CARBON FIXATION
THE LIGHT INDEPENDENT
REACTION
THE LIGHT INDEPENDENT
REACTION
Enzyme controlled (Q10  2)
 Located in the stroma of the chloroplast
 Occurs simultaneously with the light
dependent reaction
 It can continue in the dark provided the
necessary raw materials are available
(CO2, NADPH + H+ and ATP)

© 2010 Paul Billiet ODWS
Enzyme controlled reaction pathways
Ea
Ea
A
B
Ec
C
D
X = Precursor (raw
material) added here
© 2010 Paul Billiet ODWS
Ee
Ed
E
F
Enzyme controlled reaction pathways
To find out the sequence of the reactions and the
point at which X is added in, two approaches
can be used:
1. Label and trace the products formed through
time
2. Cut the supply of X and observe what
happens to the intermediates in the pathway
e.g. in studying photosynthesis,
cut the CO2 supply or
switch off the light
so cutting the supply of ATP and NADPH+H+
© 2010 Paul Billiet ODWS
Calvin and Benson 1946 to 1953


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Used 14C radioisotope
for labelling
Unicellular algae:
Chlorella and
Scenedesmus
Simple plants which
respond quickly to
changes in the
environment
So little time lag
Image Credit Scenedesmus
© 2010 Paul Billiet ODWS
The “Lollipop” vessel
A flat-sided, round flask
containing the culture of
algae
This shape:
- provided even illumination
of all the cells
- permitted careful control of
environmental conditions
(e.g. pH, temperature)
- permitted rapid mixing of
contents
- precise sampling time
© 2010 Paul Billiet ODWS
Labelling and tracing carbon using
14C



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Add NaH14CO3 solution
At timed intervals the algae are sampled and
killed by dropping in hot methanol
Two-way (2-dimensional) chromatography
used to separate the compounds
Identify radioactively labelled compounds by
autoradiography
© 2010 Paul Billiet ODWS
Two-way (2-dimensional) chromatography
A. Mixture placed
at the origin
D. 2nd run
© 2010 Paul Billiet ODWS
B.1st run
C. Rotate the paper 90°
E. Autoradiograph reveals the
compound/s which are labelled with 14C
Results
2s
Salisbury & Ross (1969) Plant Physiology Wadsworth
Result
The compound that became labelled first
was Phosphoglyceric Acid (or
Phosphoglycerate) PGA
 PGA is a 3 carbon sugar
 Therefore: Compound B + CO2  PGA
What is compound B?!!!

© 2010 Paul Billiet ODWS
Light independent pathway
Ea
Ea
A
B
Ec
PGA
D
CO2
What is compound B?!!!
© 2010 Paul Billiet ODWS
Ee
Ed
E
F
Searching for the compound that
combines with carbon dioxide
Feed the algae NaH14CO3 for over 15 min
 All the compounds involved in photosynthesis
are labelled
 Then reduce the level of NaH14CO3 rapidly
Prediction
 PGA levels should fall rapidly
 The compound that fixes CO2 should rise
sharply

© 2010 Paul Billiet ODWS
Salisbury & Ross (1969) Plant Physiology Wadsworth
Result
This compound was found to be Ribulose
Bisphosphate (RBP)
 RBP is a 5 carbon sugar
Therefore
RBP
+
CO2 
2PGA
5C
+
1C =
2x3C


The enzyme controlling this important step is
Ribulose Bisphosphate Carboxylase
(Rubisco)
© 2010 Paul Billiet ODWS
Light independent pathway
Ea
A
RUBISCO
RBP
CO2
© 2010 Paul Billiet ODWS
Ec
PGA
Ee
Ed
D
E
F
RUBISCO
A large enzyme 500 000
Daltons
Inhibited by oxygen
A relatively slow enzyme
50% of chloroplast protein
Probably the most abundant
protein on Earth
Consists of two subunits (1
large + 1 small)
The large subunit is coded
for by a gene on chlDNA
© 2010 Paul Billiet ODWS
Image Credit: Rubisco stick model
Carbon fixation and light
The light dependent
pathways produce ATP
and NADPH+H+ to be
used in the light
independent processes
 Illuminate the algae for 15
min then cut the light
Result
 PGA levels rise
 Other compounds, after
PGA in the pathway, fall

© 2010 Paul Billiet ODWS
Carbon fixation and light
The compound requiring the NADPH+H+
and ATP was found to be Glycerate 3phosphate (GP)
 GP is converted to various other three
carbon sugars or Triose Phosphates (TP)
some are used to produce hexoses such
as fructose phosphate and glucose
phosphate

© 2010 Paul Billiet ODWS
Light independent pathway
Ea
A
RUBISCO
RBP
CO2
© 2010 Paul Billiet ODWS
Ec
PGA
Ee
Ed
GP
12 ATP
12 NADPH + H+
E
Hexoses
A curious turn

When the light is cut
interestingly RBP
levels also fall

This is for two
reasons



RBP requires ATP in its
synthesis
RBP is formed from
some of the GP
In other words the
reaction pathway is a
CYCLE
© 2010 Paul Billiet ODWS
Image Credit: Salisbury & Ross (1969) Plant Physiology Wadsworth
Squaring the circle! The Calvin-Benson
Cycle
6 RBP
(5C)
6 ATP
12 PGA
(3C)
Intermediates
1 Hexose
(6C)
6CO2
(1C)
12 TP
(3C)
12 GP
(3C)
12 ATP
12 NADPH + H+
PHOTOSYNTHESIS OVERVIEW
CHLOROPLAST
H2O
Light dependent
reaction
ATP
CO2
O2
NADPH+H+
Light independent
reaction
Triose
phospates
Starch
© 2010 Paul Billiet ODWS
CYTOSOL
Sugars
Fatty acids
Amino acids