1. Light Reactions of Photosynthesis 2 H 2 O + 2 NADP + + 8 photons → O 2 + 2 NADPH + 2 H + ANIMATION

2. Integration of photosystems I and II in chloroplasts. The "Z scheme“ evolved by combining 2 bacterial RCs. Reaction center chlorophylls lie close to the exiton acceptor preventing internal conversion (fluorescence). This fixed orientation mimics the solid state. Resembles that of green sSulfur bacteria Resembles that of purple bacteria. analogous to cyt c

3. Cyclic vs. Nonclyclic PS animation

4. Photosystem II of the cyanobacterium Synechococcus elongates All electron carriers are bound to a nearly symmetric dimer. Participants are positioned like the bacterial RC in purple bacteria.

5. Light Reactions of Photosynthesis PS II 4 P680 + 4 H + + 2 PQ B + 4 photons → 4 P680 + + 2 PQ B H 2

6. The supramolecular complex of PSI and its associated antenna chlorophylls

7. Light Reactions of Photosynthesis PS I 4 P700 + 2 H + + 2 NADP + + 4 photons → 4 P700 + + 2 NADPH

8. Electron and proton flow through the cytochrome b6f complex Plastoquinol (PQH2) formed in PSII is oxidized by the cytochrome b6f complex in a series of steps like those of the Q cycle in the cytochrome Complex III of mitochondria. One electron from PQH2 passes to the Fe-S center of the Rieske protein, the other to heme bL of cytochrome b6. The net effect is passage of electrons from PQH2 to the soluble protein plastocyanin, which carries them to PSI. Heme groups f for frons

9. Localization of PSI and PSII in thylakoid membranes

10. Non cyclic electron flow (PSI + PS II) produces a proton gradient + NADPH Cyclic electron flow (PSI) produces a proton gradient only Calvin cycle requires ATP and NADPH in a ratio of 3:2 The 2 PS are physically separated to prevent exitons from leaving P680 and transferring to P700. Why would this happen? –(longer wavelength, lower energy)

11. Localization of PSI and PSII in thylakoid membranes. PSII is present almost exclusively in the appressed regions (granal lamellae [stacks], in which several membranes are in contact), and PSI almost exclusively in nonappressed (stromal lamellae) regions, exposed to the stroma. LHCII is the "adhesive" that holds appressed lamellae together.

12. Accumulation of plastoquinol stimulates a protein kinase that phosphorylates a Thr residue in the hydrophobic domain of LHCII, which reduces its affinity for the neighboring thylakoid membrane and converts appressed regions to nonappressed regions (state 2). A specific protein phosphatase reverses this regulatory phosphorylation when the [PQ]/[PQH2] ratio increases. Balancing of electron flow in PSI and PSII by state transition noncyclic cyclic

13. Water-splitting activity of the oxygen-evolving complex Water splitting complex passes 4 electrons, 1 at a time, back to P680 +. The electrons lost from the multinuclear Mn center pass one at a time to an oxidized Tyr residue in a PSII protein, then to P680+ H + released into lumen.

14. Light-Induced Redox Reactions and Electron Transfer Cause Acidification of Lumen Because the volume of the lumen is small, a few hydrogen ions dramatically change the pH: lumen = pH 5, stroma pH = 8!!! The proton-motive force across the thylakoid membrane drives the synthesis of ATP… sound familiar?? Thylakoid membrane is impermeable to hydrogen ions. Reaction centers, electron carriers and ATP synthases are located in this membrane Uncouplers decouple light absorption from ATP synthesis.

15. In vitro ATP synthesis Incubate chloroplasts in pH 4 buffer in the dark. Buffer slowly entered thylakoids lowering the pH to 4. Add ADP and P i and suddenly increase the pH to 8. (How would you do this?) IN THE DARK. !!!! ATP produced!!!

16. Proton and electron circuits in thylakoids.

17. Flow of Protons: Mitochondria, Chloroplasts, Bacteria According to endosymbiotic theory, mitochondria and chloroplasts arose from entrapped bacteria Bacterial cytosol became mitochondrial matrix and chloroplast stroma

18. Comparison of the topology of proton movement and ATP synthase orientation in the membranes of mitochondria, chloroplasts, and the bacterium E. coli.

19. Dual roles of cytochrome b6f and cytochrome c6 in cyanobacteria reflect evolutionary origins. Cyanobacteria use cytochrome b6f, cytochrome c6, and plastoquinone for both oxidative phosphorylation and photophosphorylation.

