Photosynthesis: Light and Dark Reactions

General Properties of Photosynthesis

• Carbon dioxide is fixed during photosynthesis
• 6 CO₂ and 6 H₂O yields C6H12O6 and 6O2
• The chloroplast is the location of photosynthesis
• The inner membrane encloses the stroma, a concentrated solution of enzymes, DNA, RNA, and ribosomes
• The stroma surrounds the thylakoid membrane which encloses lumen
• Granum: stack of thylakoid vesicles (sacs)
• Lamellae: Unstacked folds of thylakoid membrane
• H₂O is the ultimate electron donor for photosynthetic NADP+ reduction
• The overall reaction for CO2 fixation is 12 NADPH + 12 H+ + 18 ATP + 6 CO2 + 12 H2O yields C6H12O6 + 12 NADP+ + 18 ADP + 18 Pi
• A more general equation for photosynthesis is CO2 + 2 H2A → (CH2O)n + 2 A + H2O, where A = O or S

Light and Dark Reactions

• Light reactions utilize light energy to generate NADPH and ATP
  • Occur in the Thylakoid membrane
  • O2 evolution from H2O, a poor electron donor (Eº′ = + 0.82)
• Dark reactions use NADPH and ATP to synthesize carbohydrate from CO2 and H2O, these reactions are light-independent
  • Occur in the Stroma
  • Carbon fixation ("carbon assimilation") rxns
  • Other oxidizable substrates like (H2S) can be used instead of water

Thylakoid Membrane

• The Z scheme components, which mediate e- transport from H2O to NADPH, are organized into thylakoid membrane-bound complexes
• These complexes are linked by mobile e- carriers (PQ, PC, Fd pools)
• PSII, cytochrome b6f complex, PSI and CF1CF0-ATP synthase are major components
• Charge separation occurs across the membrane, with a negative stroma and positive lumen
• Proton translocation at Cyt b6f complex into lumen happens through chemiosmotic coupling
• The Oxygen Evolving Complex (OEC) releases H+ into the lumen

PSII in Cyanobacteria

• Similar to high plants & green algae
• Generates a strong oxidant capable of oxidizing H2O at the OEC
• 2 H2O + 2 PQ + 4 hν yields O2 + 2 PQH2 + 4 H+
• Exhibits asymmetric e- transfer through PSII

Water Oxidation in PSII

• O2 is generated through a five-stage water-splitting reaction mediated by an Mn-containing protein complex called the oxygen evolving complex (OEC)
• 2 H2O yields O2 + 4e- + 4 H+
• The OEC cycles through 5 states when flashed with light
• 4 e- are stripped away one at a time in light-driven reactions (S0 → S4)
• Recovery step (S₄ → So) is light-independent where O2 is released
• Protons from water are released into the lumen
• Mn – Cluster cycles through a series of oxidation states: Mn (II), Mn (III), Mn (IV)

Electron Pathway After Leaving Water

• H2O → P680 (PSII) → Plastoquinone Pool → Cyt b6f → Plastocyanin → P700 (PSI) → Ferrodoxin → Ferredoxin:NADP+ Reductase → NADPH

PSI in Cyanobacteria

• Similar to higher plants and algae
• Generates a strong reductant capable of reducing NADP+
• All e- transfer prosthetic groups are localized in PsaA, PsaB, and PsaC
• PsaA & PsaB form the RC heterodimer
• PsaC/PsaD serve as the docking site for ferredoxin (Fe-S protein)
• PsaF is the docking site for plastocyanin
• PSI contains 12 protein subunits and 127 cofactors

ATP Synthesis Driven by Light.

