1. Photosynthesis: Calvin Cycle Advanced Biochemistry for Biotechnology,

2. THE COLOR OF LIGHT SEEN IS THE COLOR NOT ABSORBED Chloroplasts absorb light energy and convert it to chemical energy Light Reflected light Absorbed light Transmitted light Chloroplast AN OVERVIEW OF PHOTOSYNTHESIS (Light reactions)

3. Photosynthesis is the process by which autotrophic organisms use light energy to make sugar and oxygen gas from carbon dioxide and water AN OVERVIEW OF PHOTOSYNTHESIS Carbon dioxide WaterGlucoseOxygen gas PHOTOSYNTHESIS

4. The Calvin cycle makes sugar from carbon dioxide –ATP generated by the light reactions provides the energy for sugar synthesis –The NADPH produced by the light reactions provides the electrons for the reduction of carbon dioxide to glucose Light Chloroplast Light reactions Calvin cycle NADP  ADP + P The light reactions convert solar energy to chemical energy –Produce ATP & NADPH AN OVERVIEW OF PHOTOSYNTHESIS

5. Chloroplasts: Sites of Photosynthesis Photosynthesis –Occurs in chloroplasts, organelles in certain plants –All green plant parts have chloroplasts and carry out photosynthesis The leaves have the most chloroplasts The green color comes from chlorophyll in the chloroplasts The pigments absorb light energy

6. In most plants, photosynthesis occurs primarily in the leaves, in the chloroplasts A chloroplast contains: –stroma, a fluid –grana, stacks of thylakoids The thylakoids contain chlorophyll –Chlorophyll is the green pigment that captures light for photosynthesis Photosynthesis occurs in chloroplasts

7. The location and structure of chloroplasts LEAF CROSS SECTION MESOPHYLL CELL LEAF Chloroplast Mesophyll CHLOROPLAST Intermembrane space Outer membrane Inner membrane Thylakoid compartment Thylakoid Stroma Granum StromaGrana

8. Chloroplasts contain several pigments Chloroplast Pigments –Chlorophyll a –Chlorophyll b –Carotenoids Figure 7.7

9. Chlorophyll a & b Chl a has a methyl group Chl b has a carbonyl group Porphyrin ring delocalized e - Phytol tail

10. Different pigments absorb light differently

11. Excited state ee Heat Light Photon Light (fluorescence) Chlorophyll molecule Ground state 2 (a) Absorption of a photon (b) fluorescence of isolated chlorophyll in solution Excitation of chlorophyll in a chloroplast  Loss of energy due to heat causes the photons of light to be less energetic.  Less energy translates into longer wavelength.  Energy = (Planck’s constant) x (velocity of light)/(wavelength of light)  Transition toward the red end of the visible spectrum. ee

12. Cyclic Photophosphorylation Process for ATP generation associated with some Photosynthetic Bacteria Reaction Center => 700 nm

13. Photon Water-splitting photosystem NADPH-producing photosystem ATP mill Two types of photosystems cooperate in the light reactions

14. Primary electron acceptor Electron transport chain Electron transport Photons PHOTOSYSTEM I PHOTOSYSTEM II Energy for synthesis of by chemiosmosis Noncyclic Photophosphorylation Photosystem II regains electrons by splitting water, leaving O 2 gas as a by-product

15. The O 2 liberated by photosynthesis is made from the oxygen in water (H + and e - ) Plants produce O 2 gas by splitting H 2 O

16. 2 H  + 1 / 2 Water-splitting photosystem Reaction- center chlorophyll Light Primary electron acceptor Energy to make Electron transport chain Primary electron acceptor Primary electron acceptor NADPH-producing photosystem Light NADP  1 2 3 How the Light Reactions Generate ATP and NADPH

17. Two connected photosystems collect photons of light and transfer the energy to chlorophyll electrons The excited electrons are passed from the primary electron acceptor to electron transport chains –Their energy ends up in ATP and NADPH In the light reactions, electron transport chains generate ATP, NADPH, & O 2

18. The electron transport chains are arranged with the photosystems in the thylakoid membranes and pump H + through that membrane –The flow of H + back through the membrane is harnessed by ATP synthase to make ATP –In the stroma, the H + ions combine with NADP + to form NADPH Chemiosmosis powers ATP synthesis in the light reactions

19. The production of ATP by chemiosmosis in photosynthesis Thylakoid compartment (high H + ) Thylakoid membrane Stroma (low H + ) Light Antenna molecules Light ELECTRON TRANSPORT CHAIN PHOTOSYSTEM IIPHOTOSYSTEM IATP SYNTHASE

