Lecture 7 2024 Phototrophic Metabolism PDF

Phototrophic Metabolism: Chloroplast & Photosynthesis Slides prepared by: AP Dr. Alvin Hee and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes Upon completing this lecture, you are expected to be able to: 1. Describe the s...

Phototrophic Metabolism: Chloroplast & Photosynthesis Slides prepared by: AP Dr. Alvin Hee and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes Upon completing this lecture, you are expected to be able to: 1. Describe the structure and function of chloroplasts. 2. Clarify the roles of light-dependent and light-independent reactions in photosynthesis. 3. Clarify the events that result in the absorption of light by photosynthetic pigments. 4. Explain the operation of the photosynthetic units and their reaction centres. 5. Elaborate on the events involved in the flow of electrons from water to NADP+ to form NADPH. 6. Describe the mechanism by which ATP is produced during photosynthetic electron flow. 7. Compare and contrast cyclic and non-cyclic photophosphorylation.  Learning Outcomes Upon completing this lecture, you are expected to be able to: 8. Describe the C3 pathway, its role in carbon fixation and its production of carbohydrates using the products of the light- dependent reactions. 9. Explain the functioning of Rubisco and its ability to initiate both photosynthetic and photorespiration pathways. 10. Describe briefly the process of C4 photosynthesis and adaptations that allow those C4-plants to live in dry and hot habitats. 11. Describe briefly the mechanism by which CAM plants are able to survive in very hot and dry climates; and compare it with C3 and C4 photosynthesis. 12. Describe the relationships between peroxisomes, chloroplasts and mitochondria in relation to plant physiology.  Utilization of Biocompounds & Energy Heterotrophs: Organisms that depend on an external source of organic compounds. Autotrophs: Organisms capable of surviving on CO2 as their principal carbon source. - Chemoautotrophs utilize chemical energy stored in inorganic molecules (e.g. NH3) to convert CO2 to organic compounds. - Photoautotrophs utilize radiant energy (sunlight) to perform photosynthesis, the transformation of solar energy into chemical energy that is stored in carbohydrates and other organic molecules.  Utilization of Biocompounds & Energy Photosynthesis converts low energy electrons from a donor compound into high energy electrons that can be used in anabolic reactions. The earliest photoautotrophs used H2S as an electron source. Later, a much more abundant supply of H2O was used instead, as a source of electrons, to produce molecular oxygen, O2 (cyanobacteria → aerobic metabolism). But as S in H2S has less affinity for its electrons (easier to oxidize) than O in H2O → much harder to pull electrons from H2O. Thus, to carry out oxygen-releasing photosynthesis, an organism must generate very strong oxidizing agent as part of photosynthetic metabolism to pull the electrons from H 2O.  Chloroplast Structure and Function The chloroplast (a large membranous cytoplasmic organelle) is the photosynthetic organelle in eukaryotic cells. Chloroplasts are composed of three membrane systems: - Outer membrane, - Inner membrane, separated by a narrow - Intermembrane space. Chloroplast Structure and Function Having an outer and inner membrane makes it similar to a mitochondrion. Outer membrane contains porin and permeable to large molecules. Inner membrane contains light-absorbing pigments, electron carriers and ATP- synthesizing enzymes.  Chloroplast Structure and Function Similar to the mitochondrion, a chloroplast has a double membrane: - Outer membrane containing porin and is permeable to large molecules; - Inner membrane (highly impermeable) containing light- absorbing pigments, electron carriers and ATP-synthesizing enzymes.  Chloroplast Structure and Function Inner membrane is organized (folded) into flattened membranous sacs (thylakoids) (third membrane system), and arranged like stack of coins called grana. Lumen- space within the thylakoids Stroma- matrix outside the thylakoids (containing Carbohydrate (CHO) - synthesizing enzymes). Stroma thylakoids (stroma lamella)- interconnecting the grana.  