Made for 2nd year Bioenergetics class

1. Photosynthesis: Calvin
Cycle

2. Photosynthesis grana disks 2 outer
takes place in (thylakoids) membranes
chloroplasts.
stroma
It includes light compartment
reactions and
reactions that are
not directly
energized by light. Chloroplast
Light reactions: Energy of light is conserved as
 “high energy” phosphoanhydride bonds of ATP
 reducing power of NADPH.
Proteins & pigments responsible for the light
reactions are in thylakoid (grana disc)
membranes.
Light reaction pathways will be not be presented

3. grana disks 2 outer
Calvin Cycle, (thylakoids) membranes
earlier designated
the photosynthetic stroma
"dark reactions," compartment
is now called the
carbon reactions
pathway: Chloroplast
The free energy of cleavage of ~P bonds of ATP, and
reducing power of NADPH, are used to fix and
reduce CO2 to form carbohydrate.
Enzymes & intermediates of the Calvin Cycle are
located in the chloroplast stroma, a compartment
somewhat analogous to the mitochondrial matrix.

4. 2-
H2C OPO 3
O C -O O
C
H C OH
H C OH
H C OH
H2 C OPO 32-
2-
H2C OPO 3
3-Phosphoglycerate
Ribulose-1,5-bisphosphate (3PG)
(RuBP)
Ribulose Bisphosphate Carboxylase (RuBP
Carboxylase), catalyzes CO2 fixation:
ribulose-1,5-bisphosphate + CO2  2 3-
phosphoglycerate
Because it can alternatively catalyze an oxygenase
reaction, the enzyme is also called RuBP
Carboxylase/Oxygenase (RuBisCO). It is the most

5. H 2C OPO32 H 2C OPO32 H 2C OPO32
1

O C2 O C HO C CO2
CO2
O O
H C OH C OH C O C
3
H C OH H+ H C OH H C OH H2O H C OH
4
H 2C OPO32 H 2C OPO32 H 2C OPO32 H 2C OPO32
5
ribulose-1,5- enediolate b-keto 3-phosphoglycerate
bisphosphate intermediate intermediate (2)
RuBP Carboxylase - postulated mechanism:
Extraction of H+ from C3 of ribulose-1,5-bisphosphate
promotes formation of an enediolate intermediate.
Nucleophilic attack on CO2 leads to formation of a
b-keto acid intermediate, that reacts with water and
cleaves to form 2 molecules of 3-phosphoglycerate.

6. 2 2
H2C OPO 3 H2C OPO 3
HO C CO 2 HO C CO 2
C O H C OH
H C OH H C OH
2 2
H2C OPO 3 H2C OPO 3
Proposed b-keto acid 2-Carboxyarabinitol-1,5-
intermediate bisphosphate (inhibitor)
Transition state analogs of the postulated b-keto
acid intermediate bind tightly to RuBP Carboxylase
and inhibit its activity.
Examples: 2-carboxyarabinitol-1,5-bisphosphate
(CABP, above right) & carboxyarabinitol-1-
phosphate (CA1P).

7. RuBP
Carboxylase
in plants is a
complex
(L8S8) of: RuBisCO PDB 1RCX RuBisCO PDB 1RCX
 8 large catalytic subunits (L, 477 residues, blue,
cyan)
 8 small subunits (S, 123 residues, shown in red).
Some bacteria contain only the large subunit, with the
smallest functional unit being a homodimer, L2.
Roles of the small subunits have not been clearly
defined. There is some evidence that interactions
between large & small subunits may regulate

8. PDB 1RCX
Large subunits within ribulose-1,5-
RuBisCO are arranged as bisphosphate
antiparallel dimers, with the
N-terminal domain of one
monomer adjacent to the C-
terminal domain of the other.
Each active site is at an
interface between
monomers within a dimer,
explaining the minimal 2L & 2S
subunits
requirement for a dimeric of RuBisCO
structure.
The substrate binding site is at the mouth of an ab-barrel
domain of the large subunit.
Most active site residues are polar, including some
charged amino acids (e.g., Thr, Asn, Glu, Lys).

