explain that in non-cyclic photophosphorylation: photosystem I (PSI) and photosystem II (PSII) are both involved, photoactivation of chlorophyll occurs, the oxygen-evolving complex catalyses the photolysis of water, ATP and reduced NADP are synthesis

Photosynthesis – Non‑Cyclic Photophosphorylation (Cambridge AS & A Level)

Learning Objectives

• LO 13.1 – Describe the structure‑function relationships of the chloroplast, its pigments and the two photosystems (PSII & PSI).
• LO 13.1 – Explain the sequence of events in non‑cyclic photophosphorylation (photo‑activation, water splitting, electron transport, ATP synthesis and NADP⁺ reduction).
• LO 13.1 – Contrast non‑cyclic with cyclic photophosphorylation.
• LO 13.2 – Design and interpret investigations of factors that limit the light‑dependent reactions.
• LO 13.2 – Outline the Calvin (light‑independent) cycle and the use of ATP/NADPH.

1. Chloroplast – Structure & Function (LO 13.1)

• Outer membrane – porous, protects the organelle.
• Inner membrane – encloses the stroma; contains transport proteins.
• Stroma – aqueous matrix; site of the Calvin cycle and storage of enzymes, ribosomes, DNA.
• Thylakoid system

  • Grana (stacked thylakoids) – house PSII, the oxygen‑evolving complex (OEC) and most antenna pigments.
  • Stroma lamellae (unstacked thylakoids) – contain PSI, ATP synthase and the cytochrome b₆f complex.

2. Chloroplast Pigments (LO 13.1)

• Major pigments: chlorophyll a (λ_max ≈ 430 nm & 662 nm), chlorophyll b (λ_max ≈ 453 nm & 642 nm), carotene (λ_max ≈ 470 nm), xanthophylls (λ_max ≈ 440 nm).
• Absorption spectra – show two peaks for chlorophyll a (blue & red) and a broad blue‑green peak for carotenoids. The combined absorption spectrum matches the plant’s action spectrum, i.e. the wavelengths that drive photosynthesis most efficiently.
• Chromatography of pigments (practical note) – Thin‑layer chromatography (TLC) of leaf extracts separates pigments by Rf values:

  • Chlorophyll a Rf ≈ 0.90 (bright green band)
  • Chlorophyll b Rf ≈ 0.80 (pale green band)
  • Carotene   Rf ≈ 0.60 (orange band)

  The pattern of bands is a diagnostic test for pigment composition.

3. Overview of Non‑Cyclic Photophosphorylation (LO 13.1)

Linear electron flow from water to NADP⁺ produces both chemical energy (ATP) and reducing power (NADPH). The process is divided into three linked phases:

1. Light‑dependent reactions – photo‑activation of chlorophyll, photolysis of water, electron transport.
2. Generation of a proton motive force and synthesis of ATP (photophosphorylation).
3. Reduction of NADP⁺ to NADPH.

4. Photo‑Activation of Chlorophyll (LO 13.1)

• PSII: central pigment P680 absorbs a photon → excited state P680*.
• PSI: central pigment P700 absorbs a photon → excited state P700*.
• The excited pigment donates an electron to a primary electron acceptor, initiating the electron transport chain (ETC).

5. Water Splitting at the Oxygen‑Evolving Complex (OEC) (LO 13.1)

The loss of electrons from P680 creates a charge deficit that is replenished by oxidation of water at the Mn₄CaO₅ cluster of the OEC (bound to the D1 protein of PSII).

Overall photolysis reaction:

\$2\,\text{H}2\text{O} \;\longrightarrow\; 4\,\text{H}^+ + 4\,e^- + \text{O}2\$

• 4 e⁻ replace those lost by P680.
• 4 H⁺ are released into the thylakoid lumen, contributing to the proton gradient.
• O₂ is liberated to the atmosphere.

6. Electron Transport Chain (ETC) (LO 13.1)

1. Plastoquinone (PQ) – accepts two electrons from P680⁺ and carries them to the cytochrome b₆f complex while picking up two H⁺ from the stroma.
2. Cytochrome b₆f complex – transfers electrons to plastocyanin (PC) and pumps additional H⁺ from the stroma into the lumen (Q‑cycle).
3. Plastocyanin (PC) – soluble copper protein that delivers electrons to P700⁺ in PSI.

7. ATP Synthesis – Photophosphorylation (LO 13.1)

The proton motive force (ΔpH + Δψ) generated by the ETC drives the CF₁CF₀‑ATP synthase:

\$\text{ADP} + \text{P}i + \text{H}^+{\text{out}} \xrightarrow{\text{ATP synthase}} \text{ATP} + \text{H}_2\text{O}\$

• ≈ 3 ATP are formed per pair of electrons that travel from H₂O to NADP⁺ (exact yield depends on the plant species).

