Chapter 21 Photosynthesis

1. Chapter 21Photosynthesis

2. Outline • What are the general properties of photosynthesis ? • How is solar energy captured by chlorophyll ? • What kinds of photosystems are used to capture light energy ? • What is the molecular architecture of photosynthetic reaction centers ? • What is the quantum yield of photosynthesis ? • How does light drive the synthesis of ATP ? • How is carbon dioxide used to make organic molecules ? • What is photorespiration ?

3. The Sun - Ultimate Energy 1.5 x 1022 kJ of sunlight energy falls on the earth each day • Biological energy comes from the sun. • 1% is absorbed by photosynthetic organisms and transformed into chemical energy. • ~5% uv, ~45% vis, ~50% IR • 6 CO2 + 6 H2O + energy  C6H12O6 + 6 O2. • 1011 tons of CO2 are fixed globally per year.

4. 21.1 What Are the General Properties of Photosynthesis? General Aspects • Photosynthesis occurs in thylakoid membranes of chloroplasts - structures involving paired folds (lamellae) that stack to form "grana". • The soluble portion of the chloroplast is the "stroma". • The interior of the thylakoid vesicles is the "thylakoid space" or "thylakoid lumen". • Chloroplasts possess DNA, RNA and ribosomes.

5. 21.1 What Are the General Properties of Photosynthesis? Figure 21.2 Schematic diagram of an idealized choloplast.

6. 21.1 What Are the General Properties of Photosynthesis? Figure 21.1 Electron micrograph of a chloroplast.

7. Photosynthesis Consists of Both Light Reactions and Dark Reactions • The light reactions, associated with the thylakoid membranes, capture light energy and convert it to chemical energy in the form of reducing potential (NADPH) and ATP with evolution of oxygen. HOH -- > O2 , NADP -- > NADPH + H+ and ADP + Pi -- > ATP • The dark reactions, located in the stroma,use NADPH and ATP to drive the endergonic process of hexose sugar formation from CO2 in a series of reactions. CO2 + NADPH + ATP -- > carbohydrate

8. Photosynthesis Consists of Both Light Reactions and Dark Reactions NADPH and ATP made. NADPH and ATP used. Figure 21.4 The light-dependent and light-independent reactions of photosynthesis.

9. Water is the Ultimate e- Donor for Photosynthetic NADP+ Reduction The reaction sequence for photosynthesis in green plants: 2H2O + 2NADP+ + xADP + xPi→ O2 + 2NADPH + 2H+ + xATP + xH2O Photosynthesis provides the oxygen on which we depend. ----------------------------------------------------------------------------------------------------------------------------- A More Generalized Equation for Photosynthesis: CO2 + 2H2A → (CH2O) + 2A + H2O Hydrogen Hydrogen Reduced Oxidized acceptor donor acceptor donor In photosynthetic bacteria,H2A can be H2S or other oxidizable substrates, like isopropanol:

10. 21.2 How Is Solar Energy Captured by Chlorophyll? • Chlorophyll is a photoreactive pigment with an isoprene-based sidechain. • It has a planar, conjugated ring system - similar to porphyrins. • Mg2+is coordinated in the center of the planar conjugated ring structure. • A long chain alcohol, phytol, group confers membrane solubility. • Aromaticity makes chlorophyll an efficient absorber of light. • Light absorption promotes an electron to a higher orbital, enhancing the potential for transfer of this electron to a suitable acceptor.

11. 21.2 How Is Solar Energy Captured by Chlorophyll? Figure 21.5 Structures (a) and absorption spectra (b) of chlorophyll a and b. The phytyl side chain of ring IV provides a hydrophobic tail to anchor the chlorophyll in membrane protein complexes. Since the absorption spectra for a and b differ, plants that possess both can harvest a wider spectrum of incident energy.