9. O
H
Enz-Lys +
NH3 + HCO3 Enz-Lys N C + H2O + H+
O
Carbamate Formation
with RuBP Carboxylase Activation
"Active" RuBP Carboxylase has a carbamate that
binds an essential Mg++ at the active site.
The carbamate forms by reaction of HCO3 with the
e-amino group of a lysine residue, in the presence of
Mg++.
HCO3 that reacts to form carbamate is distinct from
CO2 that binds to RuBP Carboxylase as substrate.
Mg++ bridges between oxygen atoms of the carbamate
& substrate CO .

10. Binding of either RuBP or a transition state analog
to RuBP Carboxylase causes a conformational
change to a "closed" conformation in which
access of solvent water to the active site is
blocked.
RuBP Carboxylase (RuBisCO) can spontaneously
deactivate by decarbamylation.
In the absence of the carbamate group, RuBisCO
tightly binds ribulose bisphosphate (RuBP) at the
active site as a “dead end” complex, with the
closed conformation, and is inactive in catalysis.
In order for the carbamate to reform, the enzyme
must undergo transition to the open conformation.

11. RuBP Carboxylase Activase is an ATP hydrolyzing
(ATPase) enzyme that causes a conformational
change in RuBP Carboxylase from a closed to an
open state.
This allows release of tightly bound RuBP or other
sugar phosphate from the active site, and carbamate
formation.
Since photosynthetic light reactions produce ATP, the
ATP dependence of RuBisCO activation provides a
mechanism for light-dependent activation of the
enzyme.
The activase is a member of the AAA family of
ATPases, many of which have chaperone-like
roles.
RuBP Carboxylase Activase is a large multimeric

12. Phosphoglycerate Glyceraldehyde-3-phosphate
Kinase Dehydrogenase
O O O OPO32
C ATP ADP C NADPH NADP+ CHO
H C OH H C OH H C OH
H2C OPO32 H2C OPO3
2 Pi H2C OPO3
2
3-phospho- 1,3-bisphospho- glyceraldehyde-
glycerate glycerate 3-phosphate
Glyceraldehyde-3-P Dehydrogenase catalyzes
reduction of the carboxyl of 1,3-bisphosphoglycerate
to an aldehyde, with release of Pi, yielding
glyceraldehyde-3-P.
This is like the Glycolysis enzyme running backward,
but the chloroplast Glyceraldehyde-3-P
Dehydrogenase uses NADPH as e donor, while the
cytosolic Glycolysis enzyme uses NAD+ as e

13. Continuing with Calvin Cycle:
A portion of the glyceraldehyde-3-P is converted
back to ribulose-1,5-bisP, the substrate for
RuBisCO, via reactions catalyzed by:
Triose Phosphate Isomerase, Aldolase, Fructose
Bisphosphatase, Sedoheptulose
Bisphosphatase, Transketolase, Epimerase,
Ribose Phosphate Isomerase, &
Phosphoribulokinase.
Many of these are similar to enzymes of Glycolysis,
Gluconeogenesis or Pentose Phosphate Pathway,
but are separate gene products found in the
chloroplast stroma. (Enzymes of the other pathways
listed are in the cytosol.)
The process is similar to Pentose Phosphate
Pathway run backwards.