8. NADP⁺ Reduction at PSI (LO 13.1)

• P700* transfers an electron to the primary acceptor A₀, then via phylloquinone (A₁) to the iron‑sulphur protein ferredoxin (Fd).
• Ferredoxin‑NADP⁺ reductase (FNR) catalyses:

  \$\text{NADP}^+ + 2\,e^- + \text{H}^+ \;\longrightarrow\; \text{NADPH}\$

• Two photons are required for each NADPH molecule – one absorbed by PSII and one by PSI.

9. Summary Table – Key Components (LO 13.1)

10. Cyclic Photophosphorylation – Contrast (LO 13.1)

• When it occurs: under conditions of low NADP⁺ (e.g. high light, limited CO₂) to generate extra ATP.
• Electron flow: P700* → A₀ → A₁ → ferredoxin → plastoquinone pool → cytochrome b₆f → plastocyanin → back to P700⁺.
• Key features

  • Only PSI is involved – PSII, OEC and NADP⁺ are bypassed.
  • No water splitting → no O₂ evolution.
  • Electrons are recycled; each turn yields ≈ 1 ATP (no NADPH).
  • Enhances the ATP/NADPH ratio to meet the Calvin cycle’s demand for ATP under stress.

11. Calvin Cycle – Light‑Independent Reactions (LO 13.2)

Occurs in the stroma; uses ATP and NADPH produced by the light‑dependent reactions.

1. Carbon fixation

   • Enzyme: Ribulose‑1,5‑bisphosphate carboxylase/oxygenase (Rubisco).
   • Reaction: CO₂ + RuBP → 2 × 3‑phosphoglycerate (3‑PGA).

2. Reduction phase

   • Enzymes: phosphoglycerate kinase (PGK) and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH).
   • Reactions: 3‑PGA + ATP → 1,3‑bisphosphoglycerate (1,3‑BPG); 1,3‑BPG + NADPH → glyceraldehyde‑3‑phosphate (GAP) + ADP + NADP⁺.

3. Regeneration of RuBP

   • Enzymes: triose phosphate isomerase, aldolase, fructose‑1,6‑bisphosphatase, transketolase, ribose‑5‑phosphate isomerase, phosphoribulokinase.
   • Five of the six GAP molecules are used to regenerate three RuBP molecules, consuming 3 ATP.

4. Carbohydrate synthesis

   • Every 3 CO₂ fixed yields 1 GAP that can leave the cycle to form glucose, sucrose or starch.

12. Practical Investigation of Limiting Factors (LO 13.2)

Design a simple experiment to determine which factor (light intensity, CO₂ concentration, temperature) limits the rate of O₂ evolution.

1. Materials: leaf discs (spinach), bicarbonate solution, gas syringe or dissolved‑oxygen probe, light source with variable intensity, thermostatically controlled water bath.
2. Method (example – light intensity)

   • Place leaf discs in bicarbonate solution and seal the chamber.
   • Expose the chamber to a series of light intensities (e.g. 0, 100, 200, 400, 800 µmol m⁻² s⁻¹).
   • Record the volume of O₂ produced over a fixed time (e.g. 5 min) at each intensity.

3. Data analysis

   • Plot O₂ evolution (µmol O₂ min⁻¹) against light intensity.
   • The plateau indicates the light‑saturated rate; the region before the plateau shows light‑limited conditions.
   • Repeat the experiment while varying CO₂ (by adding NaHCO₃) or temperature (30 °C, 35 °C, 40 °C) to identify the factor that causes the earliest plateau.

4. Interpretation

   • If O₂ production continues to rise with increasing light but levels off when CO₂ is altered, CO₂ is the limiting factor, and vice‑versa.
   • Link the result to the role of ATP (light‑dependent) and NADPH (light‑independent) in the Calvin cycle.

13. Key Take‑aways

• Non‑cyclic photophosphorylation is a linear electron flow that couples light energy to the synthesis of both ATP and NADPH.
• Both photosystems are essential: PSII supplies electrons by splitting water; PSI provides the high‑energy electrons needed to reduce NADP⁺.
• The oxygen‑evolving complex is the source of atmospheric O₂ and contributes protons to the lumenal gradient.
• Proton pumping by the cytochrome b₆f complex creates the chemiosmotic drive for ATP formation via CF₁CF₀‑ATP synthase.
• Cyclic photophosphorylation supplements ATP production when NADP⁺ is scarce, without generating O₂ or NADPH.
• The ATP and NADPH generated are immediately used in the Calvin cycle to fix CO₂ into carbohydrate.
• Understanding limiting factors (light, CO₂, temperature) helps explain variations in photosynthetic rate in natural environments.