12. 21.2 Large Rings Similar to Chlorophyll Heme and Corrin Structures

13. Accessory light-harvesting pigments increase the possibility for hν absorption Figure 21.6 Structures of representative accessory light-harvesting pigments in photosynthetic cells. (a) β-Carotene, an accessory light-harvesting pigment in leaves. (b) Phycocyanobilin, a blue pigment found in cyanobacteria.

14. Absorbances in the Visible Spectrum Chlorophylls and the accessory light-harvesting pigments photosynthetic cells.

15. Light Event Terms Resonance Energy Transfer: energy transfer between photoreceptor molecules. Photoexcitation: Chl -- > Chl* Energy transduction: Chl* -- > Chl+ Photooxidation: 2 HOH -- > O2 + 4 H+ + 4 e Photoreduction: NADP+ + 2 H+ + 2 e -- > NADPH + H+ Photophosphorylation: ADP + Pi -- > ATP using PSII, cyt bf and PSI Cyclic-Photophosphorylation: ADP + Pi -- > ATP using cyt bf and PSI only

16. Possible Fates for Light Energy Absorbed by Photosynthetic Pigments • Each photon represents a quantum of energy. • Each quantum of light energy has 4 possible fates: 1) Loss as heat. Energy can be dissipated as heat through redistribution into atomic vibrations within the pigment molecule. 2) Loss as light. A light-excited electron can return to a lower orbital, emitting a photon of fluorescence. 3) Resonance energy transfer. Excitation energy can be transferred to a neighboring molecule. 4) Energy transduction. The excitation energy changes the reduction potential of the pigment, making it an effective electron donor.

17. Possible Fates for Light Energy Absorbed by Photosynthetic Pigments Figure 21.7 Possible fates of the quantum of light energy absorbed by photosynthetic pigments.

18. Light energy, hv • Electromagnetic energy: E = hν= hc/λ • 1 mol of photons = 1 Einstein (varies with λ) • Example calculation in kJ/mol at 400 nm: 1 Einstein = (6.62x10-34 J-sec) (3x108 m/sec) (109 nm/m) (1/400 nm) (6.02x1023) = 300000 J/mol = 300 kJ/mol. • Likewise at 700 nm: 1 Einstein = 171 kJ/mol • The amount of energy in 1 Einstein depends on λ.

19. Emerson’s Red Drop Phenomenon - 1930 The quantum yield of photosynthesis drops for λ >680 nm. Quantum yield = O2 evolved/photon. Photosysnthesis is not linear with light intensity. There is a saturation effect limited by the number of photosynthetic units available.

20. Other Discoveries • 1937 - Hill discovered that Oxygen came from water and not CO2. hv, no CO2 Chloroplasts + H2O + Fe3+ ------- > O2 + H+ + Fe2+ • 1950 – Arnon showed that chloroplasts generated ATP. NADPH was not transported to the mitochondria and used in oxidative phosphorylation. • 1952 – Duysen postulated two photosystems, both driven by light < 680 nm but only one driven by light > 680 nm. PSI generates a weak oxidant and a strong reductant. PSII generates a strong oxidant and a weak reductant.

21. Photosynthetic Units Consist of Many Chlorophyll Molecules but Only a Single Reaction Center The photosynthetic unit (quantosome) consists of several hundred light-capturing chlorophylls, and other accessory pigments plus a pair of special chlorophylls in the reaction center. Light is captured by one of the "antenna chlorophylls" and routed from one to the other until it reaches the reaction center chlorophyll dimer that is photochemically active. See Figure 21.10. Oxidation of chlorophyll leaves a cationic radical ion, Chl•+, whose properties as an electron acceptor are important to photosynthesis.

22. Photosynthetic Units Consist of Many Chlorophyll Molecules but Only a Single Reaction Center Figure 21.9 Schematic diagram of a photosynthetic unit.

