TG

Physiology Sept. 19th (rest of it)

Catching Light and Energy Transfer

  • Harvested Photons: Light energy is initially absorbed by various pigment molecules within the Light Harvesting Complex (LHC), primarily chlorophyll a, chlorophyll b, and carotenoids. This energy is then transferred non-radiatively (via Förster Resonance Energy Transfer, FRET) from peripheral pigments to the core antenna pigments and finally to the reaction center chlorophylls, P680 (in Photosystem II, PSII) or P700 (in Photosystem I, PSI). This process is highly efficient, minimizing energy loss.

  • Light Harvesting Complex (LHC): A multi-subunit protein complex that plays a crucial role in capturing photons over a broad spectrum of wavelengths and funneling this energy to the reaction centers of PSII. Each LHCII complex contains about 14 chlorophyll a, 8 chlorophyll b, and 4 carotenoid molecules, specifically bound to an apoprotein matrix which gives the complex stability and specific absorption properties.

Photosynthetic Electron-Transport Chain

  • Oxygen Evolving Complex (OEC): Located on the lumenal side of PSII, this complex is a cluster of four manganese ions, one calcium ion, and associated chloride ions. It catalyzes the light-driven oxidation of water through a series of four redox states (S0 to S4, known as the Kok cycle), ultimately releasing four protons (H+), four electrons (e^--), and one molecule of molecular oxygen (O2) for every two molecules of water oxidized (2H2O
    ightarrow 4H^+ + 4e^- + O_2). The electrons derived from water splitting are used to reduce the oxidized P680 reaction center.

  • Electron Storage and Transfer: Following excitation by light energy, P680 becomes P680* (excited state) and rapidly transfers an electron to a nearby pheophytin molecule, initiating charge separation. This electron then moves to a tightly bound plastoquinone (QA) and subsequently to a more loosely bound plastoquinone (QB). Two electrons taken up by QB, along with two protons from the stroma, fully reduce QB to plastoquinol (PQH2). PQH2 is a mobile electron carrier that diffuses within the thylakoid membrane to the cytochrome b6f complex. The cytochrome b6f complex contains four major polypeptides (cytochrome f, cytochrome b6, a Rieske iron-sulfur protein, and subunit IV) and functions as a proton pump using a modified Q-cycle mechanism. It oxidizes PQH2, releasing its protons into the thylakoid lumen and transferring electrons to plastocyanin.

  • NADPH Production: From the cytochrome b6f complex, electrons are transferred to plastocyanin (PC), a small, soluble copper-containing protein found in the thylakoid lumen. PC carries electrons to the oxidized P700 reaction center of Photosystem I (PSI). Upon light absorption, P700 also becomes excited (P700*) and transfers an electron through a series of iron-sulfur centers (A0, A1, FX, FB, FA) to ferredoxin (Fd), a small, soluble iron-sulfur protein in the stroma. From ferredoxin, the electrons are finally transferred to NADP+ reductase (FNR), which catalyzes the reduction of NADP+ to NADPH, using two electrons and one proton from the stroma: NADP^+ + 2e^- + H^+
    ightarrow NADPH.

  • Charge Separation: This fundamental process converts light energy into chemical energy during photosynthesis.

    • At PSII: Absorption of a photon by P680 leads to its excitation (P680*), followed by the rapid transfer of an electron to an acceptor molecule, yielding an electron (e^--) and an oxidized P680 radical cation (P680^{ullet+}). The P680^{ullet+} is then reduced by electrons derived from water splitting by the OEC.

    • At PSI: Similarly, light absorption excites P700, which then transfers an electron to a series of acceptors, leading to the formation of an electron (e^--) and an oxidized P700 radical cation (P700^{ullet+}). The P700^{ullet+} is subsequently reduced by electrons from plastocyanin.

Chloroplast Architecture

  • Stroma: The aqueous fluid-filled space within the inner chloroplast membrane, analogous to the cytoplasm of a prokaryotic cell. It is the site for light-independent reactions (the Calvin cycle) and also contains chloroplast DNA, ribosomes, and various enzymes required for these processes. Carbon fixation and sugar synthesis primarily occur here.

