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Photosynthesis: Calvin Cycle

Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.

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<b>Light reactions:</b> Energy of light is conserved as

 _{“high energy” phosphoanhydride bonds of}<b>_ATP</b>

 _{reducing power of}<b>_NADPH</b>_.

Proteins & pigments responsible for the light reactions

are in <b>thylakoid</b> (grana disc) <b>membranes</b>.

Light reaction pathways will be not be presented here.
grana disks

(thylakoids)

stroma
compartment

2 outer

membranes

Chloroplast
<b>Photosynthesis </b>

takes place in

chloroplasts.
It includes light
reactions and

reactions that are
not directly

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The free energy of cleavage of ~P bonds of <b>ATP</b>, and
reducing power of <b>NADPH</b>, are used to fix and reduce

<b>CO₂</b> to form <b>carbohydrate</b>.

Enzymes & intermediates of the <b>Calvin Cycle</b> are located
in the chloroplast <b>stroma</b>, a compartment somewhat

analogous to the mitochondrial matrix.
grana disks

(thylakoids)

stroma
compartment

2 outer

membranes

Chloroplast
<b>Calvin Cycle</b>,

earlier designated
the photosynthetic
"dark reactions,"
is now called the

<b>carbon reactions</b>

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<b>Ribulose Bisphosphate Carboxylase</b> (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

(<b>RuBisCO</b>). It is the <b>most abundant</b> enzyme on earth.

Ribulose-1,5-bisphosphate
(RuBP)

OH
H₂C

C
H

C
C

OH

H

H₂C OPO₃

2-OPO₃
2-O

3-Phosphoglycerate
(3PG)

OH
H₂C

C
H

C O
O

OPO ₃

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<b>-RuBP Carboxylase</b> - postulated mechanism:

Extraction of <b>H+</b> from <b>C3</b> of ribulose-1,5-bisphosphate
promotes formation of an <b>enediolate intermediate</b>.

Nucleophilic attack on CO₂ leads to formation of a <b></b>

<b>-keto acid intermediate</b>, that reacts with water and cleaves
to form 2 molecules of <b>3-phosphoglycerate</b>.

O H
H₂C

C
H
C
C
O H
H

H₂C O P O ₃2

O P O ₃2
O

O H
H₂C

C
H

C
C

O H

H₂C O P O₃2

O P O₃2

 _O

H+ O H

H ₂C
C
H

C
C

O

H ₂C O P O₃2

O P O₃2
H O C O₂
C O₂

O H
H ₂C

C
H

C

O P O ₃2
O

O

H₂O

1

5
4
3
2

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<b>Transition state analogs</b> of the postulated -keto acid

intermediate bind tightly to RuBP Carboxylase and <b>inhibit</b>

its activity.

Examples: 2-carboxyarabinitol-1,5-bisphosphate (<b>CABP</b>,
above right) & carboxyarabinitol-1-phosphate (CA1P).

2-Carboxyarabinitol-1,5-bisphosphate (inhibitor)

OH
H₂C

C
H
C

C
OH
H

H₂C OPO₃2

OPO₃2
HO CO₂

Proposed -keto acid
intermediate

OH
H₂C

C
H

C
C

O

H₂C OPO ₃2

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 <b>_{8 large catalytic subunits}</b>₍<b>_L</b>_{, 477 residues, blue, cyan)}
 <b>_{8 small subunits}</b>₍<b>_S</b>_{, 123 residues, shown in red).}

Some bacteria contain only the large subunit, with the
smallest functional unit being a homodimer, <b>L2</b>.

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 1RCX

RuBisCO PDB 1RCX

<b>RuBP </b>

<b>Carboxylase</b>

in plants is a
complex

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Large subunits within

RuBisCO are arranged as

<b>antiparallel dimers</b>, with the
N-terminal domain of one

monomer adjacent to the
C-terminal domain of the other.
Each <b>active site</b> is at an

<b>interface</b> between monomers
within a dimer, explaining the
minimal requirement for a

dimeric structure.

The substrate binding site is at the mouth of an -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

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"Active" RuBP Carboxylase has a <b>carbamate</b> that binds an
essential <b>Mg++</b> at the active site.

The carbamate forms by reaction of <b>HCO₃</b> with the 
-amino group of a <b>lysine</b> residue, in the presence of <b>Mg++</b>.

HCO₃ that reacts to form carbamate is distinct from CO

2 that
binds to RuBP Carboxylase as substrate.

<b>Mg++</b> bridges between oxygen atoms of the carbamate &

substrate <b>CO₂</b>.