23. 21.3 What Kinds of Photosystems Are Used to Capture Light Energy? Oxygenic phototrophs have two distinct photosystems: PSI (P700) and PSII (P680) • PSI systems have a maximal red light absorption at 700 nm and use ferredoxins as terminal electron acceptors. • PSII have a maximal red absorption at 680 nm and use quinones as terminal electron acceptors. • Chloroplasts given light at 680 and 700 nm simultaneously yield more O2 than the sum of amounts when each is used alone. • All chlorophyll is protein-bound – as part of either PSI or PSII or light-harvesting complexes (LHCs).

24. PSI and PSII Participate in the Overall Process of Photosynthesis • What do PSI and PSII do ? • PSI provides reducing power in the form of NADPH. • PSII splits water, producing O2, and feeds the electrons released into an electron transport chain that couples PSII to PSI. • Electron transfer between PSII and PSI pumps protons for chemiosmotic ATP synthesis. • Essentially, electrons flow from H2O to NADP+, driven by light energy absorbed at the reaction centers. • Light-driven phosphorylation of ADP to make ATP is termed photophosphorylation.

25. PSI and PSII Participate in the Overall Process of Photosynthesis Figure 21.10 Roles of the two photosystems, PSI and PSII.

26. PSI and PSII Participate in the Overall Process of Photosynthesis Events in photosystem I (PSI) Chl700 PSI Aoox hv Aoox Aored- Aored- Chl700 ----> Chl700* ----> Chl700+ ----> Chl700 PCred PCred PCred PCox+ Aored- is a strong reductant, e to NADP+. Plastocyaninox+ is a weak oxidant, e from Cyt6f.

27. The Z Scheme Figure 21.11 The Z scheme of photosynthesis. (a) A diagrammatic representation.

28. PSI and PSII Participate in the Overall Process of Photosynthesis Events in photosystem II (PSII) Chl680 PSII Pheooxhv Pheoox Pheored - Pheored - Chl680 ----> Chl680* ----> Chl680+ ----> Chl680 Zred Zred Zred Zox+ Pheophytinred- is a weak reductant, e to PQ. Zox+ is a strong oxidant, e from HOH  O2

29. The Pathway of Photosynthetic Electron Transfer Is Called the Z Scheme The electron carriers are arranged as a chain, according to their standard reduction potentials • The arrangement resembles the letter “Z”. • Components of – the Z scheme: • Pheo = Pheophytin (2H+ replace Mg++ in Chl) • PQ = plastoquinone (two: PQA and PQB). • PC = plastocyanin (Cu++ protein ~10 kdaltons). • "F"s = ferredoxins (several, aka Fd). • Ao = a special chlorophyll a. • A1 = a special PSI quinone (phylloquinone ??). • Cytochrome b6/cytochrome f complex is a proton pump with a Q cycle similar to mito. Complex III.

30. The Z Scheme Water:plastoquinol oxidoreductase Plastocyanin:ferredoxin oxidoreductase Ferredoxin:NADP+ oxidoreductase Plastoquinol:plastocyanin oxidoreductase Figure 21.11 The Z scheme of photosynthesis. (b) The functional relationships among PSI, PSII, the cyt bf complex, and the ATP synthase in the thylakoid membrane.

31. How Does PSII Generate O2 From H2O? • The oxidation of H2O to O2 is chemically difficult. • The oxygen-evolving complex (OEC) is a large globular protein domain on the lumenal side of the D1 subunit of PSII. • The active site of the OEC contains a cube-like metal cluster of four Mn++ ions, one Caion, and five O atoms bridging the Mn atoms. • This cluster is held in place by Glu, Asp, and His residues of D1 and a Glu from CP43, a chlorophyll-containing inner subunit of PSII (see Figure 21.17). • Electron removal from H2O molecules at the cluster (one from each Mn++) facilitates O2 formation.

32. How Does PSII Generate O2 From H2O? Figure 21.17 Structure of the PSII oxygen-evolving complex (OEC). Four Mn atoms (red, A-D) and a Ca atom (green) form the water-splitting metal cluster of the OEC. Bridging O atoms are purple.