20. A Photosynthesis Road Map Chloroplast Light Stack of thylakoids ADP + P NADP  Stroma Light reactions Calvin cycle Sugar used for  Cellular respiration  Cellulose  Starch  Other organic compounds

21. Review: Photosynthesis uses light energy to make food molecules Light Chloroplast Photosystem II Electron transport chains Photosystem I CALVIN CYCLE Stroma Electrons LIGHT REACTIONSCALVIN CYCLE Cellular respiration Cellulose Starch Other organic compounds A summary of the chemical processes of photosynthesis

22. Light reactions: Energy of light is conserved as  “high energy” phosphoanhydride bonds of ATP  reducing power of NADPH. Proteins & pigments responsible for the light reactions are in thylakoid (grana disc) membranes. Light reaction pathways will be not be presented here. Photosynthesis takes place in chloroplasts. It includes light reactions and reactions that are not directly energized by light. 22

23. The free energy of cleavage of ~P bonds of ATP, and reducing power of NADPH, are used to fix and reduce CO 2 to form carbohydrate. Enzymes & intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat analogous to the mitochondrial matrix. Calvin Cycle, earlier designated the photosynthetic "dark reactions," is now called the carbon reactions pathway: 23

24. Ribulose Bisphosphate Carboxylase (RuBP Carboxylase), catalyzes CO 2 fixation: ribulose-1,5-bisphosphate + CO 2  2 3-phosphoglycerate Because it can alternatively catalyze an oxygenase reaction, the enzyme is also called RuBP Carboxylase/Oxygenase (RuBisCO). It is the most abundant enzyme on earth. 24

25. RuBP Carboxylase - postulated mechanism: Extraction of H + from C 3 of ribulose-1,5-bisphosphate promotes formation of an enediolate intermediate. Nucleophilic attack on CO 2 leads to formation of a  -keto acid intermediate, that reacts with water and cleaves to form 2 molecules of 3-phosphoglycerate. 25

26. Transition state analogs of the postulated  -keto acid intermediate bind tightly to RuBP Carboxylase and inhibit its activity. Examples: 2-carboxyarabinitol-1,5-bisphosphate (CABP, above right) & carboxyarabinitol-1-phosphate (CA1P). 26

27.  8 large catalytic subunits (L, 477 residues, blue, cyan)  8 small subunits (S, 123 residues, shown in red). Some bacteria contain only the large subunit, with the smallest functional unit being a homodimer, L 2. Roles of the small subunits have not been clearly defined. There is some evidence that interactions between large & small subunits may regulate catalysis. RuBP Carboxylase in plants is a complex (L 8 S 8 ) of: 27

28. Large subunits within RuBisCO are arranged as antiparallel dimers, with the N-terminal domain of one monomer adjacent to the C- terminal domain of the other. Each active site is at an interface 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). 28

29. "Active" RuBP Carboxylase has a carbamate that binds an essential Mg ++ at the active site. The carbamate forms by reaction of HCO 3  with the  -amino group of a lysine residue, in the presence of Mg ++. HCO 3  that reacts to form carbamate is distinct from CO 2 that binds to RuBP Carboxylase as substrate. Mg ++ bridges between oxygen atoms of the carbamate & substrate CO 2. 29

30. Binding of either RuBP or a transition state analog to RuBP Carboxylase causes a conformational change to a "closed" conformation in which access of solvent water to the active site is blocked. RuBP Carboxylase (RuBisCO) can spontaneously deactivate by decarbamylation. In the absence of the carbamate group, RuBisCO tightly binds ribulose bisphosphate (RuBP) at the active site as a “dead end” complex, with the closed conformation, and is inactive in catalysis. In order for the carbamate to reform, the enzyme must undergo transition to the open conformation. 30

31. RuBP Carboxylase Activase 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 ATP dependence of RuBisCO activation provides a mechanism for light-dependent activation of the enzyme. The activase is a member of the AAA family of ATPases, many of which have chaperone-like roles. RuBP Carboxylase Activase is a large multimeric protein complex that may surround RuBisCO while inducing the conformational change to the open state. 31

32. When O 2 reacts with ribulose-1,5-bisphosphate, the products are 3-phosphoglycerate plus the 2-C compound 2-phosphoglycolate. This reaction is the basis for the name RuBP Carboxylase/Oxygenase (RuBisCO). Photorespiration: O 2 can compete with CO 2 for binding to RuBisCO, especially when [CO 2 ] is low & [O 2 ] is high. 32

33. The complex pathway that partly salvages C 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 released as CO 2. Photorespiration is a wasteful process, substantially reducing efficiency of CO 2 fixation, even at normal ambient CO 2. Photorespiration: Diagram 33

