state that cyclic photophosphorylation and non-cyclic photophosphorylation occur during the light-dependent stage of photosynthesis

Photosynthesis – Light‑Dependent and Light‑Independent Reactions

Learning Objective

State that cyclic photophosphorylation and non‑cyclic photophosphorylation occur during the light‑dependent stage of photosynthesis, and explain why both pathways are required to meet the ATP : NADPH demand of the Calvin‑Benson cycle.

1. Structure of the Chloroplast

• Outer membrane – porous to small molecules and ions.
• Inner membrane – encloses the stroma; contains transport proteins for metabolites.
• Stroma – site of the Calvin‑Benson cycle (light‑independent reactions) and of enzymes such as Rubisco.
• Thylakoid system

  • Flattened sacs (thylakoids) stacked into grana.
  • Inter‑granal space (lumen) where a proton gradient is generated.
  • Thylakoid membrane – houses Photosystem II, Photosystem I, the cytochrome b₆f complex, plastoquinone (PQ), plastocyanin (PC), ferredoxin (Fd), ATP synthase and the oxygen‑evolving complex (OEC).

2. Pigments and Their Absorption

• Chlorophyll a – primary pigment; absorption peaks ≈ 430 nm (blue) and ≈ 662 nm (red).
• Chlorophyll b – accessory pigment; peaks ≈ 453 nm and ≈ 642 nm.
• Carotenoids (carotene, xanthophyll) – broaden the usable spectrum; absorb mainly 400–500 nm and protect against photodamage.
• Combined, these pigments allow plants to harvest a large proportion of the solar spectrum.

3. Light‑Dependent Reactions (Photophosphorylation)

3.1 Overview

These reactions take place in the thylakoid membrane. Light energy is used to (i) split water, (ii) generate a proton gradient, and (iii) produce the energy carriers ATP and NADPH.

3.2 Water Splitting – Oxygen‑Evolving Complex (OEC)

The OEC is a Mn₄CaO₅ cluster attached to the lumenal side of PSII. It catalyses the oxidation of water:

\[

2\text{H}2\text{O} \;\xrightarrow{\text{OEC}}\; \text{O}2 + 4\text{H}^+ + 4\text{e}^-

\]

• The four electrons replace those lost by the reaction‑centre chlorophyll (P680) in PSII.
• The four protons are released into the lumen, contributing to the H⁺ gradient.
• O₂ is expelled to the atmosphere.

3.3 Non‑Cyclic (Linear) Photophosphorylation

Electrons travel from water to NADP⁺, producing ATP, NADPH and O₂.

Overall electron flow:

\[

\text{H}2\text{O} \xrightarrow{\text{PSII}} \text{PQ} \rightarrow \text{Cyt}\,b{6}f \rightarrow \text{PC} \rightarrow \text{PSI} \xrightarrow{\text{Fd}} \text{FNR} \rightarrow \text{NADP}^+

\]

1. Photon absorption by PSII – excites electrons in P680.
2. Primary electron transport – electrons reduce plastoquinone (PQ → PQH₂); the cytochrome b₆f complex pumps H⁺ from stroma to lumen.
3. ATP synthesis – H⁺ flow back through ATP synthase drives phosphorylation of ADP.
4. Second excitation at PSI – P700 absorbs photons, raising electrons to a higher energy level.
5. NADPH formation – ferredoxin (Fd) transfers electrons to NADP⁺ via ferredoxin‑NADP⁺ reductase (FNR).

3.4 Cyclic Photophosphorylation

Electrons from PSI are recycled back to the plastoquinone pool, generating a proton gradient but no NADPH or O₂.

Electron flow:

\[

\text{PSI} \rightarrow \text{Fd} \rightarrow \text{PQ} \rightarrow \text{Cyt}\,b_{6}f \rightarrow \text{PC} \rightarrow \text{PSI}

\]

• Only ATP is produced (via chemiosmosis).
• Activated when the chloroplast requires more ATP relative to NADPH – for example, to satisfy the 3 ATP : 2 NADPH ratio of the Calvin‑Benson cycle.
• No water is split; therefore O₂ is not evolved.

3.5 Why Both Pathways Are Needed

The Calvin‑Benson cycle consumes ATP and NADPH in an approximate ratio of 3 ATP : 2 NADPH. Non‑cyclic photophosphorylation supplies both carriers, but the ATP generated is insufficient to meet the 3 ATP demand. Cyclic photophosphorylation provides the extra ATP required, allowing the overall ratio to be balanced.

3.6 Comparison of Cyclic and Non‑Cyclic Photophosphorylation

4. Light‑Independent Reactions – The Calvin‑Benson Cycle

The cycle occurs in the stroma and uses the ATP and NADPH produced in the light‑dependent reactions to fix CO₂ into carbohydrate.