  • Thylakoid Membranes: An intricate system of interconnected membranous sacs and tubules within the stroma. These membranes are crucial for hosting the light-dependent reactions of photosynthesis. They are organized into stacked regions called grana (singular: granum) and unstacked regions called stroma lamellae (or intergranal thylakoids). PSII and its associated Light Harvesting Complex II (LHCII) are predominantly localized in the appressed regions of the grana thylakoids, while PSI, ATP synthase, and the cytochrome b_6f complex are more evenly distributed, with PSI and ATP synthase enriched in the non-appressed stroma lamellae. This structural differentiation allows for efficient electron flow and proton gradient establishment between the thylakoid lumen and the stroma.

Proton Gradient and ATP Synthesis

  • H+ Release and Gradient Formation: The accumulation of protons (H^+) in the thylakoid lumen (the space enclosed by the thylakoid membrane) is critical for ATP synthesis. Protons are primarily released into the lumen from two sources: (1) the oxidation of water by the Oxygen Evolving Complex (OEC) on the lumenal side of PSII, and (2) the transfer of electrons from plastoquinol (PQH2) to the cytochrome b6f complex, which actively pumps protons across the thylakoid membrane from the stroma into the lumen (the Q-cycle). This differential distribution of protons creates a steep electrochemical gradient (proton-motive force, PMF) across the thylakoid membrane, with the lumen becoming significantly more acidic (lower pH) than the stroma.

  • Plastocyanin: As a soluble electron carrier in the thylakoid lumen, plastocyanin facilitates the link between the cytochrome b_6f complex and PSI by transferring electrons. Its role is purely electron transport, not involving proton movement.

  • ATP Synthase: Also known as the CF0CF1-ATP synthase, this multi-subunit enzyme complex is embedded in the thylakoid membrane. It consists of two main parts: CF0, a hydrophobic transmembrane channel that allows protons to flow down their electrochemical gradient from the lumen to the stroma, and CF1, a catalytic headpiece that protrudes into the stroma and uses the energy from proton movement to drive the phosphorylation of ADP to ATP (ADP + P_i
    ightarrow ATP). This process is known as photophosphorylation, coupling electron transport, proton gradient, and ATP synthesis according to the chemiosmotic hypothesis proposed by Peter Mitchell.

The Calvin-Benson Cycle

  • Overview: The Calvin-Benson cycle, also known as the light-independent reactions or carbon fixation cycle, is the central metabolic pathway in plants where atmospheric carbon dioxide (CO_2) is converted into organic compounds, primarily sugars like glucose. This anabolic pathway occurs in the stroma of the chloroplast and depends entirely on the ATP and NADPH produced during the light-dependent reactions.

  • Steps of Cycle: The cycle is generally divided into three main phases:

    1. Carboxylation (Carbon Fixation): In this initial step, three molecules of CO2 (one at a time) are fixed by an enzyme called Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Each CO2 molecule combines with a five-carbon sugar, Ribulose-1,5-bisphosphate (RuBP), to form an unstable six-carbon intermediate. This intermediate immediately splits into two molecules of 3-Phosphoglycerate (3-PGA), a stable three-carbon compound. Thus, for three CO_2 molecules, six molecules of 3-PGA are formed.

    2. Reduction: This phase involves converting the 3-PGA molecules into higher-energy three-carbon sugars (triose phosphates). Each 3-PGA molecule is first phosphorylated by ATP (donated by light reactions) to form 1,3-Bisphosphoglycerate, a reaction catalyzed by phosphoglycerate kinase. Subsequently, 1,3-Bisphosphoglycerate is reduced by NADPH (also from light reactions) to Glyceraldehyde-3-phosphate (G3P), a reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. For every six G3P molecules produced, one molecule exits the cycle to be used for the synthesis of glucose and other carbohydrates (e.g., sucrose in the cytoplasm, starch in the chloroplast), while the remaining five G3P molecules proceed to the regeneration phase.