Chloroplast Structure and Function Chloroplast are semiautonomous, self- replicating organelles containing their own small, double stranded circular DNAs. The thylakoid membranes contain high percentage of unsaturated neutral glycolipids that increase fluidity → facilitate protein mobility (lateral diffusion of protein complexes through the membrane during photosynthesis).  Overview of Photosynthesis Chloroplast as the site of photosynthesis. Two phases of photosynthesis: - Energy transduction 1a. Light harvesting reaction 1b. e- transport to NADPH with simultaneous H+ pumping 1c. ATP synthesis - Carbon assimilation 2a. Calvin cycle 2b. Starch synthesis 2c. Sucrose synthesis  Photosynthetic metabolism Similar to aerobic respiration except that it is a reverse process (one requiring energy). An oxidation-reduction reaction transferring an e- from H2O to CO2. CO2 + 12 H2O + light → (C6H12O6) + 6 H2O + 6 O2 Using 18O, it was demonstrated: O2 released during photosynthesis came from two molecules of H2O, not CO2. During photosynthesis, H2O oxidized → O2; During respiration, O2 reduced → H2O. - Respiration removes high energy electrons from reduced organic substrates to form ATP+NADH. - Photosynthesis boosts low energy electrons to form ATP + NADPH, which are then used to reduce CO2 to CHO.  Photosynthetic metabolism Occurs in 2 stages: 1. Light-dependent (Light) reactions - light energy from sunlight is absorbed, converted to chemical energy & stored as 2 key high-energy biological molecules (ATP, NADPH); - ATP as cell’s primary source of chemical energy - NADPH as its primary source of reducing power 2. Light-independent (Dark) reactions - CHO made from CO2 using energy stored in ATP & NADPH molecules produced by light-dependent reactions.  Absorption of Light Light energy depends on wavelength (λ) & travels in packets called photons. Absorption of light as first step in photochemical process. Absorption of photons lifts e- from inner → outer orbitals, elevating molecules from the ground state to the excited state. - The energy in a mole of photons depends on λ - The energy required to shift e- varies for different molecules. Thus, molecules absorb specific λ of light. Energy from excited molecules can be re-radiated at a lower wavelength (fluorescence) or in the case of chloroplasts, transferred to e- carriers.  Photosynthetic pigments are coloured molecules (chromophores) that absorb light of particular λ within the visible spectrum. In plants, the presence of pigments with varying absorption properties ensures that a greater percentage of incoming photons will stimulate photosynthesis action spectrum The principal photoreceptor in photosynthesis is chlorophyll. Plants contain abundance of chlorophyll, the most important light-absorbing photosynthetic pigment. Absorbs in red and blue region of the visible spectrum, thus giving the plants (leaves) green appearance. Absorption spectrum for several photosynthetic pigments of higher plants. (Visible Spectrum)  The background shows the colors that we perceive for the wavelengths of the visible spectrum. Chlorophylls absorb most strongly in the violet, blue and red regions of the spectrum Carotenoids (beta-carotene) also absorb into the green region. Red algae and cyanobacteria contain additional pigments (phycobilins) that absorb in the middle bands of the spectrum. Action Spectrum for Photosynthesis  Chlorophyll contains: 1. Porphyrin ring that absorbs light, 2. Long phytol side chain (tail), anchored to the chloroplast membranes that contributes to its hydrophobicity. The light-absorbing function of chlorophyll is due to the tetrapyrrole ring with its network of conjugated double bonds. The central metal ion is Mg2+ (not Fe).  There are many different types of chlorophyll. They differ mainly in the side chains. Chlorophyll a: all O2- producing photosynthetic organism. Chlorophyll b: higher plants and green algae. Chlorophyll c: brown algae, diatoms and protozoa.  