CHAPTER 6 Photosynthesis and the Chloroplast

Introduction (1) The earliest living organisms were heterotrophs, which survived on nutrients from the environment. Autotrophs manufacture organic nutrients from CO 2 and H 2 S. Synthesis of complex molecules from CO 2 requires a large input of energy. – Chemoautotrophs use energy from inorganic molecules. – Photoautotrophs use radiant energy to make organic compounds.

Introduction (2) Photosynthesis converts energy from sunlight into chemical energy stored in carbohydrates. – Low energy electrons are removed from a donor molecule. – First photoautotrophs used H 2 S as electron source – About 2.7 million years ago, cyanobacteria used electrons from water to produce oxygen as a waste product: light CO 2 + H 2 O  (CH 2 ) + O 2

6.1 Chloroplast Structure and Function (1) Photosynthesis in eukaryotes takes place in the chloroplast, a cytoplasmic organelle. Chloroplasts have a double membrane. – The outer membrane contains porins and is permeable to large molecules. – The inner membrane contains light-absorbing pigment, electron carriers, and ATP-synthesizing enzymes.

The functional organization of a leaf

The internal structure of a leaf

Chloroplast Structure and Function (2) The inner membrane of a chloroplast is folded into flattened sacs (thylakoids), arranged in stacks called grana. Chloroplasts are self-replicating organelles containing their own DNA. Thylakoid membranes contain a large percentage of glycolipids, which make the membrane highly fluid for diffusion of proteins complexes.

Thylakoid membranes

6.4 An Overview of Photosynthetic Metabolism (1) Photosynthesis is a redox reaction transferring an electron from water to carbon dioxide: 6 CO H 2 O  C 6 H 12 O H 2 O + 6 O 2 Experiments using 18 O showed that O 2 molecules released from photosyntheiss came from two molecules of H 2 O, not from CO 2.

An Overview of Photosynthetic Metabolism (2) Photosynthesis oxidizes water to oxygen; respiration reduces oxygen to form water. – Respiration removes high energy electrons from reduced organic substrates to form ATP and NADH. – Photosynthesis uses low energy electrons to form ATP and NADPH, which are then used to reduce CO 2 to carbohydrate.

Overview of the energetic of photosynthesis and aerobic respiration

An Overview of Photosynthetic Metabolism (3) Photosynthesis occurs in two stages: – Light-dependent reactions (light reactions)in which sunlight is absorbed, converting it into ATP and NADPH. – Light-independent reactions (dark reactions) use the energy stored in ATP and NADPH to produce carbohydrate.

6.3 The Absorption of Light (1) Absorption of photons (light “particles”) by a molecule makes them go from ground state to excited state. – Energy in the photon depends on the wavelength of light. – Energy required to shift electrons varies for different molecules. – Molecules absorb specific wavelengths of light.

The Absorption of Light (2) Photosynthetic Pigments – molecules that absorb light of particular wavelengths. – Chlorophyll contains a porphyrin ring that absorbs light and a hydrophobic tail embedding it to the photosyntheic membrane.

The Absorption of Light (3) The alternating single and double bonds along the porphyrin ring form a cloud making it a conjugated system. Conjugated bond systems absorb energy of a range of wavelengths.

The Absorption of Light (4) Besides chlorophyll, there are accessory pigments called carotenoids. Carotenoids absorb light in the blue-green region of spectrum. Various pigments allow for greater absorption of incoming photons.

6.4 Photosynthetic Units and Reaction Centers (1) Each photosynthetic unit contains several hundred chlorophyll molecules. One member of the group—the reaction- center chlorophyll—transfers electrons to an electron acceptor. Pigments that do not participate directly in the conversion of light energy, they are responsible for light absorption, and are called antenna pigments.

The transfer of excitation energy

Photosynthetic Units and Reaction Centers (2) Oxygen Formation: Coordinating the Action of Two Different Photosynthetic Systems – Two large pigment-protein complexes called photosystems act in series to raise electrons from H 2 O to NADP +. Photosystem II (PSII) boosts electrons from below energy level of water to a midpoint. Photosystem I (PSI) boosts electrons to a level above NADP +.

Overview of the flow of electrons during the light-dependent reactions of photosynthesis

Photosynthetic Units and Reaction Centers (3) Oxygen Formation (continued) – The reaction center of PSII is referred to as P680, and that of PSI as P700 standing for the wavelengths where absorption is stronger. – Electrons are transferred to a primary electron acceptor. – The flow of electrons from H 2 O to NADP + is referred to as the Z scheme.

