Carbon Fixation (dark reactions)

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• Carbon Fixation (dark reactions)

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Carbon Dioxide Fixation

• A unique ability of plants, algae, etc.
• Melvin Calvin at Berkeley in 1945 showed that
  Chlorella could take up 14CO2 and produce
  3-phosphoglycerate
• 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

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Reductive Pentose Phosphate Cycle
6CO29ATP5H20 ? 9ADP8Pi6NADP(DHAP or G3P)
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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.

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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

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Mg2 plays role in binding and activating R
1,6-BP to accept CO2
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Rubisco Rxn Mechanisms
carboxylase
oxygenase
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Reductive Pentose Phosphate Cycle
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Reduction Stage
cytosol

• Conversion of 3-phosphoglycerate 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

F 1,6-bisphophatase
aldolase
Glyceraldehyde dehydrogenase
Phosphoglycerate kinase
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Reductive Pentose Phosphate Cycle
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Regeneration Step

• Need to regenerate ribulose 1,5-phosphate 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.

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Transketolases and Aldolases are used to make 5
carbon sugars
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Formation of 5 Carbon Sugars
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F-6-P 2 G3P DHAP 3 ATP ? 3 R-1,5-BP 3 ADP
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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

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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.

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• thioreodxin ties light rxns to RPP cycle
  regulation
• In light Thioredoxin is reduced.
• Reduced thioredoxin activates RPP cycle enzymes.
• dark Rxns dont really function well in the
  dark.

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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)

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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)

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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

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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.

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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
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