Photosynthetic membranes contain several accessory pigments in addition to chlorophyll. In fact, they complement those of chlorophylls and broaden the range of light energy that can be absorbed. The carotenoids include β-carotene and related pigments such as the xanthophylls. Carotenoids absorb light in the blue and green region of the visible spectrum whilst reflecting those of yellow, orange and red regions. Produces characteristics colours of carrots, oranges and autumn leaves. Phycobilins are another group of accessory pigments. These molecules contain conjugated double bonds that allow them to absorb light. The carotenoids have two primary roles: light harvesting and photoprotection through destruction of ROS (reactive oxygen species) that arise as by-products of photoexcitation. University of Wisconsin-Madison Arboretum  Photosynthetic Units and Reaction Centres Each photosynthetic unit consists of 300 chlorophyll molecules, in which only one member of the group (reaction-centre chlorophyll) actually transfers e- to an acceptor. Most pigments do not participate directly in conversion of light to chemical energy, but for light absorption. Light-harvesting antenna that absorbs photons of various λ & transfers the energy (excitation energy) very rapidly to the pigment at the reaction centre. Polypeptides facilitate energy transfer by holding pigment molecules in fixed orientations.  Excitation energy is transferred to a reaction centre chlorophyll that passes e- to e- acceptors. Oxygenic phototrophs have 2 types of photosystems. The two photosystems act in series to boost e- from H2O to NADPH. - Photosystem II (PSII) boosts e- from below the energy level of H2O to a point midway between H2O and NADP+. Reaction centre of PSII: P680 - Photosystem I (PSI) boosts e- to a level above NADP+. Reaction centre of PS I: P700 The whole chain (how e- flows from H2O to PSII, from PSII to PSI, and from PSI to NADP+ is known as the Z scheme.  Photosynthetic Energy Transduction  Stage 1a: Light harvesting or absorption of light.  Stage 1b: NADPH synthesis Stage 1c: ATP synthesis  In Stage 1b, a series of e- carriers are used to transport excited e- from chlorophyll to the coenzyme Nicotinamide Adenine Dinucleotide Phosphate (NADP+), forming NADPH that is the reduced form of NADP+. This is known as photoreduction.  Stage 1b: NADPH Synthesis Basically covering the Z scheme. Absorption of light energy by each photosystem boosts e- to the ‘top’ of and ETS (with lower E0’ ). As the e- flows exergonically from PSII to PSI, a portion of their energy is conserved in a proton gradient across the thylakoid membrane. From PSI, e- are passed to ferrodoxin and then to NADP+, generating reducing power in the form of NADPH. Starts with the Photosystem II operations. (Difference between NADP+ and NAD+ : NADP+ is the coenzyme of choice for a large number of anabolic reactions, whilst NAD+ is usually involved in catabolic pathways)  Photosystem II Operations: Obtaining e- by Splitting H2O PSII uses the absorbed light energy to remove e- from H2O and to generate a H+ gradient. Two peptides, D1 and D2 bind the P680 chlorophyll molecule and perform reactions required to oxidize H2O. First step in PSII: Light is harvested by a pigment- protein complex called the light-harvesting complex II (LHCII). LHCII proteins bind both chlorophylls and carotenoids. Situated outside the core of the photosystem.  Flow of e- from PSII to Plastoquinone (PQ) Harvested energy is passed through a core antenna complex to P680. - Transfer of energy from P680 to a primary e- acceptor (Pheo) generates a pair of opposite charged species, P680+ and Pheo-. - The e- from H2O pass through the D1 polypeptide to P680+ while Pheo- passes its e- to PQ intermediates to the opposite side of the chloroplast membrane.  Flow of e- from H2O to PSII - e- are passed to H+ and PQ → PQH2 in the stroma forming a pH gradient in the thylakoid lumen. - The redox potential of P680 (+1.1 V) is sufficient to pull e- from H2O (+ 0.82), a process called photolysis. (H+ transported to lumen → H+ gradient). - The four e- required to form one molecule of O2 are transferred in successive cycles through P680+ to four Mn 2+ ions, allowing the O2-evolving complex of PSII to catalyze the removal of four e- from two molecules of H2O. 4 photons 2 H2O 4 H+lumen + O2 + 4 e- Functional organization of photosystem II  From PSII to PSI From PSII to PSI, e- are transferred from PQ to P700 via cytochrome (cyt) b6f and plastocyanin (PC).  