outline the three main stages of the Calvin cycle: rubisco catalyses the fixation of carbon dioxide by combination with a molecule of ribulose bisphosphate (RuBP), a 5C compound, to yield two molecules of glycerate 3-phosphate (GP), a 3C compound, GP

Photosynthesis – The Calvin (C₃) Cycle

Where the Cycle Takes Place

• Occurs in the stroma of the chloroplast, the fluid‑filled region surrounding the thylakoid membranes.
• Spatial separation from the light‑dependent reactions (which are confined to the thylakoid membranes) allows the ATP and NADPH produced by the light reactions to be used efficiently for carbon fixation without interference from the high‑energy electron transport chain.

Why the Calvin Cycle Is Important

• It is the primary pathway by which atmospheric CO₂ is converted into organic carbon (triose phosphates) – the first step in the synthesis of glucose, starch and other carbohydrates.
• Net carbon fixation (CO₂ → G3P) is opposed by the photorespiratory oxygenation reaction of Rubisco; the balance between these two reactions determines the overall efficiency of C₃ photosynthesis.
• In the wider context of the Cambridge syllabus, the Calvin cycle links the “energy‑capture” stage (light reactions) with the “energy‑use” stage (sugar synthesis) and therefore illustrates the flow of energy and matter in plants.

Overall Stoichiometry (per 3 CO₂)

• Inputs from the light‑dependent reactions: 9 ATP + 6 NADPH
• Outputs: 1 Glyceraldehyde‑3‑phosphate (G3P) net + 3 RuBP regenerated
• Carbon balance: 3 × CO₂ (3 C) → 1 × G3P (3 C)

Three Main Stages of the Calvin Cycle

1. Carbon Fixation

   • Enzyme: Ribulose‑1,5‑bisphosphate carboxylase/oxygenase (Rubisco)
   • Substrates: CO₂ + Ribulose‑1,5‑bisphosphate (RuBP, a 5‑C molecule)
   • Reaction: RuBP + CO₂ → unstable 6‑C intermediate → 2 × 3‑phosphoglycerate (3‑PGA, also called glycerate‑3‑phosphate, GP)
   • Energy input: none (reaction is driven by substrate binding)
   • Side reaction (photorespiration): Rubisco can also add O₂, producing 1 × 3‑PGA + 1 × 2‑phosphoglycolate; this wastes ATP and NADPH and is a common examination point.

2. Reduction

   • Enzymes: Phosphoglycerate kinase (PGK) and glyceraldehyde‑3‑phosphate dehydrogenase (GAP‑DH)
   • Steps:

     1. ATP phosphorylates each 3‑PGA → 1,3‑bisphosphoglycerate.
     2. NADPH reduces 1,3‑bisphosphoglycerate → glyceraldehyde‑3‑phosphate (G3P, a triose phosphate).

   • Products: 2 × G3P (one of which will be exported after three turns)
   • Energy input (per CO₂ fixed): 2 ATP + 2 NADPH

3. Regeneration of RuBP

   • Key enzymes (in order of appearance):

     • Triose‑phosphate isomerase (TP‑I)
     • Aldolase
     • Fructose‑1,6‑bisphosphate aldolase (sometimes listed as “aldolase” again)
     • Sedoheptulose‑1,7‑bisphosphatase (SBPase)
     • Trans‑ketolase (requires Mg²⁺)
     • Phosphoribulokinase (PRK)

   • Carbon rearrangement: 5 × G3P + 1 ATP → 3 × RuBP (each RuBP is 5 C)
   • Products: 3 × RuBP ready for the next turn and 1 × G3P that can leave the chloroplast for biosynthesis.
   • Energy input (per CO₂ fixed): 3 ATP

Summary Table (per CO₂ Fixed)

Link to the Light‑Dependent Reactions

• Photosystem II, Photosystem I, the cytochrome b₆f complex and ATP synthase generate the ATP and NADPH that power the reduction and regeneration phases.
• If light intensity falls, ATP and NADPH production drops, limiting the rate of carbon fixation even though Rubisco may still be saturated with CO₂.

Regulation of the Calvin Cycle (AO2)

• Activation of Rubisco: Carbamylation of a lysine residue in the active site (requires CO₂ and Mg²⁺) and binding of the co‑factor RuBP.
• Inhibition: Accumulation of 2‑phosphoglycolate (product of the oxygenation reaction) and low ATP/NADPH ratios can down‑regulate the cycle.
• Feedback control: High concentrations of G3P or sucrose signal that less RuBP needs to be regenerated, reducing the flow through the cycle.

Typical Limiting‑Factor Investigations (Cambridge Practical Context)

Candidates may be asked to design or interpret experiments that explore how the Calvin cycle responds to environmental changes.

• Light intensity: Vary illumination (e.g., 0, 200, 800 µmol m⁻² s⁻¹) and measure the rate of CO₂ uptake or O₂ evolution.
• CO₂ concentration: Use a sealed chamber with controlled CO₂ levels (e.g., 0.04 % – 2 %) and monitor photosynthetic rate.
• Temperature: Perform the assay at 10 °C, 25 °C and 35 °C to illustrate the temperature optimum and denaturation point of Rubisco.
• O₂ concentration (photorespiration): Compare rates under normal air (≈21 % O₂) with those under reduced O₂ (e.g., 2 % O₂, 98 % N₂) to highlight the oxygenation side‑reaction.
• Mg²⁺ availability: Since Mg²⁺ is required for Rubisco activation and several regeneration enzymes, varying Mg²⁺ in the medium can affect cycle speed.

Contrast with Other C₃ Pathways (Brief Note for Comparison Questions)

• C₄ plants: CO₂ is first fixed in mesophyll cells by phosphoenolpyruvate carboxylase (PEPC) to form a four‑carbon acid, which is then shuttled to bundle‑sheath cells where the Calvin cycle operates. This spatial separation reduces photorespiration.
• CAM plants: CO₂ is fixed at night into malate, stored in vacuoles, and released for the Calvin cycle during daylight. This temporal separation also limits photorespiration.
• Understanding these alternatives helps students answer higher‑order Cambridge questions that ask for advantages/disadvantages of the C₃ pathway.

Key Points to Remember

• Rubisco is the most abundant enzyme on Earth; it catalyses both carboxylation (CO₂ fixation) and oxygenation (photorespiration).
• One turn of the cycle fixes one CO₂; three turns are needed to produce one net G3P that can leave the chloroplast.
• Total energy cost per 3 CO₂ fixed = 9 ATP + 6 NADPH.
• Regeneration of RuBP is essential – without it the cycle stops even if ATP and NADPH are abundant.
• The Calvin cycle is tightly linked to the light‑dependent reactions; any factor that limits light‑energy capture (low light, shading, chlorophyll damage) indirectly limits carbon fixation.
• Regulatory mechanisms (Rubisco activation, feedback inhibition by G3P/2‑phosphoglycolate, ATP/NADPH ratios) are part of the AO2 “design/interpret investigations” requirement.