Carbamate Formation

with RuBP Carboxylase Activation

Enz-Lys NH₃+ H_N _C

O

O

+ HCO₃ ₊_H

2O + H+

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Binding of either RuBP or a transition state analog to
RuBP Carboxylase causes a <b>conformational change</b> to
a "<b>closed</b>" conformation in which access of solvent

water to the active site is blocked.

RuBP Carboxylase (RuBisCO) can spontaneously

<b>deactivate</b> by decarbamylation.

In the <b>absence of the carbamate</b> group, RuBisCO

tightly binds ribulose bisphosphate (RuBP) at the active
site as a <b>“dead end” complex</b>, with the closed

conformation, and is inactive in catalysis.

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<b>RuBP Carboxylase Activase</b> 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

<b>ATP dependence</b> of RuBisCO activation provides a

mechanism for <b>light-dependent activation</b> of the enzyme.
The activase is a member of the <b>AAA family of ATPases</b>,
many of which have <b>chaperone-like</b> roles.

RuBP Carboxylase Activase is a <b>large</b> multimeric protein

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When <b>O₂</b> reacts with ribulose-1,5-bisphosphate, the

products are 3-phosphoglycerate plus the <b>2-C</b> compound
<b>2-phosphoglycolate</b>.

This reaction is the basis for the name RuBP
Carboxylase/Oxygenase (RuBisCO).

OH
H ₂C

C

H

C O

O

OPO ₃2



H ₂C
C

OPO ₃2

O 
O

3 -p h o s p h o - p h o s p h o g ly c o la te
g ly c e ra te

<b>Photorespiration:</b>

<b>O₂</b> can <b>compete with CO₂</b>

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The complex pathway that <b>partly</b> <b>salvages</b> <b>C</b> 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 <b>released</b> as <b>CO2</b>.

<b>Photorespiration </b>is a<b> wasteful </b>process, substantially

reducing efficiency of CO2 fixation, even at normal ambient
CO2.

OH
H ₂C

C
H

C O
O

OPO ₃2



H ₂C
C

OPO ₃2

O 

O

3 -p h o s p h o - p h o s p h o g ly c o la te
g ly c e r a te

<b>Photorespiration:</b>

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 _{Most plants, designated}<b>_C₃</b>_{, fix CO}₂_{initially via RuBP}
Carboxylase, yielding the 3-C 3-phosphoglycerate.

 Plants designated <b>C₄</b> have one cell type in which
phosphoenolpyruvate (PEP) is carboxylated via the

enzyme PEP Carboxylase, to yield the <b>4-C oxaloacetate</b>.
Oxaloacetate is converted to other 4-C intermediates that

are transported to cells active in photosynthesis, where

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<b>C₄</b> <b>plants maintain a high ratio of CO₂/O₂</b> within

photosynthetic cells, thus minimizing photorespiration.
Research has been aimed at increasing expression of

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<b>Continuing with Calvin Cycle:</b>

The normal RuBP Carboxylase product,
3-phospho-glycerate is converted to glyceraldehyde-3-P.

<b>Phosphoglycerate Kinase</b> catalyzes transfer of <b>P_i</b> from

<b>ATP</b> to the carboxyl of <b>3-phosphoglycerate</b> (RuBP

Carboxylase product) to yield <b>1,3-bisphosphoglycerate</b>.

OH
H ₂ C

C
H

C O

O

OPO ₃ 2 



OH
H ₂ C

C
H

C O PO 3

2

O

OPO ₃ 2 

OH
H ₂ C

C
H

CHO

OPO ₃ 2 

A T P A D P N A D P H N A D P +

P _i

1 , 3 - b i s p h o s p h o -
g l y c e r a t e
3 - p h o s p h o -

g l y c e r a t e g l y c e r a l d e h y d e - 3 - p h o s p h a t e

P h o s p h o g l y c e r a t e
K i n a s e

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<b>Glyceraldehyde-3-P Dehydrogenase</b> catalyzes reduction
of the carboxyl of 1,3-bisphosphoglycerate to an <b>aldehyde</b>,
with release of <b>P_i</b>, yielding <b>glyceraldehyde-3-P</b>.

This is like the Glycolysis enzyme running backward, but
the chloroplast Glyceraldehyde-3-P Dehydrogenase uses

<b>NADPH</b> as e donor, while the cytosolic Glycolysis

enzyme uses NAD+ as e acceptor.