Photosystem I Operations: Reduction of NADP+ to NADPH in the Stroma Protons harvested by antenna pigments in PSI (LHCI) oxidize chlorophyll a in P700 → P700+ (occurring in the luminal side of membrane). e- formed by oxidation of P700 is then passed to another molecule of chlorophyll a acting as a primary e- acceptor (A0-) leading to A0+. The e- is then transferred out of PSI to a small, water- soluble, iron-sulfur protein called ferredoxin, associated with the stromal surface of membrane. The redox potential of the P700+/A0- pair reduces NADP+.  Photosystem I Operations: Reduction of NADP+ to NADPH in the Stroma The transfer of e- from P700+ on the luminal side, through intermediates to NADP+ on the stromal side adds to the proton gradient. NADP+ reduction to form NADPH is catalyzed by a large enzyme (ferredoxin-NADP+ reductase). e- from PSI can be diverted to reduce nitrate, ammonia or sulphate-forming compounds necessary for life. Removal of proton from stroma adds to proton gradient across the thylakoid membrane. 4 e- + 2 H+stroma + 2 NADP+ 2 NADPH Functional organization of photosystem I  Killing Weeds by Inhibiting e- Transport Herbicides are able to kill plants by: 1. Blocking e- transport through PSII. - binding to DI protein of PSII (diuron, atrazine) 2. Competing with ferredoxin for e- from PSI reaction centre. - as a result, e- are diverted to paraquat and later used to reduce O2, generating highly reactive oxygen radicals that damage the chloroplasts. Photosynthetic Energy Transduction  Stage 1a: Light harvesting or absorption of light.  Stage 1b: NADPH synthesis  Stage 1c: ATP synthesis  In Stage 1c, the potential energy now stored in the proton gradient is used to synthesize ATP from ADP and Pi. As the energy used to phosphorylate ADP originated in the sun, this process is known as photophosphorylation.  Photophosphorylation The conversion of 1 mole of CO2 to 1 mole of CH2O requires energy (3 moles of ATP and 2 moles of NADPH per mole of CH2O produced). The ATP synthase in chloroplasts is constructed of polypeptides homologous to mitochondrial enzymes and believed to function by a similar mechanism. CF0 as the basal region of ATP synthase, located in the membrane; mediated proton movement. The synthase headpieces (CF1) are aggregated in exposed regions of the grana stacks and project into the stroma so that H+ move through the synthase down the [H+] gradient from thylakoid lumen to stroma. This gradient drives the phosphorylation of ADP to ATP.  Photophosphorylation In summary, the ATP produced in chloroplasts by essentially the same mechanism used in mitochondria. Proton gradient produced by pumping protons into thylakoid lumen. Protons go back to stroma through ATP synthases to make ATP; starts with CF0 then through the head (CF1). The formation of ATP during oxygenic photosynthesis is called non-cyclic photophosphorylation since e- pass from H2O to NADP+ in a linear (non-cyclic) path.  Isolated chloroplasts were not only able to synthesize ATP from ADP, but could do so even in the absence of added CO2 or NADP+. Why? All that was needed was illumination, chloroplasts, ADP and Pi. The process (cyclic in nature) is carried out independently by PSI and begins with absorption of a quantum of light. Involves recycling of the e- from ferredoxin back to the e-- deficient reaction centre.  Cyclic Photophosphorylation The degree of cyclic vs non-cyclic photophosphorylation depends on the energy needs of the cell (for ATP, NADPH & CHO) at the particular time. Carbon Dioxide Fixation & Production of Carbohydrate Photosynthetic metabolism: 14CO light 2 + H2O (14CH2O) + O2 Carbohydrate synthesis as light-independent reactions. Two of the products of the light-dependent reactions, NADPH & ATP are used in light- independent reactions i.e. reactions of the Calvin-Benson cycle (Calvin cycle). Occurs in C3 plants.  Carbohydrate Synthesis in C3 plants Why C3? - Compound 3-phosphoglycerate (PGA) that was first identified in the Calvin cycle. This 3-C molecule also appeared as an intermediate in glycolysis. - Thus, the detection and identification of PGA led to the C3 pathway and plants that utilize this pathway were referred to as C3 plants. In this Calvin cycle, CO2 is condensed with a 5-C compound, ribulose-1,5-biphosphate (RuBP). Following this the unstable 6-C molecule is broken down rapidly to two 3-C molecules of PGA (first stable product of this light-independent reactions).  