Photosynthetic Units and Reaction Centers (4) PSII Operations: Obtaining Electrons by Splitting Water – PSII uses absorbed light energy to remove electrons and generate a proton gradient. – Two proteins, D1 and D2, bind the P680 chlorophyll and perform reactions to oxidize H 2 O. – Light is harvested by a pigment-protein complex called light-harvesting complex II (LHCII).

The functional organization of photosystem II

Photosynthetic Units and Reaction Centers (5) The Flow of Electrons from PSII to Plastoquinone – Harvested energy is passed from LHCII to inner- antenna molecules within PSII. – Excited P680 (P680*) transfers energy to an electron acceptor generating P680 + and Pheo -. – P680 + and Pheo - are transferred to opposite sides of the thylakoid membrane where Pheo - passes an electron to plastiquinone (PQ).

Photosynthetic Units and Reaction Centers (6) The Flow of Electrons from PSII to Plastoquinone (continued) – PQ passes the electron to another PQ. – The electron is then moved to the stromal side of the membrane.

Plastoquinone

Photosynthetic Units and Reaction Centers (7) The Flow of Electrons from Water to PSII – The redox potential of P680 + pulls electrons from water (photolysis). – Formation of O 2 requires four electrons from H 2 O: 2 H 2 O  4 H + + O e – – Four electrons required to form O 2 are transferred in cycles through P680 + to four Mn ions and one Ca ion that form the oxygen-evolving complex.

Measuring the kinetics of O 2 release

Photosynthetic Units and Reaction Centers (8) The Flow of Electrons from Water to PSII (continued) – Protons produced in photolysis are retained in the thylakoid lumen. – Oxygen produced is a released as a waste product into the environment.

Photosynthetic Units and Reaction Centers (9) From PSII to PSI – Production of O 2 leads to formation of two molecules of PQH 2. – Reduced PQH 2 then diffuses through thylakoid membrane and binds cytochrome b 6 f, and releases protons the lumen of thylakoid. – Electrons from cytochrome b 6 f are passed to another carrier, plastocyanin. – Plastocyanin transfers electrons to P700 +.

Electron transport between PSII and PSI

Photosynthetic Units and Reaction Centers (10) PSI Operations: The Production of NADPH – The PSI consists of a reaction core center of 12–14 different polypeptides and a complex of protein- bound pigments called LHCI. – Photons harvested by antenna pigments in PSI (LHCI) oxidizes chlorophyll a, forming P700*. – Absorption of light leads to production of P700 + and A o –. – Redox potential of P700 + /A o – reduces NADP.

The functional organization of photosystem I

Photosynthetic Units and Reaction Centers (11) PSI Operations (continued) – The reduction of NADP + to NADPH is catalyzed by ferredoxin-NADP + reductase. – Some electrons passed to ferredoxin end up reducing nitrate, ammonia or sulfate to form other important biological molecules.

Photosynthetic Units and Reaction Centers (12) An Overview of Photosynthetic Electron Transport – For every 8 photons absorbed: 2 H 2 O + 2 NADP +  O NADPH – Electron transport also produces a proton gradient across the thylakoid membrane.

Summary of the light-dependent reactions

Photosynthetic Units and Reaction Centers (13) Killing Weeds by Inhibiting Electron Transport – Common herbicides bind to a core protein of PSII. – Light reactions serve as targets of herbicides. – Some herbicide produce oxygen radicals, which are toxic to the human tissue.

6.5 Photophosphorylation (1) The machinery for ATP synthesis in a chloroplast is similar to that of mitochondrial enzymes. The ATP synthase consists of a head (CF 1 ), and a base (CF 0 ). The CF 1 heads project outward into the stroma, keeping with the orientation of the proton gradient.

ATP synthase in the chloroplast

Photophosphorylation (2) Protons move into the lumen through the CF 0 base of the synthase. Measurements of chloroplasts during ATP synthesis show an increase in the ΔpH of more than 3 units. The movement of protons during photo- synthesis does not create a significant change in the membrane potential since other ions are transported simultaneously.

