Regulation of Carbon fixation reactions

Dr. Riddhi Datta

An induction period precedes the steady state of photosynthetic CO₂ assimilation

The rate of CO₂ fixation increases with time in the first few minutes after the onset of illumination—this is called the induction period.

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Reason:

1. The activation of photosynthetic enzymes by light
2. Increase in the concentration of intermediates of the Calvin–Benson cycle

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During induction period:

Six triose phosphates formed in the carboxylation and reduction phases of the Calvin–Benson cycle are used for the regeneration of the ribulose 1,5-bisphosphate.

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At steady state:

Five of the six triose phosphates formed contribute to the regeneration of ribulose 1,5-bisphosphate, while a sixth triose phosphate is used in the chloroplast for starch formation and in the cytosol for sucrose synthesis.

Dr. Riddhi Datta

Regulation of Calvin-Benson Cycle

• The amounts of enzymes present in the chloroplast stroma are regulated by the concerted expression of nuclear and chloroplast genomes.

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• Regulatory signaling between nucleus and plastids is mostly anterograde — that is, the products of nuclear genes control the transcription and translation of plastid genes.

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• In contrast, posttranslational modifications can rapidly change the specific activity of chloroplast enzymes.

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• Light-dependent mechanisms regulate the specific activity of five pivotal enzymes within minutes of the light–dark transition:

• Rubisco

• Fructose 1,6-bisphosphatase

• Sedoheptulose 1,7-bisphosphatase

• Phosphoribulokinase

• NADP–glyceraldehyde-3-phosphate dehydrogenase

Dr. Riddhi Datta

Rubisco-activase regulates the catalytic activity of rubisco

The CO₂ molecule plays a dual role in the activity of rubisco:

• It transforms the enzyme from an inactive to an active form (activation)
• It is also the substrate for the carboxylase reaction (catalysis)

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Activation

The catalytic activities of rubisco require the formation of a lysyl-carbamate (E–NH-CO₂-) by a molecule of CO₂, called activator CO₂.

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The subsequent binding of Mg²⁺ to the carbamate stabilizes the carbamylated rubisco (E–NH–CO₂– •Mg²⁺) and converts rubisco to a catalytically competent enzyme.

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In the stroma of illuminated chloroplasts, the increase in both pH and concentration of Mg²⁺ facilitates the formation of the (E–NH– CO₂– • Mg²⁺) complex.

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Catalysis

Another molecule of CO₂ —substrate CO₂ —can then react with ribulose 1,5-bisphosphate (RuBP) at the active site of rubisco, releasing two molecules of 3-phosphoglycerate.

Dr. Riddhi Datta

Rubisco-activase regulates the catalytic activity of rubisco

• Sugar phosphates like xylulose 1,5-bisphosphate prevent activation and inhibit catalysis by binding tightly to the uncarbamylated rubisco.

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• The naturally occurring inhibitor 2-carboxyarabinitol 1-phosphate also inhibit catalysis by binding tightly to the uncarbamylated rubisco.

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• The substrate (RuBP) prevent activation by binding tightly to the carbamylated rubisco.

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• Plants and green algae overcome this inhibition with the protein rubisco activase, which removes the sugar phosphates from the uncarbamylated and carbamylated rubisco, thus allowing rubisco to be activated through carbamylation and Mg²⁺ binding.

Dr. Riddhi Datta

• Rubisco activase (blue) switches Rubisco (yellow) from an inactive to an active form.
• This involves an ATP-dependent release of tight-binding sugar phosphates such RuBP.
• A Rubisco active site that has spontaneously lost CO₂ (decarbamylation) (step 2) binds RuBP (step 3) causing conformational changes that produce a dead-end complex consisting of inactive Rubisco and tightly bound RuBP.
• Rubisco activase physically interacts with Rubisco (step 5), changing its conformation (step 6) to one that binds RuBP less tightly.
• ATP hydrolysis by rubisco activase is required for these conformational changes, perhaps for priming activase (step 4).
• Because of the lower affinity, RuBP dissociates from the active site of Rubisco (step 7), which frees the site for subsequent carbamylation (step 1) or rebinding of RuBP (step 3), but probably after dissociation of activase (step 8).

At high temperatures, the enzyme Rubisco deactivates faster (steps 2 &3) than it can be reactivated by activase due to limitations in ATP hydrolysis or the interaction between activase and Rubisco (steps 5&6).

Rubisco-activase regulates the catalytic activity of rubisco

Jensen (2000) PNAS

Dr. Riddhi Datta

Light regulates the Calvin–Benson cycle via ferredoxin–thioredoxin system

Light regulates catalytic activity of the following four enzymes directly via the ferredoxin-thioredoxin system:

• fructose 1,6-bisphosphatase
• sedoheptulose 1,7-bisphosphatase
• Phosphoribulokinase
• NADP–glyceraldehyde 3-phosphate dehydrogenase

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The following proteins are involved in the process:

• ferredoxin (reduced by the photosynthetic electron transport chain)
• ferredoxin–thioredoxin reductase
• thioredoxin

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Dr. Riddhi Datta

Light regulates the Calvin–Benson cycle via ferredoxin–thioredoxin system

• The ferredoxin–thioredoxin system links the light signal sensed by thylakoid membranes to the activity of enzymes in the chloroplast stroma.
• Light transfers electrons from water to ferredoxin via the photosynthetic electron transport system.
• Reduced ferredoxin, together with two protons, is used to reduce a catalytically active disulfide bond of the iron–sulfur enzyme ferredoxin–thioredoxin reductase, which in turn reduces the unique disulfide bond of the regulatory protein thioredoxin (Trx).
• The reduced form of thioredoxin then reduces regulatory disulfide bonds of the target enzyme, triggering its conversion to the catalytically active form.