4.1 Phases and Key Enzymes

1. Carbon fixation (3‑phosphoglycerate formation)

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

2. Reduction (formation of glyceraldehyde‑3‑phosphate)

   • Enzyme: Phosphoglycerate kinase (PGK) – phosphorylates 3‑PGA using ATP → 1,3‑bisphosphoglycerate.
   • Enzyme: Glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) – reduces 1,3‑bisphosphoglycerate using NADPH → glyceraldehyde‑3‑phosphate (G3P).
   • Enzyme: Triose phosphate isomerase (TPI) – interconverts G3P and dihydroxyacetone‑phosphate.

3. Regeneration of RuBP

   • Series of reactions using five more ATP molecules (per three CO₂ fixed) to convert five G3P molecules back into three RuBP molecules.
   • Key enzymes: Aldolase, Fructose‑1,6‑bisphosphatase, Transketolase, Ribulose‑5‑phosphate kinase, and Phosphoribulokinase (PRK).

Overall stoichiometry (per 3 CO₂ fixed):

• 9 ATP → 3 ADP + 3 Pᵢ
• 6 NADPH → 6 NADP⁺ + 6 H⁺
• Produces one net G3P molecule that can leave the cycle for carbohydrate synthesis.

5. Action Spectra & Absorption Spectra

• Absorption spectrum – plots the proportion of light absorbed by a pigment at each wavelength.
• Action spectrum – plots the rate of a photosynthetic process (e.g., O₂ evolution or CO₂ fixation) against wavelength; it mirrors the combined absorption of all pigments.
• Obtained by exposing a leaf or algal culture to monochromatic light of different wavelengths and measuring the resulting O₂ evolution or gas exchange.

6. Practical Investigations of Limiting Factors

6.1 Light Intensity

1. Place a leaf disc in a sealed gas‑evolution apparatus (e.g., a graduated syringe).
2. Vary the distance between the leaf and a white light source (e.g., 5 cm, 10 cm, 15 cm, 20 cm).
3. Record the volume of O₂ produced over a fixed time (e.g., 5 min).
4. Plot O₂ volume against light intensity; expect a hyperbolic rise to a plateau.

6.2 CO₂ Concentration (Detailed Protocol)

1. Prepare a series of sodium bicarbonate solutions (e.g., 0 mM, 5 mM, 10 mM, 20 mM) in distilled water.
2. Bubble each solution with air to equilibrate CO₂, then transfer 50 mL of the solution to a sealed gas‑evolution apparatus containing a fresh leaf disc.
3. Maintain constant light intensity (use a fixed distance from the light source).
4. Measure O₂ evolution for a set period (e.g., 5 min) and record the volume.
5. Plot O₂ volume against the known CO₂ concentration; the curve should rise linearly at low concentrations and level off at saturation.

6.3 Temperature

1. Conduct the light‑intensity experiment at three temperatures (e.g., 10 °C, 20 °C, 30 °C) using a water bath.
2. Plot O₂ evolution versus temperature; the rate increases to an optimum then declines sharply at higher temperatures.

6.4 Water Availability

1. Place leaf discs in solutions of differing osmotic potential (e.g., distilled water, 0.2 M sucrose, 0.5 M sucrose).
2. Observe stomatal behaviour and record O₂ evolution under constant light.
3. Reduced O₂ evolution indicates limitation by water stress.

7. Identification of Pigments by Chromatography

Paper chromatography is the standard method in the Cambridge syllabus.

1. Extract pigments from fresh spinach in 80 % acetone (keep in the dark).
2. Spot 2 µL of the extract onto a strip of filter paper; allow it to dry.
3. Develop the strip in a solvent mixture (e.g., petroleum ether : acetone = 70 : 30) in a chromatography chamber.
4. When the solvent front has moved ~10 cm, remove the paper, dry, and visualise the bands under white light.
5. Measure the distance each pigment band travelled and calculate its R_f value:

R_f = (distance moved by pigment) / (distance moved by solvent front)

Typical R_f values (paper chromatography, 70 : 30 solvent):

• Chlorophyll a ≈ 0.70
• Chlorophyll b ≈ 0.55
• Carotene ≈ 0.30

8. Quantitative Aspects

• Photon energy – \(E = \dfrac{hc}{\lambda}\)

  • h = 6.63 × 10⁻³⁴ J·s, c = 3.00 × 10⁸ m s⁻¹, λ = 680 nm (PSII peak).
  • Result: \(E \approx 2.9 \times 10^{-19}\) J per photon (≈ 1.8 eV).

• ATP yield per photon – Roughly 1 ATP is synthesised for every 3 photons absorbed by PSII and every 2 photons by PSI (overall ≈ 4 photons → 1 ATP).
• Overall stoichiometry (per O₂ evolved)

  • 8 photons → 4 ATP (via a combination of cyclic and non‑cyclic pathways) + 2 NADPH.
  • These carriers can fix 1 CO₂ in the Calvin‑Benson cycle (requires 3 ATP + 2 NADPH).

9. Links to Other Syllabus Topics

• The ATP and NADPH generated in the light‑dependent reactions are the energy carriers used in the Calvin‑Benson cycle (Topic 13) and also feed into cellular respiration (Topic 12) when sugars are catabolised.
• Understanding how cyclic photophosphorylation adjusts the ATP : NADPH ratio is essential for discussing metabolic integration and energy flow in living organisms.