Chapter 15 (part 3)
Carbon Fixation (dark
reactions)
Carbon Dioxide Fixation
• A unique ability of plants, algae, etc.
• Melvin Calvin at Berkeley in 1945
showed that Chlorella could take up
14CO and produce 3-phosphoglycerate
2
• What was actually happening was that
CO2 was combining with a 5-C sugar to
form a 6-C intermediate
• This breaks down to two 3-P
glycerates
Reductive Pentose Phosphate Cycle
6CO2+9ATP+5H20  9ADP+8Pi+6NADP++(DHAP or G3P)
Ribulose-1,5-Bisphosphate
carboxylase/oxygenase (rubbisco)
• Probably the world's most abundant protein
• In leaves greater than 50% of the soluble protein is
rubisco (stromal conc. 4 mM)
• Rate Limiting step in RPP cycle
• Rubisco is a slow enzyme (turnover number is 3 rxn
per second)
• Composed of 8 large subunits (LSU) (56,000 dal) and
8 small subunits (SSU) (14,000 dal). Active sites
assocaited with LSU.
• LSU encoded by chloroplast genome. SSU encoded
by nuclear genome.
Activation of Rubisco
• Rubisco cycles between active and inactive
form.
• Active form requires a bound Mg2+ ion,
light and high pH.
• A none substrate CO2 molecule
participates in Mg2+ binding to active site.
• CO2 molecule binds reversibly to lysine
residue forming carbamate adduct
• Activation facilitated by the enzyme
rubisco activase.
• In the dark, carbamate adduct
disassociates from active site. R 1,5-BP
then binds tightly to active site and
inhibits enzyme
Mg2+ plays role in binding and
activating R 1,6-BP to accept CO2
Rubisco Rxn Mechanisms
carboxylase
oxygenase
Reductive Pentose Phosphate Cycle
Reduction Stage
•Conversion of 3phosphoglycerate to glucose is
very similar to gluconeogenesis,
but glyceraldehyde
dehydrogenase uses NADPH not
NADH.
•Steps require consumption of
ATP and NADPH.
•3-phosphoglycerate could also
be exported to cytsol and be
used in normal gluconeogenesis.
•Hexoses can then be used for
energy or starch synthesis
cytosol
F 1,6-bisphophatase
aldolase
Glyceraldehyde
dehydrogenase
Phosphoglycerate
kinase
Reductive Pentose Phosphate Cycle
Regeneration Step
• Need to regenerate ribulose 1,5phosphate for subsequent rubisco
reactions
• One of the two 3-phosphoglyserates
goes towards regeneration.
• Need to generate 5 carbon sugar from 3
carbon and 6 carbon sugars.
• Most expensive part of RPP cycle.
Transketolases and Aldolases are
used to make 5 carbon sugars
Formation of 5 Carbon Sugars
F-6-P + 2 G3P + DHAP + 3 ATP  3 R-1,5-BP + 3 ADP
Regulation of RPP Cycle
• Rubisco activity is
regulated by pH
Mg2+
• Other enzymes
regulated by redox
state of
chloroplast
• All factors are
influenced by light
Thioredoxin
• 12 kD protein
• Contains Cysteine residue
that can cycle between
reduced –SH and oxidized –
S-S-.
• Reduced thioredoxin can
activate enzymes by reducing
disulfides in regulatory
domains.
• thioreodxin ties
light rxns to RPP
cycle regulation
• In light Thioredoxin
is reduced.
• Reduced thioredoxin
activates RPP cycle
enzymes.
• “dark Rxns” don’t
really function well
in the dark.
Oxygenase Activity of Rubisco
• CO2 and O2 compete for binding at active site.
• Under normal conditions the rate of
carboxylation is 3 to 4 times the rate of
oxygenation.
• Both require activation by carbamate adduct
(therefore no oxygenation w/o CO2)
• Oxygenase activity produces 3-phosphoglycerate
(normal C3 product) and 2-phosphoglycolate (C2
product)
Photorespiration
(recycling of 2-phosphoglycolate)
• 4 of five carbons from
R 1,5-BP salvaged.
• Loose one carbon as CO2
• Because O2 consumed and
CO2 released the process
is called photorespiration
• Wasteful process, loose
carbon as CO2 w/o
producing ATP or NADH
• Biochemist have been
trying to engineer better
rubisco (no luck)
Mechanisms to Avoid
Photorespiration
• C4 Photosynthesis – Spatial
separation of carbon fixation and
carbon utilization
• CAM (Crassulacean Acid Metabolism)
Photosynthesis – temporal separation
of carbon fixation and carbon
utilization
C4 Photosynthesis
• C4 cycle is way to pump CO2 into bundle sheath cells
making concentration 20 fold higher than in mesophyll
cells.
• Important in plants from hot climates.
• Under elevated temperature rubisco favors oxygenase
function causing plants to undergoe photorespiration.
• By fixing CO2 in Ms Cells with PEP carboxylase and
transferring it to the Bs Cells as a 4 carbon sugar can
concentrate CO2 and prevent photorepsiration.
CAM Photosynthesis
• Found in succulent plants
(Crassulacea).
• Drought tolerant plants.
• Gas exchange occurs by opening
pores called stoma
• What to import CO2 without
loosing water through stoma.
• CAM plants open stoma at night
to fix CO2,
• They then store it until daytime
when it is release it to rubisco
stoma