Photosynthesis Pigments

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation ANABOLISM I: Carbohydrates Exam-1 material Exam-2 material PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions energy in a photon pigments HOW Light absorbing complexes-”red-drop experiment” Reaction center Photosystems (PS) PSII – oxygen from water splitting PSI – NADPH Electron transport Chemiosmotic theory: Phosphorylation Fat Catabolism Fatty acid Catabolism Exam-3 material Mobilization from tissues (mostly adipose) Activation of fatty acids Transport; carnitine Oxidation: b-oxidation, 4 steps: Saturated FA Unsaturated FA Odd-chain FA Ketone Bodies Protein Catabolism Digestion, lysosome, Ubiquitin-Proteosome Amino-Acid Degradation Dealing with the nitrogen Proton Motive Force – ATP Overview of light reactions Carbon Assimilation – Calvin Cycle Urea Cycle Dealing with the carbon Seven Families One-carbon (1-C) metabolism; THF, SAM PLP uses Convergence with Fatty acid-odd chain Nucleic Acid & Nucleotide Degradation Nucleic Acids Nucleotides Salvage pathway Degradation of purines Degradation of pyrimidines Stage One – Rubisco Carboxylase Know mechanism Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Overview and regulation Calvin cycle connections to biosynthesis C4 versus C3 plants Kornberg cycle - glyoxylate Photorespiration 1 First Stage of Calvin Cycle Dealing with the oxygenase activity of rubisco 1 The Glycolate Pathway* • Complex ATP-consuming process for the recovery of C2 fragments from photorespiration. 1st Glycolate is made. • Uses three organelles • Loss of C as CO2 by mitochondrial decarboxylation of glycine (see Pyr family) • Two 2-PGs are converted to Ser + CO2. • The Ser is cycled back to the chloroplast to generate one 3-PGA. • Whole cycle costs an ATP per 1 3-PGA converted back 2 2 1 2 1 2 1 1 Serine OHMe transferase 2 1 N5N10CH2 THF decarboxylase* 1 *Don’t confuse with the glyOXYlate Cycle 1 Photosynthesis First Stage of Calvin Cycle Regulation of RUBISCO activity Rubisco is Inhibited by Several Criteria: 1. pH • In the stroma the pH is ~8 when light is on • pH decreases in the dark; rubisco inactive at low pH CO2 (for the carbamoylation of Lys-210; also better at higher pH) NADPH (indirectly through rubisco activase) 2. 3. 4. a “Noctural” Inhibitor • 2-carboxyarabinitol 1-phosphate inhibits carbamoylated Rubisco. – transition state analog of b-keto acid intermediate – synthesized in the dark in some plants Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes (6) (6) (6) Rubisco (10) (6) (2) (12) (12) (12) (12) (12) chloroplast isoforms (12) – 3-phosphoglycerate kinase (3PGK) (12) – glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP → 2 glyceraldehyde 3phosphate (GA3P) + 4 H+ + 12 NADP+ + 18 ADP + 16 Pi 2 Photosynthesis Second Stage of Calvin Cycle: making Glucose 3-PGA Reduced to GA3P Twelve 3-phosphoglycerate + 12 ATP + 12 NADPH + 12 H+ à 12 glyceraldehyde 3-phosphate + 12 ADP + 12 NADP+ + 12 Pi • Requires most of the ATP and NADPH from photosynthesis at ratio of 12:12 • Reverse of the key reactions in glycolysis (except NADPH used rather than NADH) – uses chloroplast isozymes of 3-phosphoglycerate kinase (3-PGK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) • Driven forward by high concentration of NADPH and ATP in the chloroplast stroma; and the pulling of GA3P and Pi by the cycle and ATP synthase ATP NADPH+H+ NADP+ ADP 3-Phosphoglycerate DG°’ = +5.5 kcal/mol –1.8 kcal/mol Photosynthesis Reverse of glycolysis: GA3P converted to DHAP by triose phosphate isomerase (TPI); GA3P and DHAP converted to fructose 1,6-bisphosphate (Fru 1,6-P2) via aldolase, then to Fru 6-P via fructose 1,6bisphosphatase, then Glc 6-P via phosphoglucoisomerase (PGI). Fru6P 12-C ’s 2 2 3 3 4 (6) 10/12 glyceraldehyde-3-phosphate products (30 carbons) MUST be used to regenerate 6 ribulose-1,5bisphosphate (30 carbons). TPI ⇌ Aldolase 5 ⇌ PGI ⇌ Fru6P 1 Rubisco 