Dr. Riddhi Datta

Light regulates the Calvin–Benson cycle via ferredoxin–thioredoxin system

• Darkness halts the electron flow from ferredoxin to the enzyme, and thioredoxin becomes oxidized.
• This ultimately inactivates the target enzymes.

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Dr. Riddhi Datta

Light-dependent ion movements modulate enzymes of the Calvin–Benson cycle

In light:

• Protons flow from the stroma to the thylakoid lumen.
• Mg²⁺ is released from the intrathylakoid space to the stroma.
• This decreases the stromal concentration of protons and increase that of Mg²⁺ which, in turn, activates enzymes of the Calvin–Benson cycle:
• Rubisco
• Fructose 1,6-bisphosphatase
• Sedoheptulose 1,7-bisphosphatase
• Phosphoribulokinase

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In darkness:

• The modifications of ionic composition of the chloroplast stroma are reversed rapidly upon darkening.

Dr. Riddhi Datta

Light controls the assembly of chloroplast enzymes into supramolecular complexes

• The formation of supramolecular complexes with regulatory proteins has important effects on the catalytic activity of chloroplast enzymes.

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• Chloroplasts contain two isoforms of glyceraldehyde-3-phosphate dehydrogenases, named A₄ and A₂B₂.

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• The A₄ isoform is a catalytically active tetramer.

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• The A and B polypeptides of the A₂B₂ isoform are similar except that a C-terminal extension of the subunit B holds two cysteines that can form a disulfide bridge.

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• Moreover, the A₂B₂ glyceraldehyde-3-phosphate dehydrogenase can form the A₈B₈ oligomer.

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Dr. Riddhi Datta

Light controls the assembly of chloroplast enzymes into supramolecular complexes

In darkness:

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• Interaction of oxidized phosphoribulokinase with the A₄-glyceraldehyde 3-phosphate dehydrogenase and oxidized CP12 stabilizes the complex.

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• Enzymes are catalytically inactive in the ternary complex.

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In light:

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• Reduced thioredoxin cleaves the disulfide bonds of CP12 and phosphoribulokinase.

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• This separates the components of the ternary complex releasing the active enzymes.

Phosphoribulokinase and A₄-glyceraldehyde 3-phosphate dehydrogenase activity dependent on CP12

Dr. Riddhi Datta

Light controls the assembly of chloroplast enzymes into supramolecular complexes

• The A₂B₂ glyceraldehyde-3-phosphate dehydrogenase forms the A₈B₈ oligomer in darkness and is catalytically inactive.

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• Reduced thioredoxin (Trx) cleaves the disulfide bond in subunit B of A₈B₈-glyceraldehyde-3-phosphate dehydrogenase.

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• The reduction converts the inactive oligomer into the active A₂B₂ -glyceraldehyde-3-phosphate dehydrogenase.

A₂B₂-glyceraldehyde 3-phosphate dehydrogenase activity dependent C-terminal extension

Dr. Riddhi Datta

Light controls the assembly of chloroplast enzymes into supramolecular complexes

A₂B₂-glyceraldehyde 3-phosphate dehydrogenase activity dependent C-terminal extension

The CP12 assembled supramolecular complex represents a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light.

Phosphoribulokinase and A₄-glyceraldehyde 3-phosphate dehydrogenase activity dependent on CP12

On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

Lucia et al. 2009, Molecular Plant

Dr. Riddhi Datta

Light reactions of photosynthesis supplies ATP and NADPH for the operation of the C₄ cycle,

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In addition, variations in photon flux density elicit changes in the activities of:

• NADP–malate dehydrogenase
• PEPCase
• pyruvate–phosphate dikinase

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Two different mechanisms are involved:

• thiol–disulfide exchange
• phosphorylation–dephosphorylation of specific amino acid residues

Light regulates the activity of key C₄ enzymes

Dr. Riddhi Datta

Regulation of NADP–malate dehydrogenase

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• NADP–malate dehydrogenase is regulated via the ferredoxin–thioredoxin system.

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• The enzyme is reduced (activated) by thioredoxin when leaves are illuminated, and is oxidized (deactivated) in the dark.

Carr et al. 1999, Cell

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Regulation of PEPCase

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• The diurnal phosphorylation of PEPCase by PEPCase kinase increases the uptake of ambient CO₂, and the nocturnal dephosphorylation by protein phosphatase 2A brings PEPCase back to low activity.

Light regulates the activity of key C₄ enzymes

Dr. Riddhi Datta

• A highly unusual enzyme regulates the dark–light activity of pyruvate–phosphate dikinase.

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• Pyruvate–phosphate dikinase is regulated by a bifunctional threonine kinase–phosphatase.

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• Darkness promotes the ADP dependent phosphorylation of pyruvate–phosphate dikinase by the regulatory kinase–phosphatase causing the loss of enzyme activity.
• In light, Pi-dependent dephosphorylation by the same enzyme restores the catalytic capacity of pyruvate–phosphate dikinase.

Light regulates the activity of key C₄ enzymes