Oxidative Phosphorylation versus. Photophosphorylation

Transcription

1 Photosynthesis

2 Oxidative Phosphorylation versus Photophosphorylation Oxidative Phosphorylation Electrons from the reduced cofactors NADH and FADH 2 are passed to proteins in the respiratory chain. In eukaryotes, oxygen is the ultimate electron acceptor for these electrons. Energy of oxidation is used to phosphorylate ADP. Photophosphorylation Water is the source of electrons that are passed via a chain of protein transporters to the ultimate electron acceptor, NADP +. Light energy is used to separate charges on chlorophyll to generate NADPH and phosphorylate ADP. Oxygen is the byproduct of water oxidation.

3 Oxidative Phosphorylation versus Photophosphorylation Oxidative Phosphorylation Photophosphorylation

4 Light Energy Is Converted to ATP in Plant Chloroplasts

5 Assimilation of CO 2 by Plants Animal cells: use 3-C intermediates (pyruvate, lactate, etc.) for synthesis of carbohydrates, amino acids, and lipids, but the 3-C intermediates must be made from degradation of a larger molecule Plant cells: can also make 3-C intermediates for further synthesis Glyceraldehyde 3-phosphate (G3P) is an important one. made from CO 2, H 2 O, plus ATP and NADPH from photosynthesis Making CO 2 into intermediates is CO 2 assimilation.

6 Assimilation of CO 2 into Biomass

7 CO 2 Assimilation Occurs in Plastids Organelles in plants and algae includes chloroplasts (for photosynthesis), amyloplasts (for starch storage), and so on Enclosed by a double membrane As in mitochondria, the inner membrane is impermeable to ions and polar molecules. Have their own small genome again, like mitochondria

8 Amyloplasts Filled with Starch (and Stained with Iodine)

9 CO 2 Assimilation It occurs in the stroma of chloroplasts via a cyclic process known as the Calvin cycle. Key intermediate ribulose 1,5-bisphosphate is constantly regenerated using energy of ATP. It produces 3-phosphoglycerate, then glyceraldehyde 3-phosphate (G3P) in eq with dihydroxyacetone phosphate (DHAP) The net result is the reduction of CO 2 with NADPH that was generated in the light reactions of photosynthesis.

10 The Three Stages of the Calvin Cycle of CO 2 Assimilation

11 Three Stages of the Calvin Cycle 1. CO 2 fixation: 3 ribulose 1,5-bisphosphate + 3 CO phosphoglycerate catalyzed by rubisco 2. 3-phosphoglycerate reduced to glyceraldehyde 3- phosphate using NADPH and ATP from photosynthesis Catalyzed by chloroplast isoforms of 3-phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase from glycolysis 3. Regeneration of ribulose 1,5-bisphosphate Overall: 3 CO NADPH + 5 H 2 O + 9 ATP glyceraldehyde 3- phosphate (G3P) + 2 H NADP ADP + 8 P i

12 The First Stage (CO 2 Fixation) Is Catalyzed by Rubisco Most plentiful enzyme on Earth Rubisco = ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo) Large Mg 2+ dependent enzyme Catalyzes the reaction: ribulose 1,5-bisphosphate + CO phosphoglycerate

13 Rubisco Exists in Two Major Forms and Is Abundant Form 1: plants, algae, cyanobacteria 8 large catalytic subunits (encoded by plastid genome) + 8 small subunits (encoded by nucleus) Form II: photosynthetic bacteria only only 2 catalytic subunits; resemble plant subunits 50% of plant enzymes are rubisco.

14 1. Creation of Enediolate Intermediate Mg 2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate forms enediolate intermediate

15 2. Nucleophilic Attack to Create -Keto Acid Intermediate Mg 2+ polarizes CO 2 to accept nucleophilic attack from enediolate, forming 6-C -keto acid intermediate

16 3. Hydroxylation at C-3 Hydroxylation at C-3 of ribulose-1,5- bisphosphate by water

17 4. Cleavage to Yield First 3-Phosphoglycerate

18 5. Protonation and Release of Second 3-Phosphoglycerate Deprotonation of Lys 175 is easily reversed at physiological ph, allowing complete reset of the active site.

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

20 Rubisco Is Activated via Covalent Modification of the Active Site Lysine Rubisco is highly regulated (as first step of CO 2 assimilation). Inactive until Lys 201 is carbamoylated by CO 2 (Lys 201 inaccessible). Rubisco activase (an enzyme sometimes triggered by light) changes rubisco conformation to expose Lys 201. Rubisco activase requires ATP.