8 Fructose-1,6- Fru 1,6P2 Pi bisphosphatase 1 G3P (2) DHAP 3 (6) (6) (10) for Stage 3 2 (6) 12-C ’ s 6-C ’s Glc6P +3.7 kcal/mol The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes DHAP 2 Glyceraldehyde 3-phosphate 1,3-Bisphosphoglycerate (12) (12) 3 (12) (12) (12) chloroplast isoforms (12) – 3-phosphoglycerate kinase (3PGK) (12) – glyceraldehyde 3-phosphate dehydrogenase (GAPDH) • 12 glyceraldehyde-3-phosphate are produced during the Calvin cycle in the assimilation of 6 carbons into Glc. • 2/12 glyceraldehyde-3-phosphate products are pulled out of the cycle and used for Glc6P, then to other carbos. 3 Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes (6) (6) (6) Rubisco (10) (6) (2) (12) (12) (12) chloroplast isoforms (12) – 3-phosphoglycerate kinase (3PGK) (12) – glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (12) (12) Photosynthesis Third Stage of Calvin Cycle Stage 3: Regeneration of Ru 1,5-P2 1. We have 3- and 6-carbon compounds and need 5-carbon compounds: put 2 together to make 6 (stop at Fru6P), then can pull 2 carbons from 6 (leaving 4) and with another 3 carbon compound make a 5-carbon sugar. DO THIS TWICE. The 2 Fru6P, plus 2 more GA3P to make 2 5-carbon sugars (Xyl5P). From 4/8 GA3P* 10àuse 6 GA3P 4 GA3P à 2 Fru 6-P 2 GA3P à 2 Xyl 5-P +2 Ery 4-P transketolase 2 2 2 2 *the other 4 go to make Glc(1) and DHAP(2) 4 Mechanism Photosynthesis Transketolase: exchange of 2C from ketose to an aldose acceptor Uses Thiamine Pyrophosphate (TPP) as the Cofactor • Contains thiazolium anion for nucleophilic attack on carbonyls of ketose • Also used by: – pyruvate dehydrogenase in acetyl CoA formation – pyruvate decarboxylase in ethanol metabolism – a-ketoglutarate dehydrogenase in CAC – Branched-chain amino acid dehydrogenase – transketolase in pentose phosphate pathway Photosynthesis Stage 3 sedoheptulose 1,7bisphosphatase aldolase ⇌ 2 2 transketolase ⇌ 2 2 2 2 6 2 Sedoheptulose 1,7-bisphosphate dephosphorylated to sedoheptulose 7phosphate (S7P) via sedoheptulose 1,7-bisphosphatase – This enzyme is unique to plants. 2 from Fru6-P 2 from Ery4-P 2+2= 2 2. We have two 4-carbon Ery 4-P and still have 2 GA3P and 2 DHAPs. Do aldol condensation with DHAP to get Sedoheptulose 1,7-bisphosphate (Sed 1,7-P2). 3. If Sed 1,7-P2 loses the C7-phosphate, as a ketose, we can pull 2 carbons off and put on our remaining 2 ketoses (GA3P) 4 6 6 Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase – This enzyme is also unique to plants and regulated by NADPH 5 Photosynthesis 2 2 Stage 3: Summary 2 2 2 2 2 ribulose-5phosphate epim erase 2 Regeneration Steps for 10/12 of Glyceraldehyde-3phosphate Products of the Calvin Cycle (Overview) 6 6 2 Stage 3: Addition of Phosphate from ATP to Ribulose-5-Phosphate Commits Product to Calvin Cycle ribulose-5phosphate epim erase 2 2 2 Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes (6) (6) (6) Rubisco (10) (6) (2) (12) (12) – – (12) 3-phosphoglycerate kinase (3PGK) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) chloroplast isoforms (12) (12) (12) (12) *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo) 6 Photosynthesis Stoichiometry and Energetics of CO2 Assimilation in the Calvin Cycle & NET Reaction for Photosynthesis: 6 CO2 + 12 NADPH + 12 H+ + 12 H2O + 18 ATP → 1 C6H12O6 + 2 H+ + 12 NADP+ + 18 ADP + 18 Pi + 18 H+ Stage 1/2 This is 3:2 ATP:NADPH! (from 8 photons) 12 Pi For every NADPH, 4 photons needed ∴ need 12x4=48 photons to make 1 Glc Stage 1/2 2 Pi Stage 2 (end) 6Ru5Pà12GA3P DG°’ = –56 kcal/mol Stage 2 (end) 2GA3Pà1Glc DG°’ = –10 kcal/mol Stage 3 Stage 3 10GA3Pà 6Ru5P 1 Pi DG°’ = –38 kJ/mol Glucose NET: –104/6 = –17.3 per CO2 Photosynthesis Photosynthesis: Control by Light and CO2 to Glucose • The assembly of one molecule of glucose requires the capture of roughly 48 photons. – H+ move from the stroma to the thylakoid – creates alkaline conditions in the stroma Accompanied by Mg2+ transport