1. Carbon Fixation (dark reactions)

2. 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

3. Reductive Pentose Phosphate Cycle
6CO2+9ATP+5H20  9ADP+8Pi+6NADP++(DHAP or G3P)

4. 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.

5. 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

6. Mg2+ plays role in binding and activating R 1,6-BP to accept CO2

7. Rubisco Rxn Mechanisms
carboxylase
oxygenase

8. Reductive Pentose Phosphate Cycle

9. 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

10. Reductive Pentose Phosphate Cycle

11. 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.

12. Transketolases and Aldolases are used to make 5 carbon sugars

13. Formation of 5 Carbon Sugars

15. F-6-P + 2 G3P + DHAP + 3 ATP  3 R-1,5-BP + 3 ADP

16. 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

17. 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.

18. 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.

20. 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)

21. 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)

22. 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

23. 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.

25. 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