14. Summary of Calvin cycle:
3 5-C ribulose-1,5-bisP (total of 15 C) are
carboxylated (3 C added), cleaved,
phosphorylated, reduced, & dephosphorylated,
yielding
6 3-C glyceraldehyde-3-P (total of 18 C). Of
these:
1 3-C glyceraldehyde-3-P exits as product.
5 3-C glyceraldehyde-3-P (15 C) are recycled
back into 3 5-C ribulose-1,5-bisphosphate.
C3 + C3  C6
C3 + C6  C4 + C5
C3 + C4  C7
C3 + C7  C5 + C5

15. Overall: TI
glyceraldehyde-3-P dihydroxyacetone-P
5 C3  3 C5
AL, FB
Enzymes: fructose-6-P
TI, TK
Triosephosphate
Isomerase xyulose-5-P + erythrose-4-P
AL, Aldolase AL, SB
FB, Fructose-1,6- sedoheptulose-7-P
TK
bisphosphatase
SB, xylulose-5-P + ribose-5-P
Sedoheptulose- EP IS
(3) ribulose-5-P
Bisphosphatase PK
TK, Transketolase (3) ribulose-1,5-bis-P

16. CHO
H C OH
Summary of O C O H2C OPO32
Calvin Cycle carbon glyceraldehyde-
dioxide 3-phosphate
3 CO2 + 9 ATP + 6 NADPH 
glyceraldehyde-3-P + 9 ADP + 8 Pi + 6 NADP+
Glyceraldehyde-3-P may be converted to other
CHO:
• metabolites (e.g., fructose-6-P, glucose-1-P)
• energy stores (e.g., sucrose, starch)
• cell wall constituents (e.g., cellulose).
Glyceraldehyde-3-P can also be utilized by plant
cells as carbon source for synthesis of other

17. grana disks 2 outer
(thylakoids) membranes
stroma
compartment
Chloroplast
There is evidence for multienzyme complexes of
Calvin Cycle enzymes within the chloroplast stroma.
Positioning of many Calvin Cycle enzymes close to
the enzymes that produce their substrates or utilize
their reaction products may increase efficiency of
the pathway.

18. Regulation of Calvin Cycle
Regulation prevents the Calvin Cycle from
being active in the dark, when it might function
in a futile cycle with Glycolysis & Pentose
Phosphate Pathway, wasting ATP & NADPH.
Light activates, or dark inhibits, the Calvin
Cycle (previously called the “dark reaction”) in
several ways.

19.  +
H2O  OH + H
 h stroma
Regulation (alkaline)
by Light.
(acid inside
Chloroplast thylakoid disks)
Light-activated e transfer is linked to pumping of H+
into thylakoid disks. pH in the stroma increases to about
8.
Alkaline pH activates stromal Calvin Cycle enzymes
RuBP Carboxylase, Fructose-1,6-Bisphosphatase &
Sedoheptulose Bisphosphatase.
The light-activated H+ shift is countered by Mg++ release
from thylakoids to stroma. RuBP Carboxylase (in
stroma) requires Mg++ binding to carbamate at the active

20. Some plants synthesize a transition-state
inhibitor, carboxyarabinitol-1-phosphate (CA1P),
in the dark.
RuBP Carboxylase Activase facilitates release of
CA1P from RuBP Carboxylase, when it is
activated under conditions of light by thioredoxin.

21. Thioredoxin f PDB 1FAA
disulfide
Thioredoxin is a small protein with a disulfide that
is reduced in chloroplasts via light-activated electron
transfer.

22. ferredoxinRed ferredoxinOx
thioredoxin
thioredoxin
S SH
|
S Ferredoxin-
SH
Thioredoxin
Reductase
During illumination, the thioredoxin disulfide is
reduced to a dithiol by ferredoxin, a constituent of
the photosynthetic light reaction pathway, via an
enzyme Ferredoxin-Thioredoxin Reductase.
Reduced thioredoxin activates several Calvin
Cycle enzymes, including Fructose-1,6-
bisphosphatase, Sedoheptulose-1,7-bisphosphatase,
and RuBP Carboxylase Activase, by reducing
disulfides in those enzymes to thiols.

23. PHOTORESPIRATION

24.  Photorespiration occurs when the CO2
levels inside a leaf become low. This
happens on hot dry days
 On hot dry days the plant is forced to close
its stomata to prevent excess water loss.
 The plant continues fix CO2 when its
stomata are closed, the CO2 will get used
up and the O2 ratio in the leaf will increase
relative to CO2 concentrations.