Carbohydrate Synthesis in C3 plants The condensation and splitting are two activities of a single enzyme, ribulose biphosphate carboxylase (Rubisco). As an enzyme responsible for converting inorganic carbon into useful biological proteins, Rubisco is one of the key proteins in the biosphere (most abundant!). Why? Rubisco can only fix about 3 molecules of CO2 per sec (worst turnover number of any enzyme- compare with it with carbonic anhydrase: turnover number of 1,000,000 molecules of CO2 [conversion of CO2 to bicarbonate ions in the erythrocytes]). To compensate for this inefficiency, up to half of the composition of leaf protein is comprised of Rubisco Carbohydrate Synthesis in C3 plants The Calvin cycle includes three basic steps: 1. Carboxylation of RuBP and splitting (hydrolisis) to form PGA. 2. Reduction of PGA to form glyceraldehyde-3-phosphate (G-3-P) using NADPH and ATP formed by e- transport. 3. Regeneration of RuBP (to allow continued carbon assimilation.  Carbohydrate Synthesis in C3 plants G-3-P can also be used in the cytosol as metabolic substrates (in exchange for Pi) G-3-P can also be converted to starch in the chloroplast where it is stored for use when photosynthesis has stopped. Note that the production of carbohydrate requires: 12 NADPH, 18 ATP for each molecule of 6- C sugar.  Photorespiration Why photorespiration? - During carbon fixation, the active site on Rubisco binds RuBP, making RuBP susceptible to attack by either CO2 or O2. - If [CO2] is low, Rubisco tend to react with O2 resulting in production of 2-phosphoglycolate leading to further metabolism and release of CO2 in the mitochondria. - It can release a significant portion of the CO2 that has been fixed by photosynthesis and thus, a disadvantage (waste) for plants performing this series of reactions. - Occurs most in plants experiencing hot and arid weather.  Photorespiration Why plants in hot and arid weather? - Forced to keep their stomata closed to reduce water loss to dry climate. - Disadvantaged as there is continued build-up of O2 in the leaf as photosynthesis progresses. - [CO2] in leaf drops due to two reasons- why? Keywords- photosynthesis & stomata... - O2/CO2 ratio in the leaf rises, increasing photorespiration and reducing rate of Calvin cycle.  Adaptations to Overcome Photorespiration Appearance of plants capable of fixing CO2 using an enzyme known as phosphoenolpyruvate carboxylase. This enzyme is responsible for linking CO2 to phosphoenol pyruvate (PEP). Catalyzes the first step of the series of reactions in the C4 pathway. Thus, plants utilizing this pathway are referred to as C4 plants. E.g. Tropical grasses (crab grass), sugar cane, corn, sorghum, etc. PEP carboxylase is insensitive to O2 levels. It adds CO2 to PEP, producing a 4-C molecule (malate or oxaloacetate). Therefore, the name C4 from the 4-C molecule.  Mesophyll cells fix CO2 to PEP. Fixation continues without interference from elevated O2 levels. Bundle sheath cells are sealed off from O2 but permits entry of C4 products. CO2 splits off from C4 skeleton and acted upon by Rubisco → Calvin cycle. Pyruvate travels back to the mesophyll cells and converted to PEP. Specialized Anatomy in C4 Plants  Adaptations to Overcome Photorespiration Desert plants such as succulents (cacti) possess a variation of C4 photosynthesis known as CAM (Crassulacean acid metabolism). Fix CO2 with PEP carboxylase but light-dependent reactions and carbon fixation are carried out at different times rather than different cells. - Stomata kept closed during the hot day to prevent water loss - During the night, as it is cooler, stomata is kept open while carbon fixation occurs. Malate transported to cell vacoule. - During the day, malate re-enters chloroplast where CO2 is released in high concentrations and fixed to RuBP. - The closure of the stomata prevents the influx of O2 from the atmosphere and massive release of CO2 from malate inhibits oxygenase activity of Rubisco and prevent photorespiration. Peroxisomes helps in the oxidation of glycolate Glycolate is formed in chloroplast, which usually pass in the peroxisomes, where it is oxidised to glyoxylate. Glyoxylate is aminated and gives rise to amino acid glycine, which enters into the mitochondria.