Photosynthesis Lecture Note

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 e– hole! ⊕ ⊝ Plastocyanin (E’o is +0.37) e– gained! Shuttling the electron quickly out: A1 (QK) A1 (QK) PSII PSI Photosystem I creates the strongest biological reductant known: A0– Ferredoxin (E’o is –0.4) Photosystem I (P700) (E’o is ~–1.4) 1 Photosynthesis NADPH is produced by the NADPH Reductase in the Stroma Photosystem I created reduced ferrodoxin (Fd) (E’o is –0.4) Fd is a 1-e– carrier, so 2 Fd transfer their electrons, one at a time, to the FAD cofactor in the reductase. The fully reduced FADH2 then transfers 2e– to NADP+ to make each NADPH. Photosynthesis In Plants, the Two Reaction Centers Work Together Cyclic Photosynthesis Can Be Utilized to Generate Excess ATP This explains the energetics, but what about the stoichiometry? 2 Photosynthesis 4 photons 1 O2 4 photons pHin ≈ 4-5 4 e– + 4 H+ 8 H+ 4 4 e– pHout ≈ 8 4 e– Organization of Photosynthetic Machinery in the Thylakoid Membrane 4 2 NADPH DG’ = RTln [H+]out ____ [H+]in + zF D! = RT2.3(pHin – pHout) + (+1)(96480)(+0.05) = 5.9(3) + 4.8 kJ/mol = 17.7 + 4.8 kJ/mol = 22.5 kJ/mol per proton per 12 H+ = 12x22.5 kJ/mol = 270 kJ/mol 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 3 Photosynthesis Net equation: 2 H2O + 2 NADP+ + 8 photons → O2 + 2 NADPH + 2 H+ CO2 + H2O ⇌ CH2O + O2 4 photons 4 photons 2 DE’o = 1.5 V 2 4 4 NADP+ +2H+ 2 2 NADPH DE’o = 1.14 V DE’o = 1.5 V 2 4 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 F0-c-rings are larger ~14 in chloroplasts, 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 4 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+. These electrons are excited and moved through the photosynthetic electron-transport chain (cytb6f). • Per oxygen produced from 4 photons in PS-II, 12 H+ are mobilized into the lumen from the H2O and cytb6f. • 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. ANABOLISM I Carbohydrates Photosynthesis and Carbohydrate Synthesis in Plants 5 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 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 NADPH that was generated in the light reactions of photosynthesis. 6 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) Overall: 6 CO2 + 12 NADPH + 10 H2O + 18 ATP → 2 glyceraldehyde 3phosphate (GA3P) + 4 H+ + 12 NADP+ + 18 ADP + 16 Pi 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 7 Photosynthesis 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 8 Photosynthesis First Stage of Calvin Cycle 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) Photosynthesis First Stage of Calvin Cycle ℗ Role of Mg2+ • 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 :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. 9 Photosynthesis First Stage of Calvin Cycle ℗ Role of Mg2+ • 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 :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. 10