate Pathway in Plants

2 ATP are
Consumed
in the cycle

The 2 carbons
in glycolate are
lost as either
CO2 or serine
 C4 and CAM Pathways

Plants in hot climates are especially susceptible to photorespiration due to

high O2:CO2 ratios under these environmental conditions. Increased rates of
photorespiration in C3 plants (normal carbon fixation) reduces growth.

In the 1960s, Marshall Hatch and Roger Slack, plant biochemists at the
Colonial Sugar Refining Company in Brisbane, Australia, used 14CO2
labeling experiments to determine what the initial products were in the
carbon fixation reactions of sugarcane plants.

To their surprise, they found that malate was more quickly labeled with 14C
than was 3-PGA. Follow up work showed that plants such as sugarcane
and corn, and weeds like crabgrass, thrive under high temperature
conditions by having very low levels of photorespiration.

The mechanism involves the carboxylation of phosphoenolpyruvate (PEP)

by the enzyme PEP carboxylase to form oxaloacetate (OAA), a four carbon
(C4) intermediate that serves as a transient CO2 carrier molecule. OAA is
then converted to malate which functions as a storage form of CO2.

C4 and CAM Pathways Limit Photorespiration in Hot Climates

Two variations of the "Hatch-Slack"

pathway have been described;

1) C4 pathway in tropical plants

utilize two separate cell types to
reduce photorespiration during the
day. In this case, CO2 is stored in
the C4 intermediate malate which is
made in one cell type and then
transported to a second cell type
containing Rubisco where it is
decarboxylated to release the CO2.

2) CAM pathway found in desert

succulents captures CO2 in the
form of malate at night when it is
cool and O2:CO2 ratios are low.
During the day when
photosynthesis is active, the
stomata are closed and malate is
decarboxylated to release the CO2
in the same cell for use by Rubisco.
 C4 Pathway in Sugarcane
Mesophyll cells are responsible for CO2 capture. Interior bundle sheath
cells (further away from atmospheric O2), use CO2 released from the C4
intermediate malate to carry out the Calvin cycle reactions.
This "separation in space" between the two cell types essentially eliminates
the oxygenase reaction in rubisco and thereby blocks photorespiration.
Two high energy phosphate bonds are required to convert pyruvate to
phosphoenolpyruvate (rxn 4) which has an energy cost to C4 plants.

3
1

Key Enzymes in the C4 Pathway in Sugarcane

PEP carboxylase (rxn 1) catalyzes a reaction combining HCO3- with
PEP to form OAA.

OAA is reduced to malate by the enzyme NADP-malate

dehydrogenase (rxn 2). Other C4 plants transaminate OAA to form
aspartate which functions as the CO2 intermediate.

Malate is then transported to the bundle sheath cells through special

protein-lined channels connecting the cells where it is oxidized and
decarboxylated in the bundle sheath cell chloroplast by NADP-malic
enzyme (rxn 3) to form CO2 and pyruvate.

The released CO2 is incorporated into RuBP by rubisco and the

resulting 3-phosphoglycerate is metabolized by the C3 pathway.

In sugarcane, pyruvate is transported back to the mesophyll cells

where it is phosphorylated by the enzyme pyruvate phosphate
dikinase in the presence of ATP (rxn 4) to yield PEP + AMP.
 CAM Pathway
First discovered in succulent plants of the Crassulaceae family
therefore called Crassulacean Acid Metabolism (CAM) pathway.

The CAM pathway functions to concentrate CO2 levels in the

chloroplast stroma to limit the oxygenase activity of rubisco. CAM
plants like the saguaro cactus use a temporal separation.

During the night when the stomata are open to obtain moisture,
CO2 is captured by the mesophyll cells and incorporated into OAA
by PEP carboxylase. OAA is then reduced by NAD-malate
dehydrogenase to form malate.

During the day, the CO2 is released from malate by NADP-malic

enzyme, allowing the Calvin Cycle to fix the CO2 when ATP and
NADPH are plentiful from photosynthesis.

Importantly, tropical plants obtain moisture and sunlight during the

day, so spatial separation is optimal, whereas, cacti only open
stomata at night, so temporal separation allows photosynthesis to
take place during the day even with the stomata closed.

CAM Plants Temporally Separate CO2 Uptake from CO2 Fixation

1. PEP Carboxylase
2. Malate Dehydrogenase

3. NADP-Malic Enzyme

Buchanon et al., Fig. 12.51

C3 plants have an advantage in mild temperatures, whereas,
C4 plants have an advantage in high temperatures

Springtime (20º C) Summertime (30º C)

Turf (C3) outgrows Crabgrass (C4) outgrows
crabgrass (C4) because of turf (C3) because of
increased energy efficiency reduced photorespiration