Photosynthesis: Regulation of Carbon fixation reactions

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In photosynthetic organisms, the carbon fixation reactions are regulated by light to ensure that the production of carbohydrates is synchronized with the availability of light energy. This regulation involves several mechanisms, including the activation of key enzymes, changes in the concentration of metabolic intermediates, and the formation of supramolecular complexes.

Induction Period in Carbon Fixation

After the onset of illumination, there is an induction period during which the rate of CO2​ fixation increases gradually before reaching a steady state. This initial lag is because the photosynthetic enzymes need time to be activated by light, and the concentration of Calvin-Benson cycle intermediates needs to increase.

• During the induction period, six triose phosphates produced in the Calvin-Benson cycle are used for the regeneration of ribulose 1,5-bisphosphate (RuBP).

• At the steady state, five of the six triose phosphates regenerate RuBP, while the remaining one is either used for starch formation in the chloroplast or sucrose synthesis in the cytosol.

Regulation of the Calvin-Benson Cycle

The regulation of the Calvin-Benson cycle involves both long-term and short-term mechanisms.

Long-term regulation involves changes in the amount of enzymes, which is controlled by the expression of nuclear and chloroplast genes. Regulatory signaling between nucleus and plastids is mostly anterograde — that is, the products of nuclear genes control the transcription and translation of plastid genes. 

Short-term regulation involves post-translational modifications that can rapidly alter the activity of enzymes.

Light-dependent mechanisms quickly regulate the specific activity of five key enzymes:

1. Rubisco

2. Fructose 1,6-bisphosphatase

3. Sedoheptulose 1,7-bisphosphatase

4. Phosphoribulokinase

5. NADP–glyceraldehyde-3-phosphate dehydrogenase

Rubisco: Activation and Catalysis

Rubisco, a crucial enzyme for carbon fixation, is regulated by Rubisco-activase. 

A CO2​ molecule has a dual role in Rubisco's activity: it acts as an activator and a substrate.

• Activation: The activation process begins when an activator CO2​ molecule forms a lysyl-carbamate with the enzyme. The subsequent binding of Mg2+ stabilizes this complex, making Rubisco catalytically active. In the light, an increase in both the stroma's pH and Mg2+ concentration facilitates this activation.

• Catalysis: A separate CO2​ molecule, the substrate CO2​, then reacts with RuBP at the active site, yielding two molecules of 3-phosphoglycerate.

Activation of RuBisCo by CO2 and Mg2+

Rubisco-activase overcomes several types of inhibition. Sugar phosphates like xylulose 1,5-bisphosphate and 2-carboxyarabinitol 1-phosphate (naturally occurring inhibitor) can bind tightly to uncarbamylated Rubisco, preventing its activation and inhibiting catalysis. The substrate RuBP can also bind to the carbamylated enzyme, preventing activation. Rubisco-activase removes these inhibitors, allowing the enzyme to be activated by activator CO2 and Mg2+. 

Formation of the Dead-End Complex

• A Rubisco active site that has lost its activating CO2​ molecule (a process called decarbamylation) can bind to RuBP.

• This binding leads to a conformational change in Rubisco, which creates an inactive, dead-end complex where RuBP is tightly bound to the enzyme.

How Rubisco-Activase Restores Activity

Rubisco-activase removes the tightly bound RuBP in an ATP dependent process. The steps are as follows: 

1. Activase Priming: ATP hydrolysis by Rubisco-activase is required for the conformational changes that prime the activase.

2. Physical Interaction: The primed Rubisco-activase physically interacts with the Rubisco-RuBP dead-end complex.

3. Conformational Change: This interaction changes the conformation of Rubisco, reducing its affinity for RuBP.

4. RuBP Dissociation: With a lower affinity, the tightly bound RuBP dissociates from the active site of Rubisco.

5. Reactivation: Once the active site is free, Rubisco-activase likely dissociates, allowing the site to be reactivated through carbamylation by CO2​. This allows the enzyme to resume its role in carbon fixation.

At high temperatures, Rubisco deactivates faster than Rubisco-activase can reactivate it, leading to a loss of function.

Activation of RuBisCo by RuBisCo activase (adapted from Jensen (2000) PNAS)

The Ferredoxin-Thioredoxin System

The ferredoxin-thioredoxin system links the light-dependent reactions of photosynthesis to the regulation of Calvin-Benson cycle enzymes. Light transfers electrons from water to ferredoxin. This reduced ferredoxin is then used to reduce ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin (Trx). The reduced thioredoxin activates target enzymes by reducing their regulatory disulfide bonds.