20. CHAPTER 20 Carbohydrate Biosynthesis in Plants and Bacteria –CO 2 assimilation in photosynthetic organisms –Photorespiration in C 3 plants –Avoiding photorespiration in C 4 plants Key topics:

21. Introduction to Anabolic Pathways Anabolism: how to build biomolecules Plants are extremely versatile in biosynthesis –Can build organic compounds from CO 2 –Can use energy of sunlight to support biosynthesis –Can adopt to a variety of environmental situations

22. Plant versatility Autotrophic Nonmobile/motile CHO synthesis occurs in plastids Plants synthesize thick cell walls exterior to the cell containing the bulk of the cell’s CHO—how do they do this???

23. Assimilation of CO 2 by Plants

24. Plants and Photosynthetic Microorganisms Support the Life of Animals and Fungi Plants capture the energy from the ultimate energy source and make it available via carbohydrates to animals and fungi

25. Photosynthetic organisms use the energy of sunlight to manufacture glucose and other organic products, which heterotrophic cells use as energy and carbon sources.

26. Biological reproduction occurs with near-perfect fidelity (although no 2 zebras have exactly the same stripes!). Zebras are herivores.

27. CO 2 Assimilation Occurs in Plastids Self-reproducing organelles found in plants and algae Enclosed by a double membrane Have their own small genome Most plastid proteins are encoded in the nuclear DNA The inner membrane is impermeable to ions such as H +, and to polar and charged molecules

28. Amyloplasts filled with starch (dark granules) are stained with iodine in this section of Ranunculus (buttercups) root cells. Amyloplasts are pastids without the internal membrane or pigments.

29. Origin and Differentiation of Plastids Plastids were acquired during evolution by early eukaryotes via endosymbiosis of photosynthetic cyanobacteria Plastids reproduce asexually via binary fission The undifferentiated protoplastids in plants can differentiate into several types, each with a distinct function –Chloroplasts for photosynthesis –Amyloplasts for starch storage –Chromoplasts for pigment storage –Elaioplasts for lipid storage –Proteinoplasts for protein storage

30. Proplastids in nonphotosynthetic tissues (such as root) give rise to amyloplasts, which contain large quantities of starch. All plant cells have plastids, and these organelles are the site of other important processes, including the synthesis of essential amino acids, thiamine, pyridoxal phosphate, flavins, and vitamins A, C, E, and K. internal membranes lost

31. CO 2 Assimilation The assimilation of carbon dioxide occurs in the stroma of chloroplasts via a cyclic process known as the Calvin cycle The key intermediate, ribulose 1,5-bisphosphate is constantly regenerated using energy of ATP The key enzyme, ribulose 1,5-bisphosphate carboxylase / oxygenase (Rubisco), is probably the most abundant protein on Earth The net result is the reduction of CO 2 with NADPH that was generated in the light reactions of photosynthesis

32. Early studies of the Calvin cycle Design an experiment to discover the pathway for carbon assimilation First intermediate recognized was 3-PGA Search for a 2 carbon acceptor---FAILURE Actual acceptor….

33. CO 2 Assimilation The assimilation of carbon dioxide occurs in the stroma of chloroplasts via a cyclic process known as the Calvin cycle The key intermediate, ribulose 1,5-bisphosphate is constantly regenerated using energy of ATP The key enzyme, ribulose 1,5-bisphosphate carboxylase / oxygenase (Rubisco), is probably the most abundant protein on Earth The net result is the reduction of CO 2 with NADPH that was generated in the light reactions of photosynthesis

34. The Calvin Cycle

35. The Structure and Function of Rubisco Rubisco is a large Mg ++ -containing enzyme that makes a new carbon-carbon bond using CO 2 as a substrate

36. Structure of ribulose 1,5-bisphosphate carboxylase (rubisco). Ribbon model of form II rubisco from the bacterium Rhodospirillum rubrum. The subunits are in gray and blue. A Lys residue at the active site that is carboxylated to a carbamate in the active enzyme is shown in red. The substrate, ribulose 1,5-bisphosphate, is yellow; Mg2+ is green.

37. Central role of Mg2+ in the catalytic mechanism of rubisco. Mg2+ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on Lys201; two in the carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of the substrate, ribulose 1,5- bisphosphate; and one in the other substrate, CO 2.