34.  Most plants, designated C 3, fix CO 2 initially via RuBP Carboxylase, yielding the 3-C 3-phosphoglycerate.  Plants designated C 4 have one cell type in which phosphoenolpyruvate (PEP) is carboxylated via the enzyme PEP Carboxylase, to yield the 4-C oxaloacetate. Oxaloacetate is converted to other 4-C intermediates that are transported to cells active in photosynthesis, where CO 2 is released by decarboxylation. 34

35. C 4 plants maintain a high ratio of CO 2 /O 2 within photosynthetic cells, thus minimizing photorespiration. Research has been aimed at increasing expression of and/or inserting genes for C 4 pathway enzymes, such as PEP Carboxylase, in C 3 plants. 35

36. Continuing with Calvin Cycle: The normal RuBP Carboxylase product, 3-phospho- glycerate is converted to glyceraldehyde-3-P. Phosphoglycerate Kinase catalyzes transfer of P i from ATP to the carboxyl of 3-phosphoglycerate (RuBP Carboxylase product) to yield 1,3-bisphosphoglycerate. 36

37. Glyceraldehyde-3-P Dehydrogenase catalyzes reduction of the carboxyl of 1,3-bisphosphoglycerate to an aldehyde, with release of P i, yielding glyceraldehyde-3-P. This is like the Glycolysis enzyme running backward, but the chloroplast Glyceraldehyde-3-P Dehydrogenase uses NADPH as e  donor, while the cytosolic Glycolysis enzyme uses NAD + as e  acceptor. 37

38. Continuing with Calvin Cycle: A portion of the glyceraldehyde-3-P is converted back to ribulose-1,5-bisP, 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, Gluconeogenesis or Pentose Phosphate Pathway, but are separate gene products found in the chloroplast stroma. (Enzymes of the other pathways listed are in the cytosol.) The process is similar to Pentose Phosphate Pathway run backwards. 38

39. Summary of Calvin cycle: 3 5-C ribulose-1,5-bisP (total of 15 C) are carboxylated (3 C added), cleaved, phosphorylated, reduced, & dephosphorylated, yielding 6 3-C glyceraldehyde-3-P (total of 18 C). Of these: 1 3-C glyceraldehyde-3-P exits as product. 5 3-C glyceraldehyde-3-P (15 C) are recycled back into 3 5-C ribulose-1,5-bisphosphate. C 3 + C 3  C 6 C 3 + C 6  C 4 + C 5 C 3 + C 4  C 7 C 3 + C 7  C 5 + C 5 Overall 5 C 3  3 C 5 39

40. Overall: 5 C 3  3 C 5 Enzymes: TI, Triosephosphate Isomerase AL, Aldolase FB, Fructose-1,6- bisphosphatase SB, Sedoheptulose- Bisphosphatase TK, Transketolase EP, Epimerase IS, Isomerase PK, Phospho- ribulokinase 40

41. 3 CO 2 + 9 ATP + 6 NADPH  glyceraldehyde-3-P + 9 ADP + 8 P i + 6 NADP + Glyceraldehyde-3-P may be converted to other CHO: metabolites (e.g., fructose-6-P, glucose-1-P) energy stores (e.g., sucrose, starch) cell wall constituents (e.g., cellulose). Glyceraldehyde-3-P can also be utilized by plant cells as carbon source for synthesis of other compounds such as fatty acids & amino acids. Summary of Calvin Cycle 41

42. There is evidence for multienzyme complexes of Calvin Cycle enzymes within the chloroplast stroma. Positioning of many Calvin Cycle enzymes close to the enzymes that produce their substrates or utilize their reaction products may increase efficiency of the pathway. 42

43. Regulation of Calvin Cycle Regulation prevents the Calvin Cycle from being active in the dark, when it might function in a futile cycle with Glycolysis & Pentose Phosphate Pathway, wasting ATP & NADPH. Light activates, or dark inhibits, the Calvin Cycle (previously called the “dark reaction”) in several ways. 43

44. Light-activated e  transfer is linked to pumping of H + 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 Mg ++ release from thylakoids to stroma. RuBP Carboxylase (in stroma) requires Mg ++ binding to carbamate at the active site. Regulation by Light. 44

45. Some plants synthesize a transition-state inhibitor, carboxyarabinitol-1-phosphate (CA1P), in the dark. RuBP Carboxylase Activase facilitates release of CA1P from RuBP Carboxylase, when it is activated under conditions of light by thioredoxin. 45

46. Thioredoxin is a small protein with a disulfide that is reduced in chloroplasts via light-activated electron transfer. 46

47. During illumination, the thioredoxin disulfide is reduced to a dithiol by ferredoxin, a constituent of the photosynthetic light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase. Reduced thioredoxin activates several Calvin Cycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase Activase, by reducing disulfides in those enzymes to thiols. 47