    3. Regeneration: The primary goal of this phase is to regenerate the initial CO_2 acceptor, RuBP, using the remaining five G3P molecules. This is a complex series of reactions involving several enzymes and various intermediate sugars (e.g., dihydroxyacetone phosphate, fructose-6-phosphate, erythrose-4-phosphate, xylulose-5-phosphate, sedoheptulose-7-phosphate, sedoheptulose-1,7-bisphosphate). These rearrangements ultimately produce three molecules of RuBP, consuming three additional ATP molecules along the way. This regeneration ensures the continuous operation of the cycle.

  • End Products: One G3P molecule that exits the cycle is the precursor for glucose, which can then be used to synthesize: (1) Sucrose, a disaccharide synthesized in the cytosol and transported to other parts of the plant as the primary form of sugar transport, and (2) Starch, a polysaccharide synthesized and stored within the chloroplast as a long-term energy reserve.

Rubisco: Key Enzyme in Carbon Fixation

  • Structure: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a large, complex enzyme, typically consisting of 16 subunits: eight large subunits (Lsu) encoded by chloroplast DNA and eight small subunits (Ssu) encoded by nuclear DNA. The active sites are located on the large subunits.

  • Significance and Dual Activity: RuBisCO is arguably the most abundant protein on Earth, highlighting its pivotal role in carbon fixation. However, despite its abundance, RuBisCO is kinetically slow and has a dual catalytic activity: it can bind either CO2 (carboxylation) or O2 (oxygenation) at its active site. The carboxylation reaction forms two 3-PGA molecules, which feed into the Calvin cycle. The oxygenation reaction, however, leads to photorespiration, forming one molecule of 3-PGA and one molecule of 2-phosphoglycolate. 2-phosphoglycolate is a metabolic dead-end and must be converted back to 3-PGA through a salvage pathway in peroxisomes and mitochondria, which consumes ATP and releases CO2. This process is considered wasteful as it reduces the efficiency of photosynthesis by consuming energy without fixing carbon. The ratio of carboxylation to oxygenation depends on the relative concentrations of CO2 and O2 at the active site and the enzyme's specificity for each substrate (KM values for CO2 and O2). In hot, dry conditions, stomata close, limiting CO2 uptake and increasing O2 concentration due to photosynthesis, thereby favoring photorespiration.

Steps of Carbon Fixation in Detail

  • Overall Stoichiometry for one triose phosphate (G3P) molecule: To produce one net G3P molecule for sugar synthesis, the Calvin cycle must process three CO_2 molecules.

    • Carbon Fixation: 3 imes CO_2 + 3 imes RuBP
      ightarrow 6 imes 3-PGA. (Catalyzed by RuBisCO)

    • Reduction of 3-PGA: For these six 3-PGA molecules, 6 imes ATP (for phosphorylation) and 6 imes NADPH (for reduction) are consumed to produce 6 imes G3P. One G3P exits the cycle (as a building block for sugars), and five G3P molecules remain in the cycle.

    • Regeneration of RuBP: The remaining 5 imes G3P molecules are rearranged through a series of complex reactions involving transketolase, aldolase, and isomerase enzymes, to regenerate 3 imes RuBP. This process consumes an additional 3 imes ATP molecules. Thus, the overall energy cost to fix three CO_2 molecules and produce one G3P is 9 imes ATP and 6 imes NADPH.

Interaction Between Processes

  • Balancing Act: The light-dependent and Calvin-Benson cycle reactions are tightly interdependent. A mismatch in the rates of electron transport (producing ATP and NADPH) and carbon fixation (consuming ATP and NADPH) can lead to imbalances. For example, if light energy exceeds the capacity of the Calvin cycle to utilize ATP and NADPH (e.g., under low temperatures that reduce enzyme activity or CO_2 limitations), the electron transport chain can become over-reduced, leading to the formation of harmful reactive oxygen species (ROS).

  • Temperature Effects: Photosynthetic processes have differential sensitivities to temperature:

    • Fast processes (e.g., light absorption, electron transfer kinetics within reaction centers, and diffusion of molecules) are largely temperature-independent within physiological ranges, as they primarily involve physical events.

    • Enzyme-catalyzed processes, particularly those of the Calvin cycle, vary significantly with temperature. Optimal temperature ranges exist for each enzyme, and deviations can lead to reduced efficiency or denaturation, thus impacting overall photosynthetic rates.

