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 – Energy Transfer Processes (Cambridge A‑Level 9700)

Learning Objectives

• Describe the structure of the chloroplast and locate the light‑dependent and light‑independent reactions.
• Identify the main photosynthetic pigments, their absorption peaks, and how they are separated by chromatography.
• Explain the complete mechanism of non‑cyclic (linear) photophosphorylation, including the oxygen‑evolving complex (OEC), all electron carriers, and the synthesis of ATP and NADPH.
• Describe cyclic photophosphorylation, when it occurs, and why it supplies only ATP.
• Outline the Calvin‑Benson cycle, the enzymes involved and the consumption of ATP/NADPH.
• Contrast cyclic and non‑cyclic electron flow and state the physiological conditions that favour each.
• Design investigations to determine the limiting factors of photosynthesis (light intensity, wavelength, CO₂ concentration, temperature).
• Link photosynthesis to related topics such as cellular respiration and energy carriers.

1. Chloroplast Structure

Chloroplasts are double‑membrane organelles with three distinct compartments:

• Outer membrane – porous to small molecules.
• Inner membrane – encloses the stroma.
• Stroma – aqueous matrix that contains the Calvin‑Benson cycle enzymes, chloroplast DNA, ribosomes and the machinery for thylakoid formation.

The thylakoid system is organised into:

• Grana – stacks of flattened thylakoid discs. Each disc houses the two photosystems (PSII and PSI) together with their light‑harvesting complexes.
• Stroma lamellae (inter‑granal thylakoids) – unstacked thylakoids that interconnect the grana and contain the majority of ATP synthase complexes.

Thus, light‑dependent reactions occur in the thylakoid membranes (PSII in the grana, PSI in both grana and stroma lamellae) whereas the Calvin‑Benson cycle proceeds in the stroma.

2. Photosynthetic Pigments, Absorption & Action Spectra

Action spectrum (relative photosynthetic rate)

The action spectrum mirrors the combined absorption of the pigments, explaining why blue and red light drive photosynthesis most efficiently.

3. Pigment Chromatography

Paper (or thin‑layer) chromatography separates pigments according to polarity. A typical solvent system is petroleum ether : acetone (85 : 15).

4. Non‑Cyclic (Linear) Photophosphorylation

Linear electron flow transfers electrons from water to NADP⁺, producing both ATP and NADPH – the energy carriers required for the Calvin‑Benson cycle.

4.1 Step‑by‑Step Sequence

1. Light absorption by PSII (P680) – chlorophyll a is photo‑activated:
   Chl + hν → Chl*
2. Charge separation & primary electron acceptor – the excited electron is transferred to pheophytin, then to the plastoquinone pool (Q_A → Q_B).
3. Water splitting (OEC) – the oxygen‑evolving complex (Mn₄CaO₅ cluster) donates electrons to replace those lost from P680⁺:
   2 H₂O → 4 H⁺ + 4 e⁻ + O₂
4. Electron transport & proton pumping – electrons travel via plastoquinone to the cytochrome b₆f complex. This complex pumps H⁺ from the stroma into the thylakoid lumen, establishing a proton motive force.
5. Plastocyanin (PC) delivers electrons – the reduced PC carries electrons from cytochrome b₆f to PSI (P700).
6. Light absorption by PSI (P700) – a second photon excites P700, and the electron is passed to the iron‑sulphur protein A₀ and then to ferredoxin (Fd).
7. NADP⁺ reduction – ferredoxin‑NADP⁺ reductase (FNR) catalyses:
   NADP⁺ + H⁺ + 2 e⁻ → NADPH
8. ATP synthesis (chemiosmosis) – the H⁺ gradient drives ATP synthase (CF₁CF₀) located in the stroma lamellae:
   ADP + Pᵢ + H⁺out → ATP + H⁺in

4.2 Overall Stoichiometry

2 H₂O + NADP⁺ + ADP + Pᵢ + 4 hν → O₂ + NADPH + ATP

Four photons are required (two for PSII, two for PSI) to split two water molecules and reduce one NADP⁺.

4.3 Key Exam Points

• Both photosystems are essential – PSII supplies electrons, PSI supplies the energy to reduce NADP⁺.
• The OEC (Mn₄CaO₅) provides the electrons that replace those lost from P680⁺ and produces O₂.
• Proton pumping by cytochrome b₆f creates the electrochemical gradient that powers ATP synthase.
• Resulting products: O₂, NADPH, and ATP (≈ 3 ATP : 2 NADPH per water molecule).