25.  When the CO2 levels inside the leaf drop to
around 50 ppm, Rubisco starts to combine
O2 with RuBP instead of CO2
 The net result of this is that instead of
producing 2 3C PGA molecules, only one
molecule of PGA is produced and a toxic 2C
molecule called phosphoglycolateis
produced.

27. phosphoglycolate
 The plant must get rid of the
phosphoglycolate since it is highly toxic.
 It converts the molecule to glycolic acid.
 The glycolic acid is then transported to the
peroxisome and there converted to glycine.

28. phosphoglycolate
Glycolic acid
In peroxisomes
Glycine
In mitochondria
Serine

29. • The serine is then used to make other
organic molecules.
• All these conversions cost the plant
energy and results in the net loss of CO2
from the plant
• To prevent this process, two specialized
biochemical additions have been evolved
in the plant world: C4 and CAM
metabolism.

30. The C4 PATHWAY

32. The C4 pathway is designed to efficiently fix
CO2 at low concentrations and plants that
use this pathway are known as C4 plants.
 These plants fix CO2 into a four carbon
compound (C4) called oxaloacetate. This
occurs in cells called mesophyll cells.

33. 1. CO2 is fixed to a three-carbon compound
called phosphoenolpyruvate to produce the
four-carbon compound oxaloacetate.
The enzyme catalyzing this reaction, PEP
carboxylase, fixes CO2 very efficiently so the
C4 plants don't need to to have their stomata
open as much.
The oxaloacetate is then converted to
another four-carbon compound called malate
in a step requiring the reducing power of
NADPH

34. 2. The malate then exits the mesophyll
cells and enters the chloroplasts of
specialized cells called bundle sheath
cells.
Here the four-carbon malate is
decarboxylated to produce CO2, a three-
carbon compound called pyruvate, and
NADPH.
The CO2 combines with ribulose
bisphosphate and goes through the Calvin

35. 3.The pyruvate re-enters the mesophyll
cells, reacts with ATP, and is converted
back to phosphoenolpyruvate, the starting
compound of the C4 cycle.

36. The CAM PATHWAY

37.  CAM plants live in very dry condition
and, unlike other plants, open their
stomata to fix CO2 only at night.
 Like C4 plants, the use PEP carboxylase to
fix CO2, forming oxaloacetate.
 The oxaloacetate is converted to malate
which is stored in cell vacuoles. During the
day when the stomata are closed, CO2 is
removed from the stored malate and enters
the Calvin cycle

39. Differences between calvin (C3)
and C4
C3 C4
 Temp 15-250 C  Temp 30-350 C
 Absence of malate  Presence of malate
 One carboxylation  2 carboxylation reactions
reaction  HCO3 is the substrate
 CO2 is the substrate  Closed stomata to reduce
water loss and
 Usual leaf structures concentrating CO2 in the
bundle sheet cells
 Additional ATP is required

41. Comparison between C3, C4, and CAM
C3 C4 CAM
product G3P Malate Malate
Day Day &night Night only
&night
Anatomy No bundle Bundle No bundle
sheet cell sheet cell sheet cell
No of stomata 2000- 10000- 100-800
31000 16000
Photorespirati Up to 40% Not Not
on detectable detectable
Species Wheat, Sugar cane Pineapple,
rice, vanilla, cacti
potato

42. Factors affecting
photorespiration
 O2: CO2Ratio
 If Cells Have Low O2 but Higher CO2,
Normal photosynthesis i.e. Calvin Cycle
Dominates
 C4Plants Have Little Photorespiration
because They Carry the CO2to the
bundle Sheath Cells and can Build up
High [CO2]

43. • Calvin Cycle Reactions always Favored
over Photorespiration
• If Cells Have Higher O2and Lower CO2,
Photorespiration Dominates
• Temperature
Photorespiration Increases with
temperature