• Mechanism of photophosphorylation at CF1CF0-ATP synthase is chemiosmotic
• Protons at OEC, Cyt b6f, Q cycle, removal of H+ on stromal side by FNR as NADPH is produced
• The proton gradient drives ATP synthesis at CF₁CF0 - ATP synthase
• 1.5 protons translocated is equal to 1 quantum of light, 8 quanta is equal to 12 H+
• ApH = 3 pH units
• Stroma pH 8
• Lumen pH 5

Non-Cyclic and Cyclic Photophosphorylation

• Two fates for e- generated at PSI:
  • Reduction of NADP+ → NADPH and synthesis of ATP – Non-cyclic photophosphorylation
  • Returns to PSI (P700+) via cytochrome b6 & plastoquinone & plastocyanin - No NADP+ reduction, no O2 evolution, and proton translocation at Cyt b6f during ATP synthesis, this is cyclic photophosphorylation
• More productive ATP generation compared to non-cyclic photophosphorylation
• Cyclic is only ~10% of the rate of non-cyclic photophosphorylation
• Provides a means to overcome ATP deficiency for CO2 fixation

Electron Pathway through PSII & PSI

• Photosynthesis occurs through the actions of two photosynthetic reaction centers connected in series to produce O₂
• There is electron flow from H2O to NADP+
• When a molecule absorbs a photon of light, then valence e- fill inner shell and valence e- jumps to outer shell.

Capturing Solar Energy with Chlorophyll

• Chlorophyll is a Mg2+—containing substituted tetrapyrrole
• A principle photoreceptor in photosynthesis
• Contains a cyclopentenone ring (V) fused to a tetrapyrrole
• Chlorophyll a (Chl a) has R₁₁ = - CH3
• Chlorophyll b (Chl b) has R₁₁ = - CHO
• Complimentary absorption spectra with 2X as much Chl a than Chl b in plants

Light Absorption and Transduction Model

• Photooxidation of Chl to Chl* (excited state) with strong e- donor
• Electron transfer: Chl* + A → Chl+. + A-, where A represents an electron acceptor
• This produces a Cationic free radical intermediate
• A- + NADP+ → A + NADPH in Electron Transfer Pathway
• Electron transfer: B + Chl+· → Chl + B+
• Electron Transfer Pathway: B+ + H2O → B + O2 from Original state
• Proton translocation occurs as electron transfer occurs

Light Energy Capture in Photosynthesis

• Light absorbed by antenna chlorophylls is transferred to photosynthetic reaction centers (RCs)
• Each quantum of light energy has 4 fates: Loss as heat Fluorescence Resonance energy transfer Photooxidation
• Antenna chlorophylls are light harvesting pigments Many resonance energy transfer of light energy
• Reaction center " special Pair" of chlorophylls
• The Reaction Center (RC) Traps light energy because its lowest excited state is lower than the other antenna chlorophylls
• RC are photochemically active

Other Light Harvesting Pigments

• Accessory Light-Harvesting Pigments Carotenoids – Light harvesting & photoprotection through destruction of reactive oxygen species ẞ - carotene (orange) & Lutein (yellow)
• Phycobilins - Light harvesting pigments in cyanobacteria & red algae Phycoerythrin (red) & phycocyanin (blue)

Photosystems

• Photosystem I (PSI)
  • Generates strong reductant that reduces NADP+
  • Ferrodoxins are terminal e- acceptors
  • P700 (Chl a special pair) - RC
  • Produces NADPH
• Photosystem II (PSII)
  • Generates strong oxidant that oxidizes H2O
  • Quinones are terminal e- acceptors
  • P680 (Chl a special pair) - RC
  • Light-driven O2 evolution by oxidizing H2O
• Oxygenic phototrophs have two photosystems
• Photosynthetic bacteria contain only one reaction center with no PSI or PSII
• Electron transfer occurs between PSII and PSI to pump protons for chemiosmotic ATP synthesis
• Photophosphorylation – Light driven phosphorylation of ADP → ATP