33. Oxygen Evolution Requires the Accumulation of Four Oxidizing Equivalents in PSII • When isolated chloroplasts that have been held in the dark are illuminated with very brief flashes of light, O2 evolution reaches a peak on the third flash and every fourth flash thereafter. • The interpretation of this observation: The O2 evolving complex of PSII (P680) cycles through five oxidation states. • 1 e- and 1 proton are removed in each step • Oxygen (O2) is released in the fifth step and two more molecules of H2O are bound. • The net of this cycle: 2H2O oxidized to O2 + 4H+.

34. Oxygen Evolution Requires the Accumulation of Four Oxidizing Equivalents in PSII Figure 21.12 Oxygen evolution requires the accumulation of four oxidizing equivalents in PSII (a) O2 evolution after brief light flashes. (b) The cycling of the PSII reaction center through five different oxidation states, S0 to S4. One e- is removed photochemically at each light flash.

35. Electrons Flow From Pheophytin in the Z Scheme Via Plastoquinones Figure 21.13 The structures of plastoquinone A and its reduced form, plastohydroquinone (or plastoquinol). Plastoquinone A has nine isoprene units and is the most abundant plastoquinone in plants and algae. Plastoquinone (PQ) exists as a pool within the chloroplast membrane. Because of its lipid nature, plastoquinone is mobile within the membrane and shuttles electrons from PSII to the cytochrome b6f complex.

36. Electrons from PSII Are Transferred to PSI via the Cytochrome b6f Complex • The cytochrome b6f complex is a large multimeric protein possessing 26 transmembrane α-helices. • This complex is homologous to the cytochrome bc1 complex of mitochondria. • The purpose of this complex is to mediate the transfer of electrons from PSII to PSI and to pump protons across the thylakoid membrane. • Plastocyanin (PC in the Z scheme, Figure 21.11) is a small copper-containing protein that carries electrons from cytochrome b6f to PSI. • The copper in PC cycles between the reduced Cu+ and oxidized Cu2+ states in this transfer.

37. Electrons from PSII Are Transferred to PSI via the Cytochrome b6f Complex Structure of the cyanobacterial cytochrome b6f complex. The heme groups are red; the iron-sulfur clusters are blue. The upper bundle of α-helices defines the transmembrane domain.

38. How Do Green Plants Carry Out Photosynthesis ? • The structure of a light-harvesting complex (LHC) has been determined. • LHC1 is a supercomplex of 16 distinct protein subunits and 200 prosthetic groups. • Four LHC1 subunits form an arc around one side of the PSI reaction center. • A second LHC (LHC2) binds to another side. • The many Chl molecules and other light-harvesting molecules of the supercomplex form an integrated network for highly efficient transfer of light energy into P700.

39. How Do Green Plants Carry Out Photosynthesis? Figure 21.19 View of the plant PSI-LHC1 supercomplex, from the stromal side of the membrane. Chlorophylls are green, lipids are red.

40. 21.5 What Is the Quantum Yield of Photosynthesis? • The quantum yield of photosynthesis is defined as the amount of product formed per equivalent of light input. • i.e., the amount of O2 evolved per photon. • Four photons per reaction center - 8 total - drive the evolution of 1 O2, reduction of 2 NADP+, and the translocation of 12 H+. • Current estimates suggest that 3 ATPs are formed for every 14 H+ translocated. • Thus (12/14)3 = 2.57 ATP should be synthesized from an input of 8 photons of light.

41. Building the H+ Gradient • pH gradient: 3-3.5 ΔpH (~8.0 stroma:4.9 lumen) • potential gradient: 0.05-0.10 v • Due to a Mg++/Cl- antiport most of the charge gradient is lost and ΔpH is the major contributor. • Calc ΔGwhere ΔpH = 3.1 and Δψ = 0.05 v at 25o. • ΔG = 2.3(8.314)(298)(3.1) + (1) (96480)(0.05) • ΔG = (5698)(3.1) + (96480)(0.05) • ΔG = 17664 + 4824 = +22.5 kJ/mol • This is similar to the value found in the mito.