5. Cyclic Photophosphorylation

Cyclic flow occurs when the plant needs extra ATP but NADPH is already sufficient (e.g., under high light intensity, low NADP⁺ availability, or when the Calvin cycle demands more ATP than NADPH).

5.1 Sequence of Events

1. Light excites P700 → P700*.
2. Electron is transferred to A₀ → ferredoxin (Fd).
3. Instead of reducing NADP⁺, ferredoxin donates the electron back to the plastoquinone pool.
4. Electrons re‑enter the cytochrome b₆f complex, which pumps additional H⁺ into the lumen.
5. The enlarged proton gradient drives ATP synthase to produce ATP only.

5.2 Overall Equation

ADP + Pᵢ + 2 hν → ATP

No O₂ is evolved and NADPH is not formed.

5.3 When It Occurs

• High light intensity where the supply of electrons exceeds the demand for NADPH.
• Low NADP⁺ concentration (e.g., after a rapid burst of linear flow).
• During the later stages of the light period when the Calvin cycle requires a higher ATP : NADPH ratio.

6. Light‑Independent Reactions (Calvin‑Benson Cycle)

The Calvin cycle fixes CO₂ into carbohydrate using the ATP and NADPH generated in the light‑dependent reactions.

6.1 Three Main Phases (per three‑carbon molecule, RuBP)

1. Carbon fixation – Rubisco catalyses:
   3 CO₂ + 3 RuBP (5‑C) → 6 3‑PGA
2. Reduction – ATP phosphorylates 3‑PGA; NADPH reduces it to glyceraldehyde‑3‑phosphate (G3P):
   6 3‑PGA + 6 ATP + 6 NADPH → 6 G3P + 6 ADP + 6 Pᵢ + 6 NADP⁺
3. Regeneration of RuBP – Five G3P molecules are rearranged (using 3 ATP) to regenerate three RuBP molecules, allowing the cycle to continue.

6.2 Net Reaction (per 6 CO₂)

6 CO₂ + 12 ATP + 12 NADPH + 6 H₂O → C₆H₁₂O₆ + 12 ADP + 12 Pᵢ + 12 NADP⁺

6.3 Key Enzymes

• Rubisco – carboxylates RuBP; also catalyses oxygenation (photorespiration).
• Phosphoglycerate kinase – ATP‑dependent phosphorylation of 3‑PGA.
• Glyceraldehyde‑3‑phosphate dehydrogenase – NADPH‑dependent reduction to G3P.
• Transketolase & aldolase – rearrange carbon skeletons during RuBP regeneration.

7. Investigating Limiting Factors of Photosynthesis

Cambridge examinations expect students to design simple, quantitative experiments that test which factor limits the rate of photosynthesis.

7.1 Typical Experimental Set‑ups

• Leaf‑disc flotation (oxygen evolution) – discs from Elodea or spinach are placed in a bicarbonate solution; the time taken for each disc to rise is recorded under different conditions.
• Closed gas‑exchange apparatus – measures O₂ evolution or CO₂ uptake with a digital gas sensor.
• Chlorophyll fluorescence (PAM fluorometer) – provides a rapid estimate of photosynthetic efficiency under varying light or temperature.

7.2 Example Data Table

7.3 Expected Conclusions

• Rate rises with light intensity until a light‑saturated plateau is reached.
• Blue and red light give the highest rates; green light is least effective, matching the action spectrum.
• Within physiological limits, increasing CO₂ accelerates the Calvin cycle until Rubisco becomes saturated.
• Temperature shows an optimum (≈ 25 °C for most temperate plants); rates fall at lower or higher temperatures due to enzyme kinetics and membrane fluidity.

8. Links to Other Topics

• Cellular respiration – NADH and FADH₂ from glycolysis, pyruvate oxidation and the Krebs cycle feed the mitochondrial electron transport chain, a process analogous to the chloroplast’s photophosphorylation.
• Energy carriers – ATP, NADPH and NADH are interchangeable “currency” molecules; the ATP : NADPH ratio produced by the light reactions (≈ 3 : 2) matches the demand of the Calvin cycle.
• Photorespiration – occurs when Rubisco fixes O₂ instead of CO₂; it reduces overall efficiency and is directly linked to the O₂ released in the light reactions.

9. Summary Table – Light‑Dependent Reactions

10. Suggested Diagram for Revision