  • Light Conditions:

    • Under changing light conditions, especially from low to high light, the rate of electron transport can rapidly increase, sometimes overwhelming the Calvin cycle. This can lead to an accumulation of excited chlorophyll and an excess of electrons in the electron transport chain, promoting ROS buildup and photo-oxidative stress.

    • Conversely, under very low light, ATP and NADPH production may be insufficient to support the Calvin cycle adequately.

  • Regulatory Mechanisms: Plants employ various mechanisms to balance these processes, including the ferredoxin-thioredoxin system (which redox-regulates several Calvin cycle enzymes), changes in stroma pH and magnesium ion concentration (affecting RuBisCO activity), and feedback inhibition of electron transport components.

Photoprotection Mechanisms

  • Non-Photochemical Quenching (NPQ): A critical process for dissipating excess absorbed light energy as heat, thereby protecting the photosynthetic apparatus from photodamage, particularly in PSII. NPQ involves several mechanisms, primarily qE (energy-dependent quenching), which is activated by the low pH in the thylakoid lumen during high light conditions. This low pH induces conformational changes in certain LHCII subunits (e.g., PsbS protein) and activates the xanthophyll cycle enzymes, facilitating the rapid dissipation of excitation energy before it can form harmful ROS.

  • Xanthophyll Cycle: A key component of NPQ. This cycle involves the enzymatic conversion of xanthophyll pigments. Under high light intensity, the acidic lumen activates violaxanthin de-epoxidase (VDE), which converts violaxanthin (a di-epoxide, yellow) to antheraxanthin (a mono-epoxide) and then to zeaxanthin (a non-epoxide, yellow). Zeaxanthin interacts with LHCII proteins to induce conformational changes that promote energy dissipation as heat. Under low light, zeaxanthin epoxidase (ZE) converts zeaxanthin back to violaxanthin, replenishing the pool of light-harvesting pigments. This reversible conversion allows plants to rapidly adjust their light-harvesting efficiency.

  • Damage Repair: Photosystem II (PSII) is particularly vulnerable to photodamage, especially its D1 protein subunit, which is directly involved in water oxidation and electron transfer. To counteract this, plant cells have evolved efficient repair mechanisms. Damaged D1 protein is rapidly removed, degraded, and replaced by newly synthesized D1 protein, ensuring the continuous function of PSII. This active repair cycle is essential for maintaining photosynthetic efficiency under fluctuating light conditions.

Reactive Oxygen Species (ROS) Management

  • ROS Formation: Reactive Oxygen Species (ROS) are highly reactive molecules containing oxygen, such as superoxide radical (O2^{ullet-}), hydrogen peroxide (H2O2), and hydroxyl radical (OH^{ullet}). They are generated when the photosynthetic electron transport chain is over-reduced (e.g., under high light, low CO2, or low temperatures). Under these conditions, electrons can escape from the electron transport chain and directly reduce molecular oxygen (O2), a process known as the Mehler reaction (or water-water cycle), leading to the formation of O2^{ullet-} via PSI, which can then be converted to other more damaging ROS.

  • Antioxidants: Plants possess sophisticated enzymatic and non-enzymatic antioxidant systems to scavenge ROS and protect cellular components from oxidative damage.

    • Enzymatic Antioxidants: Include superoxide dismutase (SOD, converts O2^{ullet-} to H2O2), catalase (CAT, converts H2O2 to H2O and O_2), and the enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase).

    • Non-Enzymatic Antioxidants: Include carotenoids (e.g., eta-carotene and xanthophylls, quench singlet oxygen and other ROS, and dissipate excess energy), ascorbate (Vitamin C, a major water-soluble antioxidant), and tocopherols (Vitamin E, lipid-soluble antioxidants that protect membranes).

  • Impact of High Light: The over-accumulation of ROS, especially under prolonged high light stress, can lead to severe oxidative damage to photosynthetic pigments (e.g., chlorophylls), proteins (e.g., RuBisCO, D1 protein), membrane lipids (leading to lipid peroxidation), and DNA. This damage results in photoinhibition (a decline in photosynthetic efficiency), reduced plant growth, and ultimately, cell death. Effective ROS management is crucial for plants to adapt and survive in diverse and often stressful environmental conditions.