Download Calvin Cycle

Molecular Biochemistry II
Photosynthesis: Calvin Cycle
Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.
Photosynthesis
takes place in
chloroplasts.
It includes light
reactions and
reactions that are
not directly
energized by light.
2 outer
membranes
grana disks
(thylakoids)
stroma
compartment
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 here.
Calvin Cycle,
earlier designated
the photosynthetic
"dark reactions,"
is now called the
carbon reactions
pathway:
2 outer
membranes
grana disks
(thylakoids)
stroma
compartment
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.
H2C
O
OPO32-
C
-O
O
C
H
C
OH
H
H
C
H2C
OH
OPO3
C
H2C
OH
OPO32-
2-
Ribulose-1,5-bisphosphate
(RuBP)
3-Phosphoglycerate
(3PG)
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.
H 2C
1
O
OPO32

C2
H
C
OH
H
C
OH
3
4
H2C5
H 2C
OPO32
ribulose-1,5bisphosphate
O
H+ H
OPO32
C
CO2
C
OH
C
OH
H 2C
OPO32
enediolate
intermediate
H 2C
HO
C
OPO32
CO2
O
H
C
O
C
OH H2O
H 2C
OPO32
H
C
C
H 2C
O
OH
OPO32
b-keto
3-phosphoglycerate
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.
HO
H
OPO3
HO
C
CO2
O
H
C
OH
OH
H
C
OH
OPO3
C
CO2
C
C
H2C
2
H2 C
H2C
OPO3
2
Proposed b-keto acid
intermediate
H2 C
2
OPO32
2-Carboxyarabinitol-1,5bisphosphate (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).
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 catalysis.
ribulose-1,5Large subunits within
bisphosphate
RuBisCO are arranged as
antiparallel dimers, with the
N-terminal domain of one
monomer adjacent to the Cterminal domain of the other.
Each active site is at an
interface between monomers
within a dimer, explaining
the minimal requirement for
a dimeric structure.
PDB 1RCX
2L & 2S
subunits
of RuBisCO
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).
O
Enz-Lys
+
NH3 +
HCO3
Enz-Lys
H
N
+ H2O + H+
C
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 CO2.
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.
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.
O
O
Photorespiration:
O2 can compete with CO2
for binding to RuBisCO,
especially when [CO2] is
low & [O2] is high.
C
O
O
H
C
H2C
OH
OPO 3
3-phosphoglycerate
C
2
H2C
OPO 3
2
phosphoglycolate
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).
O
O
C
O
O
H
Photorespiration:
Diagram
C
H2C
OH
OPO 3
3-phosphoglycerate
C
2
H2C
OPO 3
2
phosphoglycolate
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.

O2C
C

CH2 + HCO3
OPO32


O2C
C
CH2
CO2
+ Pi
O
phosphoenolpyruvate
oxaloacetate
(PEP)
PEP Carboxylase
 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.
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.
O
O
Phosphoglycerate
Kinase
C
H
C
H2 C
ATP ADP
OPO 3
3-phosphoglycerate
O
C
H
OH
2
Glyceraldehyde-3-phosphate
Dehydrogenase
C
H2 C
OPO 32
NADPH NADP+
OH
OPO 32
1,3-bisphosphoglycerate
CHO
H
Pi
C
H2 C
OH
OPO 32
glyceraldehyde3-phosphate
Continuing with Calvin Cycle:
The normal RuBP Carboxylase product, 3-phosphoglycerate 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.
O
O
Phosphoglycerate
Kinase
C
H
C
H2 C
ATP ADP
OPO 3
3-phosphoglycerate
O
C
H
OH
2
Glyceraldehyde-3-phosphate
Dehydrogenase
C
H2 C
OPO 32
NADPH NADP+
OH
OPO 32
1,3-bisphosphoglycerate
CHO
H
Pi
C
H2 C
OH
OPO 32
glyceraldehyde3-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 acceptor.
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.
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
TI
Overall:
glyceraldehyde-3-P
dihydroxyacetone-P
5 C 3  3 C5
AL, FB
Enzymes:
fructose-6-P
TI, Triosephosphate
TK
Isomerase
xyulose-5-P + erythrose-4-P
AL, Aldolase
FB, Fructose-1,6AL, SB
bisphosphatase
sedoheptulose-7-P
SB, SedoheptuloseTK
Bisphosphatase
TK, Transketolase
xylulose-5-P + ribose-5-P
EP, Epimerase
EP
IS
IS, Isomerase
(3) ribulose-5-P
PK, PhosphoPK
(3) ribulose-1,5-bis-P
ribulokinase
CHO
H
Summary of
Calvin Cycle
O
C
carbon
dioxide
O
C
H2C
OH
OPO 32
glyceraldehyde3-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 compounds such as
fatty acids & amino acids.
2 outer
membranes
grana disks
(thylakoids)
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.
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.

H2O  OH + H
+
 h
Regulation
by Light.
Chloroplast
stroma
(alkaline)
(acid inside
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 site.
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.
Thioredoxin f
PDB 1FAA
disulfide
Thioredoxin is a small protein with a disulfide that
is reduced in chloroplasts via light-activated electron
transfer.
ferredoxinOx
S
|
S
FerredoxinThioredoxin
Reductase
thioredoxin
thioredoxin
ferredoxinRed
SH
SH
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.