Photosynthesis Carbon Assimilation - Calvin Cycle

ANABOLISM OUTLINE: Overview of Photosynthesis Key experiments: Light causes oxygen, which is from water splitting (Hill) NADPH made (Ochoa) Separate from carbohydrate biosynthesis (Rubin & Kamen) Light Reactions energy in a photon pigments Carbon Assimilation – Calvin Cycle HOW Stage One – Rubisco Light absorbing complexes Carboxylase Reaction center Oxygenase Glycolate cycle Photosystems (PS) Stage Two – making sugar PSI – oxygen from water splitting Stage Three - remaking Ru 1,5P2 PSII – NADPH Proton Motive Force – ATP Overview of light reactions Overview and regulation Calvin cycle connections to biosyn. C4 versus C3 plants Kornberg cycle - glyoxylate Photosynthesis Rubisco Carboxylase Mechanism― Overview Mg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolate intermediate Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water Recall another C-C cleavage of a b-keto acid? C3-tetrahedral intermediate 1 Photosynthesis Rubisco Carboxylase Mechanism― Overview Mg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolate intermediate Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water Recall another C-C cleavage of a b-keto acid? C3-tetrahedral intermediate Photosynthesis First Stage of Calvin Cycle Oxygenase Activity of Rubisco • O2 competes with CO2 for the active site. ~1 in every 3 or 4 turnovers, O2 binds • The reactive nucleophile in the rubisco reaction is the electron-rich enediol form of ribulose 1,5-bisphosphate. • The nucleophile adds to O2 to form 3phosphoglycerate (same as in Calvin cycle) and 2-phosphoglycolate (2-PG). –2-PG is metabolically difficult. –Salvaging its Carbons requires energy –As it is produced in appreciable amounts, an elaborate pathway has been cobbled together. This process is called PHOTORESPIRATION 2 Photosynthesis 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 2 1 Serine OHMe transferase 1 N5N10CH2 THF decarboxylase* 1 *called Glycine Cleavage Enzyme in bacteria 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 3 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 *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo) 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 3-Phosphoglycerate NADPH+H+ NADP+ ADP Glyceraldehyde 3-phosphate 1,3-Bisphosphoglycerate DG°’ = +5.5 kcal/mol –1.8 kcal/mol +3.7 kcal/mol 4 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 *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo) 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, the to Fru 6-P via fructose 1,6bisphosphatase, then Glc 6-P via phosphoglucoisomerase (PGI). The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes (6) (6) (6) Rubisco (10) (6) 10/12 glyceraldehyde-3-phosphate products (30 carbons) MUST be used to regenerate 6 ribulose-1,5bisphosphate (30 carbons). (2) TPI DHAP ⇌ Aldolase ⇌ Fructose-1,6- Fru 1,6P2 Pi bisphosphatase PGI ⇌ Fru6P Glc6P (12) (12) (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. 5 Photosynthesis Third Stage of Calvin Cycle Stage 3: Regeneration of Ru 1,5-P2 1. We have 3-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. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P). From 4 GA3P 10àuse 6 GA3P 4 GA3P à 2 Fru 6-P 2 GA3P à 2 Xyl 5-P transketolase 2 2 2 2 Photosynthesis Third Stage of Calvin Cycle Stage 3: Regeneration of Ru 1,5-P2 1. We have 3-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. It will use 4 GA3P to make 2 Fru6P, then 2 more GA3P to make 2 5-carbon sugars (Xyl5P). From 4 GA3P 10àuse 6 GA3P 4 GA3P à 2 Fru 6-P 2 GA3P à 2 Xyl 5-P transketolase 2 2 2 2 6 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 e.g., fructose 6-phosphate • Also used by: – pyruvate dehydrogenase in acetyl CoA formation – pyruvate decarboxylase in ethanol metabolism – a-ketoglutarate dehydrogenase in CAC – transketolase in pentose phosphate pathway e.g., glyceraldehyde 3-phosphate e.g., xyulose 5-phosphate use 2 GA3P Photosynthesis 42à à0 GA3P left Stage 3 sedoheptulose 1,7bisphosphatase aldolase ⇌ 2 Aldose product e.g., erythrose 4-phosphate 2 transketolase ⇌ 2 2 2 2 2 2. We have two 4-carbon Ery 4-P and still have 4/12 GA3Ps. Do it again; 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) 6 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=4 6 6 Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase – This enzyme is also unique to plants and regulated by NADPH 7 use 2 GA3P Photosynthesis 42à à0 GA3P left 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 4/12 GA3Ps. Do it again; 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 2 2 Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase – This enzyme is also unique to plants and regulated by NADPH Photosynthesis Stage 3: Summary 2 ribulose-5phosphate epim erase 2 Regeneration Steps for 10/12 of Glyceraldehyde-3phosphate Products of the 2 Calvin Cycle (Overview) 6 6 2 ribulose-5phosphate epim erase 2 2 Stage 3: Addition of Phosphate from ATP to Ribulose-5-Phosphate Commits Product to Calvin Cycle 8 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+ Movement Assist thylakoid to stroma in Activation of Photosynthesis • Enzyme for photosynthesis and CO2 and CO2 assimilation more active in the alkaline & Assimilation high [Mg2+] conditions of the stroma • from • 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 9 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 Photosynthesis Assimilation of CO2 into Biomass • 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 10