38. First stage of CO 2 assimilation: rubisco's carboxylase activity.

39. Another ene-diol intermediate!!!

44. Catalytic Role of Mg ++ in Rubisco’s Carboxylase Activity Notice that Mg ++ is held by negatively charged side chains of glutamate, aspartate, and carbamoylated lysine Mg ++ brings together the reactants in a correct orientation, and stabilizes the negative charge that forms upon the nucleophilic attack of enediolate to CO 2

46. Rubisco is Activated via Covalent Modification of the Active Site Lysine

48. Synthesis of Glyceraldehyde-3 Phosphate (First Stage) Three rounds of the Calvin cycle fix three CO 2 molecules and produce one molecule of 3- phosphoglycerate

50. Fate of Glyceraldehyde 3- phosphate (Second Stage) Converted to starch in the chloroplast Converted to sucrose for export Recycled to ribulose 1,5-bisphosphate

52. Interconversion of Triose Phosphates and Pentose Phosphates This is how ribulose 1,5-bisphosphate is regenerated in the third stage of the Calvin cycle

53. Sugar interconversions

56. Transketolase Reactions

59. Transketolase Uses Thiamine Pyrophosphate as the Cofactor

61. Stoichiometry and Energy Cost of CO 2 Assimilation Fixation of three CO 2 molecules yields one glyceraldehyde 3-phosphate Nine ATP molecules and six NADPH molecules are consumed

63. Photosynthesis: From Light and CO 2 to Glyceraldehyde 3- phosphate The photosynthesis of one molecule of glyceraldehyde 3-phosphate requires the capture of roughly 24 photons

64. ATP and NADPH produced by the light reactions are essential substrates for the reduction of CO 2

65. Enzymes in the Calvin Cycle are Regulated by Light Target enzymes are –ribulose 5-phosphate kinase, –fructose 1,6-bisphosphatase, –seduloheptose 1,7-bisphosphatase, and –glyceraldehyde 3-phosphate dehydrogenase

66. Light activation of several enzymes of the Calvin cycle

67. Photorespiration So far, we saw that plants oxidize water to O 2 and reduce CO 2 to carbohydrates during the photosynthesis Plants also have mitochondria where usual respiration with consumption of O 2 occurs in the dark In addition, a wasteful side reaction catalyzed by Rubisco occurs in mitochondria This reaction consumes oxygen and is called photorespiration; unlike mitochondrial respiration, this process does not yield energy

68. Oxygenase Activity of Rubisco The reactive nucleophile in the Rubisco reaction is the electron-rich enediol form of ribulose 1,5- bisphosphate The active site meant for CO 2 also accommodates O 2 Mg ++ also stabilizes the hydroperoxy anion that forms by electron transfer from the enediol to oxygen

70. Salvage of 2- Phosphoglycerate Complex ATP-consuming process for the recovery of C 2 fragments from the photorespiration Requires oxidation of glycolate with molecular oxygen in peroxisomes, and formation of H 2 O 2 Involves a loss of a carbon as CO 2 by mitochondrial decarboxylation of glycine

71. Glycolate pathway

72. Rubisco in C 3 Plants Cannot Avoid Oxygen Plants that assimilate dissolved CO 2 in the mesophyll of the leaf into three-carbon 3- phosphoglycerate are called the C 3 plants Our atmosphere contains about 21% of oxygen and 0.038% of carbon dioxide The dissolved concentrations in pure water are about 260  M O 2 and 11  M CO 2 (at the equilibrium and room temperature) The K m of Rubisco for oxygen is about 350  M

73. Separation of CO 2 capture and the Rubisco Reaction in C 4 Plants Many tropical plants avoid wasteful photorespiration by a physical separation of CO 2 capture and Rubisco activity CO 2 is captured into oxaloacetate (C 4 ) in mesophyll cells CO 2 is transported to bundle-sheath cells where Rubisco is located The local concentration of CO 2 in bundle-sheath cells is much higher than the concentration of O 2

74. Carbon assimilation in C4 plants

75. Chapter 20: Summary ATP and NADPH from light reactions are needed in order to assimilate CO 2 into carbohydrates Assimilations of three CO 2 molecules via the Calvin cycle leads to the formation of one molecule of 3-phosphoglycerate 3-Phosphoglycerate is a precursor for the synthesis of larger carbohydrates such as fructose and starch The key enzyme of the Calvin cycle, Rubisco, fixes carbon dioxide into carbohydrates Low selectivity of Rubisco causes a wasteful incorporation of molecular oxygen in C 3 plants; this is avoided in C 4 plants by increasing the concentration of CO 2 near Rubisco In this chapter, we learned that: