Calvin Cycle & Photorespiration

Describe the main features and phases of the Calvin cycle (carbon fixation / reduction / regeneration).
Recognise the overall balanced reaction for carbon fixation and where it occurs in the leaf.
Identify key substrates (e.g., $CO_2$ , $RuBP$ , $ATP$ , $NADPH$ ) and key products (e.g., triose-P, glucose).
Appreciate Rubisco’s dual activity (carboxylase + oxygenase) and the consequences for photorespiration.
Understand, in outline, the photorespiratory pathway and its energetic / metabolic costs.

Photosynthesis consists of two integrated modules:
- Light-dependent reactions (lecture 1) ⟶ generate $ATP$ and $NADPH$ in the thylakoid membrane.
- Light-independent reactions (Calvin cycle, this lecture) ⟶ use those reductants/energy in the chloroplast stroma to fix carbon.
All spent cofactors ( $ADP$ , $NADP^+$ ) loop back to the electron-transport chain for re-reduction.

A fully balanced form for one glucose:
$6CO2 + 12NADPH + 18ATP + 12H^+ \;\longrightarrow\; C6H{12}O6 + 12NADP^+ + 18ADP + 18Pi + 6H2O$
Six complete turns of the Calvin cycle (one $CO_2$ fixed per turn) are required to net one hexose.

Calvin cycle enzymes reside in the chloroplast stroma (outside grana/thylakoid stacks).
Light reactions remain embedded in thylakoid membranes; spatial separation helps avoid futile redox loops.

Name: Rubulose-1,5-bisphosphate carboxylase/oxygenase.
Quaternary structure: 16 subunits
- $8$ large (L) subunits – encoded by the chloroplast genome.
- $8$ small (S) subunits – encoded by nuclear genes; protein imported post-translation.
Functional implications:
- Integration of two genetic compartments is essential for Rubisco biogenesis.
- Enzyme possesses both carboxylase and oxygenase active sites (same catalytic pocket) → efficiency/ specificity trade-off.

Substrate: RuBP (ribulose-1,5-bisphosphate, 5C, 2 P groups).
Mechanism (simplified):
1. Formation of an enediol intermediate on $C_2$ of RuBP.
2. Addition of $CO_2$ → transient β-keto 6-carbon intermediate.
3. Hydrolytic cleavage (requires $H_2O$ ) → 2 × 3-phosphoglycerate (3-PGA).
Net reaction:
$RuBP \;(5C) + CO2 + H2O \xrightarrow{Rubisco}\; 2\;\times\; 3\text{-PGA (3C)}$

Each 3-PGA is sequentially:
1. Phosphorylated by phosphoglycerate kinase (consumes $ATP$ ) → 1,3-bisphosphoglycerate.
2. Reduced by glyceraldehyde-3-phosphate dehydrogenase (consumes $NADPH$ ) → glyceraldehyde-3-phosphate (G3P).
Stoichiometry per 1 turn (1 $CO_2$ ): $2ATP + 2NADPH$ .
Fate of G3P:
- Minor fraction exits cycle → sucrose/starch synthesis & central metabolism.
- Majority proceeds to regeneration.

A complex network of 5C/4C/3C sugar phosphate rearrangements (transketolase, aldolase, aldolase, etc.).
Key end points:
- Produce ribulose-5-phosphate (Ru5P).
- Phosphoribulokinase uses $ATP$ to add a second phosphate → RuBP (ready for another turn).
Energetics per 1 turn:
- Additional $ATP$ cost → total cycle demand $3ATP + 2NADPH$ per $CO_2$ .

1940s-50s: fed algae $^{14}CO_2$ pulses + rapid quenching → chromatographically separated labelled intermediates.
Mapped the entire sequence of carbon transfers, earning the trio the 1961-64 Nobel Prize in Chemistry (officially awarded to Melvin Calvin, though Benson & Bassham made equal contributions).
Innovation: combined radiotracer kinetics & paper chromatography – foundation for modern metabolic flux analysis.

Competing reaction:
$RuBP + O_2 \xrightarrow{Rubisco}\; 3\text{-PGA (3C)} + 2\text{-phosphoglycolate (2C)}$
Consequences:
- Carbon & energy loss; 2-phosphoglycolate is toxic/inhibitory to multiple chloroplast enzymes.
- Necessitates an auxiliary salvage pathway – photorespiration – to recycle carbon and detoxify.

Chloroplast
- 2-phosphoglycolate ⟶ (dephosphorylation) ⟶ glycolate.
Peroxisome
- Glycolate + $O2$ ⟶ glyoxylate + $H2O_2$ (via glycolate oxidase).
- Transamination with glutamate ⟶ glycine.
Mitochondrion
- 2 glycine ⟶ serine + $CO2$ + $NH3$ (requires $NAD^+$ → $NADH$ ).
Peroxisome (return)
- Serine ⟶ glycerate (reduces $NADH$ ).
Chloroplast (final)
- Glycerate + $ATP$ ⟶ 3-PGA (re-enters Calvin cycle).

Energetic / material cost (per oxygenation):

Evolutionary backdrop:
- Rubisco arose when atmospheric $O_2$ was low; selection pressure for specificity minimal.
Biochemical constraints:
- $CO2$ and $O2$ are small, non-polar and similar in size – difficult to discriminate without slowing catalysis.
Potential functional upsides of photorespiration:
- Acts as an energy sink protecting photosystems from photoinhibition under high light / low $CO_2$ .
- Intersects nitrogen metabolism (e.g., glycine ⇄ serine cycle), possibly aiding nitrate reduction balance.

Six turns; cumulative demand:
$\underline{18ATP + 12NADPH + 6CO2 \;\rightarrow\; C6H{12}O6 + 18ADP + 18P_i + 12NADP^+}$

Rubisco composition: $8L + 8S = 16$ subunits.
ATP / NADPH cost: $3ATP + 2NADPH$ per $CO_2$ (⇒ $18ATP + 12NADPH$ per glucose).
Calvin cycle phases: Fixation → Reduction → Regeneration.
Location sequence for photorespiration: Chloroplast → Peroxisome → Mitochondria → Peroxisome → Chloroplast.

Links back to electron-transport (lecture 1): photochemical energy conversion drives stromal carbon chemistry.
Forward link to lectures 3 & 4: diversity in carbon-fixation strategies (C₄, CAM) evolved to suppress photorespiration under specific environments.
Practical implications: Rubisco specificity & photorespiratory loss currently cap crop photosynthetic efficiency; bioengineering aims (e.g., introducing faster algal Rubiscos or bypass pathways) target these bottlenecks.
Ethical / social angle: Enhancing Calvin-cycle efficiency could boost global food security but raises ecological trade-offs (e.g., land-use, biodiversity).

“Sensible” video recap of the Calvin cycle (link provided in lecture slides).
Musical version for auditory/kinesthetic learners – demonstrates steps through song/animation.