Photosynthesis Lecture Biology

OUTLINE: ANABOLISM I: Carbohydrates Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation Electron transport Chemiosmotic theory: Phosphorylation energy in a photon pigments HOW Light absorbing complexes-”red-drop expt” Reaction center Photosystems (PS) PSII – oxygen from water splitting PSI – NADPH Fat Catabolism Fatty acid Catabolism 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 Proton Motive Force – ATP Carbon Assimilation – Calvin Cycle Amino-Acid Degradation Dealing with the nitrogen Urea Cycle Dealing with the carbon Stage One – Rubisco Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Seven Families One-carbon (1-C) metabolism; THF, SAM PLP uses Convergence with Fatty acid-odd chain Nucleic Acid & Nucleotide Degradation Nucleic Acids Nucleotides Overview and regulation Calvin cycle connections to biosynthesis C4 versus C3 plants Kornberg cycle - glyoxylate Salvage pathway Degradation of purines Degradation of pyrimidines –1.7 > hn –0.32 V +P 700 (+0.4) ⊝ (–0.4) DEo for Mito (–1.14V) PS-I ⟿ (+1.45) ⟿ > > > > > > > > > > hn +P 680 ⧛ ⧛ ⧛ ⧛ ⧛ ⧛ (+0.37) > > > > > > > > > > 2x PC 2x Fd Need >1.8 V >> >> >> > ⟿ ⟿ ⟿ PQ (+0.08) Cytochrome b6f Complex ⧛ ⧛ ⧛ ⧛ ⧛ ⧛ ⧛ NADP+ NADPH QA (–0.05) ⟿ NADH Reductase A0 (–1.4) > > > > > > > > > > > > > > Photosynthesis PS-II +1.6 +0.82 V Note: to get ½O2 you need 2 photons for EACH PS, or 4 photons. You need 8 for each dioxygen ⊕ 1 Photosynthesis In Plants, the Two Reaction Centers Work Together [Not scaled right] Cyclic Photosynthesis Can Be Utilized to Generate Excess ATP This explains the energetics, but what about the stoichiometry? Photosynthesis 4 photons 1 O2 in 4 photons pHin ≈ 4-5 4 e– + 4 H+ 8 H+ 4 4 e– 4 H+ pHout ≈ 8 ⊕⊕⊕⊕ D! = –0.05V 2 PQH2 ⊝⊝⊝⊝ 4 e– out 4 Organization of 2 NADPH Photosynthetic Machinery [H ] in the Thylakoid Membrane DG = RTln ____ + zF D! ’ + out [H+]in H+in ⇌ H+out = RT2.3(pHin – pHout) + (+1)(96480)(–0.05) = 5.9(–3) – 4.8 kJ/mol Enough for ~8 ATP = –17.7 – 4.8 kJ/mol = –22.5 kJ/mol per proton per 12 H+ = 12 x –22.5 kJ/mol = –270 kJ/mol 2 Photosynthesis pHin ≈ 4-5 Thylakoid Lumen pHout ≈ 8 Stroma DG’ = RTln [H+]out ____ [H+]in + zF D! = –17.7 kJ/mol – 4.8 kJ/mol pH gradient Membrane potential Photosynthesis Net equation: 2 H2O + 2 NADP+ + 8 photons → O2 + 2 NADPH + 2 H+ CO2 + H2O ⇌ CH2O + O2 DE’o = 1.8 V 4 photons 4 photons 2 NADP+ 2 4 4 NADPH +2H+ 2 2 DE’o = 1.14 V 2 4 DE’o = 1.5 V ~3 8 4 Light-Induced Redox Reactions Cause Acidification of Lumen 3 Photosynthesis Light Energy is Converted to ATP • In contrast to oxidative phosphorylation, protons are pumped into the lumen, building up a large concentration gradient quickly in the small space. • The proton-motive force across the thylakoid membrane drives the synthesis of ATP. • The number of c subunits in F0-rings is larger in chloroplasts (~12-14), so synthesis of ATP costs more H+ per ATP (~4-5) than in oxidative phosphorylation. • For the same light, the ratio of ATPto-NADPH synthesized is 3:2 Photosynthesis Summary of light reactions: • Photosynthetic organisms capture energy from light at a variety of wavelengths using accessory pigments and funnel it via excitons to reaction centers: P680 & P700. • In plants, electrons are freed from H2O, which generates O2 and H+. The electrons popping out of the reaction centers reduce PQ, which moves them through the photosynthetic electron-transport chain (cytb6f). • Per oxygen (O2) produced from 4 photons in PS-II, 12 H+ are mobilized into the lumen from the 2H2O and cytb6f (Q-cycle). This produces a proton-motive force sufficient for 3 ATPs • The final electron acceptor for linear photosynthesis is NADP+, which requires two electrons for conversion to NADPH. Per 8 photons (4 each to PS-I & PS-II), 2 NADPH are produced. • When ATP stores are low, the electrons can be transported to cytochrome b6f and cycled through the second half of photosynthesis. This cyclic photosynthesis resulting in an increased proton gradient and more ATP. 4 ANABOLISM I Carbohydrates Photosynthesis and Carbohydrate Synthesis in Plants Photosynthesis Assimilation of CO2 by Plants • Taking CO2 into biological, more reduced, carbon intermediates is CO2 assimilation. • Question was: What is the first compound made by plants when they assimilated CO2? Calvin and Benson used the 14 C radioisotope to determine the sequence of reactions in CO2 fixation. They exposed Chlorella to 14 CO2, then extracted the organic compounds and separated them by paper chromatography. 3 min 30 min 5 Photosynthesis • Plant cells: can also make 3-C intermediates for further synthesis – made from CO2, H2O, plus ATP and NADPH from photosynthesis • 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, which is rapidly converted to glyceraldehyde 3-phosphate (GA3P) & dihydroxyacetone phosphate (DHAP), which are important intermediates for all other compounds. • The net result is the reduction of CO2 with the NADPH that was generated in the light reactions of photosynthesis. 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) 6 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 7 Photosynthesis First Stage of Calvin Cycle CO2 Fixation Is Catalyzed by Rubisco • Most plentiful, most important, enzyme on Earth (4 tons/yr) – Low turnover # of kcat = 3 s–1 at 25 C° – means a LOT of the enzyme needed! – 50% of plant enzymes are rubisco. • Large (560 KDa; a8b8)(L=56 kDa, S=14 kDa); Mg2+– dependent enzyme; activated by CO2 carbamylation • pH dependence is sharp, optimal activity at pH=8 • Catalyzes the reaction: – ribulose 1,5-bisphosphate + CO2 à 2 3-phosphoglycerate – DG°’ = –35.1 kcal/mol 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) 8 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 9 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 10