Calvin cycle (dark reactions / light-independent reactions / C3 cycle / Calvin-Benson-Bassham cycle) occurs in the stroma of chloroplasts. Uses ATP and NADPH from light reactions to fix CO₂ into organic compounds. Three stages: (1) Carboxylation: CO₂ fixed by RuBisCO. (2) Reduction: 3-PGA reduced to G3P using ATP + NADPH. (3) Regeneration: RuBP regenerated using ATP. For one glucose: 6 CO₂ fixed → requires 18 ATP + 12 NADPH. Net: 2 G3P → 1 glucose (via gluconeogenesis). 10 G3P → 6 RuBP (for cycle continuation). Discovery: Melvin Calvin, James Bassham, Andrew Benson (1950s) using radioactive ¹⁴CO₂ and chromatography. Calvin received Nobel Prize in Chemistry (1961).

RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase): most abundant enzyme on Earth (~500 million tonnes globally; ~0.5 kg per m² of leaf). Large, complex enzyme: hexadecamer = 8 large subunits (L) + 8 small subunits (S) = L₈S₈. Large subunit: encoded by chloroplast genome (rbcL gene). Small subunit: encoded by nuclear genome (rbcS gene). Two activities: Carboxylase (useful): RuBP + CO₂ → 2 × 3-PGA. Requires CO₂ and Mg²⁺. Oxygenase (wasteful): RuBP + O₂ → 3-PGA + 2-phosphoglycolate → photorespiration (releases CO₂, consumes ATP). At high CO₂/low O₂: mostly carboxylase. At low CO₂/high O₂: more oxygenase → photorespiration. Temperature increase: oxygenase activity increases faster → photorespiration more at high T. Solution in C4 plants: concentrate CO₂ around RuBisCO → suppress oxygenase → no photorespiration.

Stage 1 — Carboxylation: CO₂ + Ribulose-1,5-bisphosphate (RuBP, 5C) → (RuBisCO) → unstable 6C intermediate → 2 × 3-phosphoglycerate (3-PGA, 3C). Per 6 CO₂: 12 × 3-PGA produced (6 CO₂ + 6 RuBP → 12 × 3-PGA). Stage 2 — Reduction: 3-PGA → 1,3-bisphosphoglycerate (by kinase, uses ATP). → Glyceraldehyde-3-phosphate (G3P) (by NADP-glyceraldehyde-3-phosphate dehydrogenase, uses NADPH). Net: 12 ATP + 12 NADPH for 12 G3P. Stage 3 — Regeneration of RuBP: 10 of 12 G3P → 6 RuBP (via complex series of reactions including phosphoribulokinase). Uses 6 ATP. Remaining 2 G3P: used for synthesis of glucose, sucrose, starch, amino acids, fatty acids. Total per glucose: 18 ATP + 12 NADPH.

C4 pathway discovered by Hatch and Slack (1966) in sugarcane. Two-cell system: Mesophyll cells: CO₂ + PEP (3C) → OAA (4C) [by PEP carboxylase, no O₂ reaction]. OAA → malate (NADPH used) or aspartate. Transported to bundle sheath cells. Bundle sheath cells: malate/aspartate decarboxylated → CO₂ (high concentration) + pyruvate/alanine. CO₂ enters Calvin cycle (RuBisCO here). Pyruvate returns to mesophyll → regenerated to PEP (using ATP: pyruvate phosphate dikinase). Three types of C4 decarboxylation: NADP-ME type (maize, sugarcane): malate → pyruvate + CO₂ in bundle sheath chloroplast. NAD-ME type (millet, amaranth): aspartate → OAA → malate → pyruvate. PCK type: aspartate → OAA → PEP + CO₂. Advantage: CO₂ concentrated 10-20× around RuBisCO → suppresses oxygenase → no photorespiration → higher efficiency.

CAM (Crassulacean Acid Metabolism): photosynthetic pathway in succulent plants adapted to arid environments. Examples: Cacti, agave, pineapple (Ananas comosus), Aloe, many orchids, Sedum. Night-time: stomata OPEN (cooler, less water loss). CO₂ + PEP → OAA → malate (stored in vacuole as malic acid). Day-time: stomata CLOSED (hot, prevent water loss). Malate decarboxylated → CO₂ + pyruvate. CO₂ enters Calvin cycle (RuBisCO). Advantage: stomata closed during hot day → minimal water loss → water use efficiency very high. Disadvantage: slow growth (stomata closed most of day). CAM plants can switch between CAM and C3 (facultative CAM) when water is available. Similarities to C4: both use PEP carboxylase for initial fixation. Difference: C4 = spatial separation (two cell types); CAM = temporal separation (night/day).

PEP Carboxylase: found in cytoplasm of mesophyll cells (C4 and CAM plants). Substrate: PEP (phosphoenolpyruvate) + CO₂ → OAA. Has NO oxygenase activity → never fixes O₂ → no photorespiration. Very high affinity for CO₂ (Km for CO₂ much lower than RuBisCO) → works even at low CO₂. This is why C4 plants can fix CO₂ efficiently even when stomata are partially closed. RuBisCO: found in stroma of chloroplasts. Substrate: RuBP + CO₂ → 3-PGA. Has BOTH carboxylase AND oxygenase activity → photorespiration possible. Lower affinity for CO₂ → needs high CO₂ for efficient operation. In C4/CAM: PEP carboxylase pre-concentrates CO₂ → supplies high-CO₂ environment to RuBisCO → suppresses oxygenase activity.

Photorespiration occurs because RuBisCO has oxygenase activity. RuBP + O₂ → 3-PGA + 2-phosphoglycolate. 2-phosphoglycolate is toxic → recycled through the 'C2 cycle' or photorespiratory pathway: Chloroplast → Peroxisome → Mitochondria → back. In peroxisome: phosphoglycolate → glycolate → glyoxylate → glycine. In mitochondria: 2 glycine → serine + CO₂ + NH₃. The CO₂ released reduces net photosynthesis. The NH₃ must be re-assimilated (using ATP). Net result of photorespiration: loss of ~25% of photosynthetically fixed carbon in C3 plants. No useful energy produced. Increases with: high temperature, high O₂/low CO₂ ratio. Agricultural implication: suppressing photorespiration would dramatically increase C3 crop yields (wheat, rice, soybean). Engineering low-photorespiration RuBisCO or introducing C4 mechanisms into C3 crops (e.g., C4 rice project) are active research areas.

The light reactions provide ATP and NADPH for the Calvin cycle. Z-scheme of electron transport: H₂O (donor) → PSII → PQ → Cytb6f → PC → PSI → Fd → NADP⁺ reductase → NADPH. O₂ released from water splitting at PSII (by oxygen-evolving complex with Mn₄Ca cluster). ATP synthesis by chemiosmosis: H⁺ gradient built across thylakoid membrane (from water splitting + PQ cycling) → H⁺ flows through CF₁CF₀ ATP synthase → ADP + Pi → ATP. Cyclic photophosphorylation: PSI only → only ATP (no O₂, no NADPH). Provides extra ATP when needed. Ratio: Calvin cycle needs 3 ATP per CO₂ and 2 NADPH. Non-cyclic photophosphorylation provides ~2.5 ATP per NADPH → not enough ATP. Cyclic flow supplements. Quantum yield: ~8 photons needed per CO₂ fixed (4 each for PSII and PSI in non-cyclic pathway). Efficiency of photosynthesis: ~1-3% of total sunlight energy stored as chemical energy.