Quantum Yield of Photosynthesis

• Quantum yield: Amount of product formed per equivalent of light input
• Fixation of CO2 to form hexose requires considerable energy as 12 NADPH and 18 ATP
• 4 hν per rxn. center pair (8 quanta) drives the evolution of 1 O2, reduction of 2 NADP+ to 2 NADPH, translocation of 12 H+ & synthesis of 2.57 ATP
• Einstein (E): the amount of energy in Avagadro’s number of photons: E = Nhc / 
• 1 Einstein (of 700 nm light) = 170 kJ
• Eight Einsteins of energy (1360 kJ) yields 2 moles of NADPH, 2.57 moles of ATP, and 1 mole of O2

Carbon Dioxide Usage

• Ribulose bisphosphate carboxylase/oxygenase (RuBisCO)
• RuBisCO catalyzes addition of CO2 (as in Calvin cycle) or O2 (as in photorespiration)

Calvin Cycle

• Light reactions use solar energy to make NADPH and ATP
• Dark reactions use NADPH and ATP to convert CO2 to Carbohydrates
• The Net Reaction is 6 CO2 + 18 ATP + 12 NADPH + 12 H+ + 12 H2O → Glucose + 18 ADP + 18 Pi + 12 NADP+
• Consists of two stages, Production and Recovery
• The Production Phase net synthesis of 2 G3P for 6 CO2 and is driven by ATP and NADPH
• 2 G3Ps used in the biosynthesis of glucose
• The Recovery phase has remaining carbon atoms shuffled to reform RuBP

Calvin Cycle Roles

• Predominant CO2 fixation pathway in nature
• Reduces 3-phophoglycerate to glyceraldehyde-3-phosphate for easier synthesis of carbohydrates
• Catalyzes reactions that transform 3-carbon compounds into 4-, 5-, 6-, and 7- carbon compounds

Calvin Cycle Regulation

• A light-sensitive control mechanism to prevent the Calvin cycle from the catabolic products in glycolysis and the TCA cycle
• Light stimulates Calvin cycle from CO2 fixation
• No Light inhibits the Calvin cycle and CO2 fixation
• CO2 fixation is activated as the stromal pH increases -Activates RuBisCO, RuBisCO activates fructose-1,6 bisphosphatase, ribulose-5-phosphate kinsae, and glyceraldehyde-3-phosphate dehydrogenase
• The Reducing power, which consists of NADPH and reduced Ferrodoxin, is generated by light -Activates reduced forms of fructose-1,6- bisphosphatase, sedoheptulose-1,7-bisphosphatase, and ribulose-5-phosphate kinase
• Light induces movement of Mg2+ into the stroma -Activates RuBisco & fructose-1,6-bisphosphatase
• Fructose bisphosphatase is a key control point in the Calvin Cycle

Photorespiration Limit

• Oxygenase reaction diminishes plant productivity because it leads to a loss of RuBP, which leads to - No RuBP, no CO2 acceptor in Calvin cycle
• O2 competes with CO2 as a substrate for RuBisCO
• Light-related uptake of O2 and release of CO2 -At high O2 levels -Photorespiration dissipates ATP & NADPH, limites growth rate of plants
• The levels are depleted on bright hot days
• all RuBisCOs show oxygenase activity, its thought evolutionary photosynthesis evolved, so it was adapted for a time when Earth’s atmosphere had high CO 2 and very little O2

Hatch-Slack (C4 Cycle) Pathway

• The C4 cycle concentrates CO2 in certain plant species’ to prevent photorespiration
• It functions as a CO2 delivery system, carrying CO2 from a leaf's surface to the interior, where O2 levels are lower
• This protects compartments from high [O2] and photorespiration
• The C4 plants consist of sugarcane, corn, weeds, and tropical grass
  • They make up around 5% of terrestrial plants
• 4-carbon CO2 carrier = malate or aspartate
• Intracellular transport of CO2 costs 2 ATP as ATP + Pyruvate + Pi yields AMP + PPi + PEP
• CAM plants like Cacti, have stomata that open at night to take up CO2 and stores this CO2 as malate in the vucuoles at night and avoid of heat loss during the day and open