42. 21.6 How Does Light Drive the Synthesis of ATP? Photophosphorylation • The transduction of the electrochemical gradient into the chemical energy of ATP is carried out by the chloroplast ATP synthase, more properly called the CF1CF0-ATP synthase. • Electron transfer through the proteins of the Z scheme drives the generation of a proton gradient across the thylakoid membrane. • Protons pumped into the lumen of the thylakoids flow back out, driving the synthesis of ATP. • CF1-CFo ATP synthase is similar to the mitochondrial ATP synthase.

43. 21.6 How Does Light Drive the Synthesis of ATP? Figure 21.20 Mechanism of photophosphorylation. Photosynthetic electron transport establishes a proton gradient that is tapped by the CF1-CF0 ATP synthase to drive ATP synthesis.

44. Cyclic Photophosphorylation Generates ATP but Not NADPH or O2 • The dark reactions need 2 NADPH : 3 ATP to effect carbohydrate synthesis. So, extra ATP is needed beyond that provided by PSII and PSI. • ATP provided by cyclic photophosphorylation depends only on PSI, not on PSII. • In cyclic photophosphorylation, the photo-excited electron removed from P700 returns to P700 in a pathway indicated by the curved line in Figure 21.21. • It diverts the activated electron lost from PSI back through the PQ pool, the cytochrome b6f complex, and plastocyanin to re-reduce P700+.

45. Cyclic Photophosphorylation Generates ATP but Not NADPH or O2 Figure 21.21 The pathway of cyclic photophosphorylation by PSI.

46. 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? Fixation of CO2 is a unique ability of plants, algae, etc. • Melvin Calvin at Berkeley in 1945 showed that Chlorella could take up 14CO2 and produce 3-phosphoglycerate. • What was actually happening was that CO2 was combining with a 5-C sugar to form a 6-C intermediate. • This breaks down to two 3-P-glycerates. • Ribulose-1,5-bisphosphate is the CO2 acceptor in CO2 Fixation.

47. Ribulose-1,5-Bisphosphate The CO2 Acceptor • Fixation is accomplished by ribulose bisphosphate carboxylase (oxygenase) = RUBISCO. • Probably the world's most abundant protein. • Figure 21.22, 550000 Daltons • Study the reaction in Figure 21.23. • Rubisco is activated when carbamylated (CO2 added to Lys201) and with Mg++ bound. • RuBP (substrate!) is inhibitor and must be released from inactive rubisco by rubisco activase. Carbamylation and Mg then activate.

48. Ribulose-1,5-Bisphosphate is the CO2 Acceptor in CO2 Fixation Figure 21.22 Molecular graphic of ribulose bisphosphate carboxylase, Rubisco. The enzyme consists of 8 equivalents each of two subunits. Clusters of four small subunits (orange and red) are located at each end of the symmetric octamer formed by the L subunits (light and dark green). The active sites are revealed in the ribbon diagram by bound ribulose-1,5-bisphosphate (yellow).

49. Ribulose-1,5-Bisphosphate is the CO2 Acceptor in CO2 Fixation The ribulose bisphosphate carboxylase-oxygenase (Rubisco) reaction. Mg2+ at the active site aids in stabilizing the 2,3-enediol transition state (I) for CO2 addition and in facilitating the C-C bond cleavage that leads to product formation.

50. Ribulose-1,5-Bisphosphate is the CO2 Acceptor in CO2 Fixation The Rubisco reaction, cont.: Ribulose bisphosphate + CO2 yields two molecules of 3-phosphoglycerate. This pathway is referred to as a three carbon pathway since the first compound isolated after CO2 fixation contains three carbon atoms.