21 Rubisco Is also Inhibited by a Noctural Inhibitor 2-carboxyarabinitol 1-phosphate inhibits carbamoylated rubisco. transition state analog of -keto acid intermediate synthesized in the dark in some plants

22 Stage 2: 3-phosphoglycerate Reduced to Glyceraldehyde 3-Phosphate Six 3-phosphoglycerate + 6 ATP + 6 NADPH + 6 H + 6 glyceraldehyde 3-phosphate + 6 ADP + 6 NADP P i Requires NADPH and ATP from photosynthesis Part of gluconeogenesis (except NADPH used rather than NADH) uses enzymes 3-phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase Driven forward by high concentration of NADPH and ATP in the chloroplast stroma

23 Stage 3: Regeneration of Ribulose 1,5- Bisphosphate Six glyceraldehyde-3-phosphate are produced during the Calvin cycle. Ribulose-1,5-bisphosphate must be regenerated in order to restart the cycle. 5/6 glyceraldehyde-3-phosphate products are regenerated into ribulose-1,5-bisphosphate. 1/6 glyceraldehyde-3-phosphate products are pulled out of the cycle and used for building carbohydrates.

24 Stage 3: Fate of One Glyceraldehyde-3- Phosphate in Carbohydrate Biosynthesis Glyceraldehyde-3- phosphate is an intermediate in glycolysis and gluconeogenesis and can be used in anabolic processes to create starch and sucrose as well as catabolic processes to create pyruvate and acetyl-coa.

25 Stage 3: Regeneration Steps for 5/6 of Glyceraldehyde-3-Phosphate Products of the Calvin Cycle 1. G3P converted to DHAP by triose phosphate isomerase 2. G3P and DHAP converted to fructose 6- phosphate (F6, 6C) via aldolase and fructose 1,6- bisphosphatase 3. 2C removed from F6P to yield erythrose 4- phosphate (E4P, 4C); catalyzed by transketolase 4. Transketolase transfers 2C from step 3 to G3P, yielding xylulose 5-phosphate (Xu5P, 5C)

26 5. E4P and DHAP combine to sedoheptulose 1,7- bisphosphate (S1,7BP; 7C) by aldolase. 6. Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7-phosphate (S7P) via sedoheptulose 1,7-bisphosphatase This enzyme is unique to plants. 7. S7P has 2C removed by transketolase to yield ribose 5-phosphate (R5P); 2C on transketolase transferred to G3P to yield another Xu5P

27 8. R5P converted into ribulose 5-phosphate (Ru5P) by phosphopentose isomerase 9. Also, from step 7, Xu5P converted to RuP by phosphopentose epimerase 10. Finally, RuP phosphorylated to ribulose 1,5- bisphosphate by phosphoribulokinase This enzyme is also unique to plants.

28 Stage 3: Regeneration Steps for 5/6 of Glyceraldehyde- 3-phosphate Products of the Calvin Cycle (Overview)

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31 Stage 3: Addition of Phosphate from ATP to Ribulose-5- Phosphate Commits Product to Calvin Cycle

32 Transketolase Uses Thiamine Pyrophosphate as the Cofactor Contains thiazolium anion for nucleophilic attack on carbonyls Also used by: pyruvate dehydrogenase in acetyl CoA formation pyruvate decarboxylase in ethanol metabolism -ketoglutarate dehydrogenase in CAC transketolase in pentose phosphate pathway

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34 Stoichiometry of CO 2 Assimilation in the Calvin Cycle Fixation of three CO 2 molecules yields one glyceraldehyde 3- phosphate for use in anabolic processes. Nine ATP molecules and six NADPH molecules are consumed.

35 Fate of the P i from ATP Hydrolysis in Stage 2 Eight of the nine P i combine with ADP to regenerate ATP. The ninth is incorporated into G3P. requires that P i be transferred from cytosol uses a special P i -triose antiport

36 The P i -Triose Phosphate Antiporter Is Needed for Sucrose Synthesis Unlike starch (made in the chloroplast stroma), sucrose is made in the cytosol! used for transport to distant plant tissues But the inner chloroplast membrane is impermeable to phosphorylated compounds. So, the antiporter exchanges G3P or DHAP for one phosphate (P i ), thus sending the triose phosphates to the cytosol for sucrose synthesis.

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38 Photosynthesis: From Light and CO 2 to Glyceraldehyde 3-Phosphate The assembly of one molecule of glyceraldehyde 3- phosphate requires the capture of roughly 24 photons. H + move from the stroma to the thylakoid creates alkaline conditions in the stroma Accompanied by Mg 2+ transport from thylakoid to stroma Enzyme for photosynthesis and CO 2 assimilation more active in the alkaline, high [Mg 2+ ] conditions of the stroma