Photosynthesis - Light Absorbing Complexes Red-drop

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation ANABOLISM I: Carbohydrates PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions Exam-1 material Exam-2 material energy in a photon pigments HOW Light absorbing complexes-”red-drop expt” Reaction center Photosystems (PS) PSII – oxygen from water splitting Cytochrome b6f Complex 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 Urea Cycle Dealing with the carbon Know mechanism Seven Families One-carbon (1-C) metabolism; THF, SAM PLP uses Convergence with Fatty acid-odd chain Nucleic Acid and Nucleotide Degradation Nucleic Acids Nucleotides Salvage pathway Degradation of purines Degradation of pyrimidines Proton Motive Force – ATP Overview of light reactions Carbon Assimilation – Calvin Cycle Stage One – Rubisco (carbox., oxgen., Stage Two – making sugar Stage Three - remaking Ru 1,5P2 glycolate) Overview and regulation Calvin cycle connections to biosynthesis C4 versus C3 plants Kornberg cycle - glyoxylate Photosynthesis First Stage of Calvin Cycle b(S) a(L) b(S) a(L) Structure of Rubisco (Form II) There are two distinct forms of rubisco. Form I is found in vascular plants, algae, and cyanobacteria; Form II is confined to certain photosynthetic bacteria. Structure of Rubisco (Form I) 1 Photosynthesis Role of Mg2+ First Stage of Calvin Cycle ℗ • Notice that Mg2+ is held by negatively charged side chains of: • Glu, Asp, and carbamoylated Lys • Mg2+ brings together the reactants in a correct orientation and stabilizes the negative charge that forms upon the nucleophilic attack by the enediolate on CO2. ℗ endiol intermediate b-ketoacid intermediate :His-294 CO2 carbamoylation • Rubisco is inactive until Lys201 is carbamoylated by CO2. But, in the apo-enzyme Lys201 inaccessible). • Rubisco activase (an enzyme sometimes triggered by light) changes Rubisco conformation, in an ATP-dependent manner, to expose Lys201. Photosynthesis Rubisco Carboxylase Mechanism― Overview – Mg2+ facilitates interaction of carbamoylated Lys to ribulose 1,5-bisphosphate à forms enediolate intermediate O C O Hydroxylation at C-3 of ribulose-1,5-bisphosphate by water Recall another C-C cleavage of a b-keto acid? O C O O C O C3-tetrahedral intermediate 2 • The first catalytic intermediate is the endiolate RUBISCO formation (Ribulose 1,5-bisphosphate • Not covered in text is the tautomerization to convert carboxylase/oxygenase) the C2-endiolate to the C3-endiolate • Immediately after CO2 binding, the nucleophilic attack Carboxylase by the C2-enol forms the b-keto acid intermediate Mechanism H-carbamate Tautomerization & CO2 binding :Carbamate This graphic shows how close the carbamate is to both the C2 and C3 carbons endiolate :His294 <2 Å Mg⊕⊕ carbamate b-keto acid endiolate 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 Very temperature dependent • The reactive nucleophile in the rubisco reaction is the electron-rich enediolate 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 3 RUBISCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase) Carboxylase Mechanism Here are the two overall reactions RUBISCO catalyzes Both RUBISCO reactions require activation by carbamoylation 2-phospho- H2C O P • The (C2) carbon-(C3)carbon bond cleavage RUBISCO is initiated by water attack of the carbonyl (Ribulose 1,5-bisphosphate carboxylase/oxygenase) Oxygenase Mechanism endiolate b-keto acid Gem-diol :Lys175 – 2-phosphglycolate :Lys175 b-keto peroxide 4 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 2 1 Serine OHMe transferase *Don’t confuse with the glyOXYlate Cycle 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 5 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 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 6 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 10 enzymes (6) G3P 4 2 TPI DHAP ⇌ Aldolase 5 3 ⇌ 3 (12) PGI 1 ⇌ Fru6P 1 (12) (12) Fructose-1,6- Fru 1,6P2 Pi bisphosphatase 2 (6) 10/12 glyceraldehyde-3-phosphate products (30 carbons) MUST be used to regenerate 6 ribulose-1,5bisphosphate (30 carbons). 8 (2) for Stage 3 3 Rubisco (10) 12-C ’ s 6-C ’s 3 (6) (6) DHAP 2 The Three Stages of the Calvin Cycle of CO2 Assimilation chloroplast isoforms (12) – 3-phosphoglycerate kinase (3PGK) (12) – glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (12) Glc6P (12) • 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. 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* 12àuse 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 2 GA3P left *the other 4 go to make Glc(1) and DHAP(2) 7 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 Photosynthesis 2 2 Stage 3: Summary 2 2 2 Finally, RuP phosphorylated to ribulose 1,5-bisphosphate by phosphoribulokinase – This enzyme is also unique to plants and regulated by NADPH 2 2 ribulose-5phosphate epim erase 2 2 6 ribulose-5phosphate epim erase 2 2 2 Regeneration Steps for 10/12 of Glyceraldehyde-3phosphate Products of the Calvin Cycle (Overview) 6 Stage 3: Addition of Phosphate from ATP to Ribulose-5-Phosphate Commits Product to Calvin Cycle 8 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: Aldose product e.g., erythrose 4-phosphate e.g., fructose 6-phosphate – pyruvate dehydrogenase in acetyl CoA formation – pyruvate decarboxylase in ethanol metabolism – Glycine cleavage enzyme in glycolate – a-ketoglutarate dehydrogenase in CAC – Branched-chain amino acid dehydrogenase – transketolase in pentose phosphate pathway e.g., glyceraldehyde 3-phosphate e.g., xyulose 5-phosphate 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) 9 Photosynthesis The Three Stages of the Calvin Cycle of CO2 Assimilation 10 enzymes (6) (6) (6) Rubisco (10) (6) (2) (12) (12) – – 3-phosphoglycerate kinase (3PGK) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (12) chloroplast isoforms (12) (12) (12) (12) *Ribulose 1,5-bisphosphate carboxylase/oxygenase (also RuBisCo) 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) 6RuBPà12GA3P DG°’ = –56 kcal/mol Stage 2 (end) 2GA3Pà1Glc DG°’ = –10 kcal/mol Stage 3 Stage 3 10GA3Pà 6Ru5P DG°’ = –38 kJ/mol NET: –104/6 = –17.3 per CO2 1 Pi Glucose 1