calvin cycle operates in stroma chloroplasts,also known as photosynthetic carbon reduction cycle.

1. Calvin CycleCalvin Cycle
and its regulationand its regulation
Iqbal rashid mir
Msc botany 3rd
sem
HT. NO.
100714502053

2. 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
here.
Photosynthesis
takes place in
chloroplasts.
It includes light
reactions and
reactions that are
not directly
energized by light.

3. 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.
Calvin Cycle,
earlier designated
the photosynthetic
"dark reactions,"
is now called the
carbon reactions
pathway:

4. 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
abundant enzyme on earth.
Ribulose-1,5-bisphosphate
(RuBP)
OH
H2C
CH
C
C
OHH
H2C OPO3
2-
OPO3
2-
O
3-Phosphoglycerate
(3PG)
OH
H2C
CH
C
OO
OPO3
2-
-

5. 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
β-keto acid intermediate, that reacts with water
and cleaves to form 2 molecules of 3-
phosphoglycerate.
OH
H2C
CH
C
C
OHH
H2C OPO3
2−
OPO3
2−
O
OH
H2C
CH
C
C
OH
H2C OPO3
2−
OPO3
2−
−
O
H+
OH
H2C
CH
C
C
O
H2C OPO3
2−
OPO3
2−
HO CO2
−
CO2
OH
H2C
CH
C
OPO3
2−
O−
O
H2O
1
5
4
3
2
ribulose-1,5- enediolate β-keto 3-phosphoglycerate
bisphosphate intermediate intermediate (2)

6. Transition state analogs of the postulated β-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).
2-Carboxyarabinitol-1,5-
bisphosphate (inhibitor)
OH
H2C
CH
C
C
OHH
H2C OPO3
2−
OPO3
2−
HO CO2
−
Proposed β-keto acid
intermediate
OH
H2C
CH
C
C
O
H2C OPO3
2−
OPO3
2−
HO CO2
−

7.  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
catalysis.
RuBisCO PDB 1RCXRuBisCO PDB 1RCX
RuBP
Carboxylase
in plants is a
complex
(L8S8) of:

8. Large subunits within
RuBisCO are arranged as
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
requirement for a dimeric
structure.
The substrate binding site is at the mouth of an αβ-barrel
domain of the large subunit.
Most active site residues are polar, including some
charged amino acids (e.g., Thr, Asn, Glu, Lys).
ribulose-1,5-
bisphosphate
PDB 1RCX
2L & 2S
subunits
of RuBisCO

9. "Active" RuBP Carboxylase has a carbamate that binds
an essential Mg++
at the active site.
The carbamate forms by reaction of HCO3
−
with the
ε-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 CO2
.
Carbamate Formation
with RuBP Carboxylase Activation
Enz-Lys NH3
+ H
N C
O
O−
+ HCO3
−
+ H2O + H+Enz-Lys

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 protein
complex that may surround RuBisCO while inducing the
conformational change to the open state.

12. When O2
reacts with ribulose-1,5-bisphosphate, the
products are 3-phosphoglycerate plus the 2-C
compound 2-phosphoglycolate.
This reaction is the basis for the name RuBP
Carboxylase/Oxygenase (RuBisCO).
OH
H2C
CH
C
OO
OPO3
2−
−
H2C
C
OPO3
2−
O−
O
3-phospho- phosphoglycolate
glycerate
Photorespiration:
O2
can compete with CO2
for binding to RuBisCO,
especially when [CO2
] is
low & [O2
] is high.

13. The complex pathway that partly salvages C from
2-phosphoglycolate, via conversion to 3-
phosphoglycerate, involves enzymes of chloroplasts,
peroxisomes & mitochondria.
This pathway recovers 3/4 of the C as 3-
phosphoglycerate.
The rest is released as CO2.
Photorespiration is a wasteful process, substantially
reducing efficiency of CO2 fixation, even at normal
ambient CO2.
OH
H2C
CH
C
OO
OPO3
2−
−
H2C
C
OPO3
2−
O−
O
3-phospho- phosphoglycolate
glycerate
Photorespiration:
Diagram

14.  Most plants, designated C3, fix CO2 initially via RuBP
Carboxylase, yielding the 3-C 3-phosphoglycerate.
 Plants designated C4 have one cell type in which
phosphoenolpyruvate (PEP) is carboxylated via the
enzyme PEP Carboxylase, to yield the 4-C
oxaloacetate.
Oxaloacetate is converted to other 4-C intermediates
that are transported to cells active in photosynthesis,
where CO2 is released by decarboxylation.
phosphoenolpyruvate oxaloacetate
(PEP)
PEP Carboxylase
→

15. C4 plants maintain a high ratio of CO2
/O2
within
photosynthetic cells, thus minimizing
photorespiration.
Research has been aimed at increasing expression
of and/or inserting genes for C4 pathway enzymes,
such as PEP Carboxylase, in C3 plants.

16. Continuing with Calvin Cycle:
The normal RuBP Carboxylase product, 3-phospho-
glycerate is converted to glyceraldehyde-3-P.
Phosphoglycerate Kinase catalyzes transfer of Pi from
ATP to the carboxyl of 3-phosphoglycerate (RuBP
Carboxylase product) to yield 1,3-
bisphosphoglycerate.
OH
H2C
CH
C
OO
OPO3
2−
−
OH
H2C
CH
C
OPO3
2−
O
OPO3
2−
OH
H2C
CH
CHO
OPO3
2−
ATP ADP NADPH NADP+
Pi
1,3-bisphospho-
glycerate
3-phospho-
glycerate
glyceraldehyde-
3-phosphate
Phosphoglycerate
Kinase
Glyceraldehyde-3-phosphate
Dehydrogenase

17. 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−
acceptor.
OH
H2C
CH
C
OO
OPO3
2−
−
OH
H2C
CH
C
OPO3
2−
O
OPO3
2−
OH
H2C
CH
CHO
OPO3
2−
ATP ADP NADPH NADP+
Pi
1,3-bisphospho-
glycerate
3-phospho-
glycerate
glyceraldehyde-
3-phosphate
Phosphoglycerate
Kinase
Glyceraldehyde-3-phosphate
Dehydrogenase

18. 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.

19. 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
Overall 5 C3
3 C5

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

21. 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
compounds such as fatty acids & amino acids.
glyceraldehyde-
3-phosphate
OH
H2C
CH
CHO
OPO3
2−
OCO
carbon
dioxide
Summary of
Calvin Cycle

22. 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.
grana disks
(thylakoids)
stroma
compartment
2 outer
membranes
Chloroplast

23. 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.

24. 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
site.
stroma
(alkaline)
Chloroplast
H2O  OH
−
+ H
+
hν
(acid inside
thylakoid disks)
Regulation
by Light.

25. 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.

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

27. 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.
thioredoxin
−S
−S
thioredoxin
−SH
−SH
|
ferredoxinRed ferredoxinOx
Ferredoxin-
Thioredoxin
Reductase