OH
H ₂ C

C
H

C O

O

OPO ₃ 2 



OH
H ₂C

C
H

C O PO 3

2 

O

OPO ₃2 

OH
H ₂ C

C
H

CHO

OPO ₃ 2 

A T P A D P N A D P H N A D P +

P _i

1 , 3 - b i s p h o s p h o -
g l y c e r a t e
3 - p h o s p h o -

g l y c e r a t e g l y c e r a l d e h y d e - 3 - p h o s p h a t e

P h o s p h o g l y c e r a t e
K i n a s e

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<b>Continuing with Calvin Cycle:</b>

A portion of the <b>glyceraldehyde-3-P</b> is converted <b>back to </b>

<b>ribulose-1,5-bisP</b>, 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,

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Summary of Calvin cycle:

<b>3 </b> <b>5-C</b> ribulose-1,5-bisP (total of <b>15 C</b>) are carboxylated
(<b>3 C</b> added), cleaved, phosphorylated, reduced, &

dephosphorylated, yielding

<b>6 </b> <b>3-C</b> glyceraldehyde-3-P (total of <b>18 C</b>). Of these:
<b>1</b> <b>3-C</b> glyceraldehyde-3-P exits as <b>product</b>.

<b>5</b> <b>3-C</b> glyceraldehyde-3-P (<b>15 C</b>) are recycled back
into <b>3</b> <b>5-C</b> ribulose-1,5-bisphosphate.

<b>C₃ + C₃</b> <b> C₆</b>

<b> C₃ + C₆</b> <b> C₄ + C₅</b>

<b> C₃</b> <b>+ C₄</b> <b> C₇</b>

<b> C₃ + C₇</b>  <b>C₅ + C₅</b>

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Overall:

<b> 5 C₃</b>  <b>3 C₅</b>

Enzymes:

TI, Triosephosphate
Isomerase

AL, Aldolase

FB,
bisphosphatase

SB,
Bisphosphatase

TK, Transketolase

EP, Epimerase

IS, Isomerase

PK,

ribulokinase

TK

EP

PK

glyceraldehyde-3-P dihydroxyacetone-P

fructose-6-P

xyulose-5-P + erythrose-4-P

sedoheptulose-7-P

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<b>3 CO₂ + 9 ATP + 6 NADPH </b>

<b> glyceraldehyde-3-P + 9 ADP + 8 P_i + 6 NADP+</b>

<b>Glyceraldehyde-3-P</b> may be converted to <b>other CHO</b>:

• _{metabolites (e.g., fructose-6-P, glucose-1-P)}
• _{energy stores (e.g., sucrose, starch)}

• cell wall constituents (e.g., cellulose).

<b>Glyceraldehyde-3-P </b>can also be utilized by plant cells as
carbon source for synthesis of <b>other compounds</b> such as
fatty acids & amino acids.

g l y c e r a l d e h y d e -
3 - p h o s p h a t e

OH
H ₂ C

C
H

CHO

OPO ₃ 2 

O
C

O

c a r b o n
d i o x i d e

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There is evidence for <b>multienzyme complexes</b> of Calvin
Cycle enzymes within the chloroplast stroma.

<b>Positioning</b> of many Calvin Cycle enzymes <b>close</b> <b>to</b> the
enzymes that produce their substrates or utilize their

reaction products may <b>increase efficiency</b> of the pathway.
grana disks

(thylakoids)

stroma
compartment

2 outer

membranes

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Regulation of Calvin Cycle

<b>Regulation</b> <b>prevents</b> the Calvin Cycle from being

<b>active in the dark</b>, when it might function in a

<b>futile cycle</b> with Glycolysis & Pentose Phosphate
Pathway, wasting ATP & NADPH.

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<b>Light-activated</b> e transfer is linked to <b>pumping of H+</b>

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 <b>Mg++</b> release

from thylakoids <b>to stroma</b>. RuBP Carboxylase (in stroma)

requires Mg++ binding to carbamate at the active site.

stroma

(alkaline)

Chloroplast

H2O  OH



+ H+

_h

(acid inside
thylakoid disks)

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Some plants <b>synthesize a transition-state inhibitor</b>,
carboxyarabinitol-1-phosphate (CA1P), <b>in the dark</b>.

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disulfide

Thioredoxin f PDB 1FAA

<b>Thioredoxin</b> is a small protein with a <b>disulfide</b> that
is reduced in chloroplasts via light-activated

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During illumination, the <b>thioredoxin</b> disulfide is <b>reduced</b> to

a dithiol by <b>ferredoxin</b>, a constituent of the photosynthetic

light reaction pathway, via an enzyme
Ferredoxin-Thioredoxin Reductase.

<b>Reduced thioredoxin</b> <b>activates</b> several <b>Calvin Cycle </b>

<b>enzymes</b>, including Fructose-1,6-bisphosphatase,

Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase

Activase, by reducing disulfides in those enzymes to thiols.

th

io

re

do

xi

n

 _S
 _S

th

io

re

do

xi

n

 _SH
 _SH

|

ferredoxin_Red ferredoxin_Ox

Ferredoxin-
Thioredoxin

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