How H+ and Mg2+ • from Movement Assist thylakoid to stroma in Activation of Photosynthesis • Enzyme for photosynthesis and CO2 and CO2 assimilation more active in the alkaline & Assimilation 2+ high [Mg ] conditions of the stroma • This is a source of coordinated regulation by several enzymes: • • • • • Rubisco fructose 1,6-bisphosphatase seduloheptose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenase ribulose 5-phosphate kinase (phosphoribulokinase) How are these regulated by NADPH? Glucose 100 Relative Activity Rubisco, Fru1,6P 2ase, Sed1,7P2ase – Target enzymes regulated by NADPH, Mg2+, and pH, or all three are: 60 30 7 Photosynthesis Photosynthesis: Control from Light and CO2 to Glucose Target enzymes regulated by Fd:NADPH: fructose 1,6-bisphosphatase seduloheptose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenase ribulose 5-phosphate kinase (phosphoribulokinase) Rubisco activase If oxidized (Cys residues in Cys-Cys disulfide form) à enzymes are inactive. In light, photosystem I sends e– to ferredoxin, which sends them to thioredoxin, which donates them to disulfide bonds to reduce them to free Cys. Excess reduced Fd is only available after its use for NADPH production Light Activation of FOUR Calvin Cycle Enzymes via Electron-Driven Reduction of Cys-Cys Crosslinks fructose 1,6-bisphosphatase sedoheptulose 1,7-bisphosphatase glyceraldehyde 3-phosphate dehydrogenase ribulose 5-phosphate kinase Calvin Cycle ACTIVE Photosynthesis Assimilation of CO2 into Biomass cytosol chloroplast cytosol • Plant cells: use 3-C intermediates for further synthesis – Glyceraldehyde 3-phosphate (G3P) is the most important one. – made from CO2, H2O, plus ATP and NADPH from photosynthesis 8 Photosynthesis The C4 Pathway C4 versus C3 Plants; Benefits of C4 Plants: Heat and Drought Resistance • C4 plants (tropical, hot climates) have an earlier step, in different cells, that isolate Rubisco from the air. – In heat, the Rubisco’s oxidase is favored. – C4 plants spatially separate CO2 fixation from rubisco activity, resulting in less reaction of rubisco with oxygen and avoidance of the costly glycolate pathway. • Physical separation of reactions: – – CO2 is captured into oxaloacetate in mesophyll cells of the leaf. Oxaloacetate then passes into bundle-sheath cells where CO2 is released for Rubisco. • The C4 pathway has a higher energy cost than the glycolate cycle on a stoichiometric basis, but its all about the ratio of carboxylase:oxidase. This pathway has overall increased efficiency in heat. • Another pathway to avoid photorespiration was first discovered in Crassulacae (Crassulacean Acid Metabolism (CAM)) in high, dry conditions – Stomata open/close; the CO2 from C4 fixation is stored as malate in vacuoles. Photosynthesis Recall in animals: GlyOXylate Cycle Kornberg Cycle Plants Use Fats and Proteins for Carbohydrate Synthesis: • In the TCA cycle, in the glyoxysome, instead of burning isocitrate, it short circuits TCA, taking isocitrate directly to succinate • The result is the glyoxylate intermediate • Re-cycle this glyoxylate by making malate from more acetyl CoA in a similar reaction as citrate synthase We’ll come back to this later…………. 9 Photosynthesis: Carbon Fixation Summary We learned that: • ATP and NADPH from photosynthesis are needed in order to assimilate CO2 into carbohydrates by Rubisco • This key enzyme of the Calvin cycle fixes carbon dioxide as well as oxygen. • assimilations of six CO2 molecules via the Calvin cycle lead to the formation of one molecule of glucose for use in anabolic reactions • enzymes of Calvin Cycle have common regulation mechanisms via pH, Mg2+, and/or NADPH (Fd) • low selectivity of rubisco causes a wasteful incorporation of molecular oxygen in C3 plants. C4 and CAM plants have evolved separate methods for reducing this waste. • plants can convert acetyl-CoA into carbohydrates via the Kornberg Cycle (glyoxylate cycle) 10