Photophosphorylation (3) The movement of electrons during the formation of oxygen is called noncyclic photophosphorylation because ions move in a linear path. Cyclic vs. noncyclic photophosphorylation: – Cyclic photophosphorylation is carried out by PSI independently of PSII. – Cyclic photophosphorylation is thought to provide additional ATP required for carbohydrate synthesis

Cyclic photophosphorylation

6.6 Carbon Dioxide Fixation and the Synthesis of Carbohydrate (1) The movement of carbon in the cell can be followed during photosynthesis using [ 18 C]O 2 as a tracer. Extracts of cells are then analyzed by autoradiography by identifying radiolabeled compounds compared to known standards.

Chromatogram after incubation with [ 18 C]O 2

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (2) Carbohydrate Synthesis in C 3 Plants – C 3 plants are those that produce a three-carbon intermediate (3-phosphoglycerate, PGA) as the first compound to be identified during carbon dioxide fixation. – CO 2 is condensed with a five-carbon compound, ribulose 1,5-bisphosphate (RuBP), to form a six- carbon molecule which then splits into two molecules of PGA.

Converting CO 2 into carbohydrate

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (3) The condensation of RuBP and the splitting of the six-carbon molecule are catalyzed by one enzyme, ribulose bisphosphate carboxylase (Rubisco). Rubisco is the most abundant protein on Earth, and has a very low turnover number. Rubisco fixes ~3 molecules of CO 2 per second.

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (4) Carbohydrate Synthesis in C 3 Plants – The C 3 pathway is known as the Calvin cycle and includes: Carboxylation of RuBP to form PGA. Reduction of PGA to glyceraldehyde 3-phosphate (GAP) using NADPH and ATP from light reactions. Regeneration of RuBP.

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (5) Carbohydrate Synthesis in C 3 Plants – The GAP molecules can be exported into the cytosol in exchange for phosphate ions and used to synthesize sucrose. – GAP can also remain in the chloroplast where it is converted to starch.

Overview of various stages of photosynthesis

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (6) Carbohydrate Synthesis in C 3 Plants – It is an expensive process. – Conversion of 6 molecules of CO 2 to 1 six-carbon sugar molecules requires 12 molecules of NADPH and 18 molecules of ATP. – Expenditure due to CO 2 being the most highly oxidized form of carbon.

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (7) Redox Control is light-dependent. – Key enzymes of Calvin cycle are only active when ATP and NADP are produced by photosynthesis. – Some electrons used to reduce NADP + are transferred to thioredoxin, which are then accepted to reduce disulfide bridges (-S-S-) in selected Calvin cycle enzymes. – In the dark, thioredoxin reduction ceases and enzymes go back to oxidized state (-S-S-) and are inactivated.

Redox control of the Calvin cycle

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (8) Photorespiration – uptake of O 2 and release of CO 2. – Rubisco also catalyzes the attachment of O 2 to RuBP to produce 2-phosphoglycolate. – Glycolate is then transferred to the peroxisome and leads to release of CO 2. – It accounts for the loss of up to 50% of fixed CO 2. – Rate of photorespiration depends on the CO 2 /O 2 ratio.

The reactions of photorespiration

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (9) Peroxisomes and Photorespiration – Glycolate produced during photorespiration is shuttled to the peroxisome. – Peroxisomal enzymes convert glycolate to glyoxylate and then glycine, resulting in the loss of CO 2.

The cellular basis of photorespiration

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (10) Carbohydrate Synthesis in C 4 Plants – The C 4 pathway involves the production of phosphoenolpyruvate (PEP), which then combines with CO 2 to produce 4-carbon compounds oxaloacetate or malate. – Plants utilizing this pathway are C 4 plants, usually tropical grasses.

Structure and function in C 4 plants

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (11) C 4 Plants (continued) – In a hot, dry environment C 4 plants get enough CO 2 for photosynthesis while keeping their stomata partially closed to prevent water loss. – C 4 plants have anatomical adaptations to transport C 4 products into the bundle sheath cells, where fixed CO 2 can be split from the 4-carbon carrier producing a high CO 2 level suitable for fixation by Rubisco.

Carbon Dioxide Fixation and the Synthesis of Carbohydrate (12) Carbohydrate Synthesis in CAM Plants – CAM plants carry out light reactions and CO 2 fixation at different times of the day using the enzyme PEP carboxylase. – CAM (crassulacean acid metabolism) plants keep their stomata closed during the day to reduce water loss.