This system regulates the activity of several enzymes, including:

• Fructose 1,6-bisphosphatase

• Sedoheptulose 1,7-bisphosphatase

• Phosphoribulokinase

• NADP–glyceraldehyde-3-phosphate dehydrogenase

When it's dark, electron flow from ferredoxin stops, and thioredoxin becomes oxidized, ultimately inactivating the target enzymes.

Regulation via ferredoxin-thioredoxin system

Light-Dependent Ion Movements and Enzyme Activity

Light also regulates Calvin-Benson cycle enzymes through the movement of ions. When illuminated, protons flow from the stroma into the thylakoid lumen, while Mg2+ is released from the thylakoid space into the stroma. This increases the stromal pH and Mg2+ concentration, which activates enzymes like Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and phosphoribulokinase. These ionic changes are reversed in the dark, deactivating the enzymes.

Regulation of Chloroplast Enzymes via Supramolecular Complexes

Another mechanism for light-dependent regulation of the Calvin cycle involves the formation of supramolecular complexes. This process primarily centers on a small regulatory protein called CP12 and involves regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK).

A2B2 and A4 Forms of GAPDH

The regulation of GAPDH is complex, involving two major isoforms in higher plants: 

• the heterotetramer A2B2
• the homotetramer A4

Both are assembled from different subunits and play distinct roles in the light-dark regulation of photosynthesis. 

A4 Homotetramer

• Composition: The A4 form consists of four identical GapA subunits. It is the catalytically active homotetramer.

• Regulation: The A4 homotetramer is regulated by its interaction with the small protein CP12.

A2B2 Heterotetramer

• Composition: The A2B2 form is made of two GapA subunits and two GapB subunits. This is the most abundant and active form of GAPDH in land plants.

• Regulation: Its regulation is largely autonomous, primarily controlled by the C-terminal extension (CTE) of the GapB subunit. 

A4-GAPDH Regulation

The CP12-Mediated Complex:

In the dark, when there is no need for active carbon fixation, the regulatory protein CP12, which is oxidized by the absence of light, binds to two key Calvin cycle enzymes:

• A4 isoform of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

• Phosphoribulokinase (PRK)

This binding forms a ternary supramolecular complex (GAPDH/CP12/PRK). The formation of this complex effectively sequesters and inactivates both GAPDH and PRK, switching off the Calvin-Benson cycle and preventing the wasteful consumption of ATP and NADPH that would otherwise occur in the dark.

Dissociation in the Light:

When light becomes available, the thylakoid electron transport chain produces reducing power in the form of NADPH. This is used to reduce ferredoxin, which then reduces thioredoxin. The reduced thioredoxin acts on the CP12-mediated complex.

Reduced thioredoxin cleaves the disulfide bonds within the oxidized CP12. This reduction of CP12 causes the entire supramolecular complex to dissociate. The dissociation releases the A4 isoform of GAPDH and PRK, which become catalytically active.

This mechanism ensures that the Calvin cycle is tightly coupled to the availability of light, providing a quick and efficient way to regulate carbon fixation in response to light-dark transitions.

Regulation by CP12-Mediated Complex (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

A2B2-GAPDH Regulation

The A2B2 isoform is a heterotetramer made of A and B polypeptides. The B subunit contains a C-terminal extension with two cysteines that can form a disulfide bridge. The A2B2 form can also assemble into a larger, inactive A8B8 oligomer.

• In Darkness: The A2B2-GAPDH forms the inactive A8B8 oligomer.

• In Light: Reduced thioredoxin (Trx) cleaves the disulfide bond in the B subunit of the A8B8 oligomer. This reduction converts the inactive oligomer into the active A2B2-GAPDH. This autonomous redox-modulation is particularly suited for conditions of very active photosynthesis.

Regulation by C-terminal extension (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

GAPDH Isoform	Composition	Regulation Mechanism	Active State	Inactive State
A4	A4 homotetramer	Forms a ternary complex with CP12 and PRK in the dark.	Free, active enzymes.	Inactive ternary complex.
A2B2	A2B2 heterotetramer	Forms an A8B8 oligomer in the dark; regulated by disulfide bond in subunit B.	Active A2B2 tetramer.	Inactive A8B8 oligomer.

Regulation of C4 Enzymes

In C4 plants, light also regulates key enzymes through thiol-disulfide exchange and phosphorylation-dephosphorylation.

• NADP–malate dehydrogenase (MDH) is activated in the light by the ferredoxin-thioredoxin system (reduced by thioredoxin) and deactivated in the dark (oxidized).

Regulation of MDH (Carr et al. 1999, Cell)

• Phosphoenolpyruvate (PEP) carboxylase is regulated by diurnal phosphorylation. Phosphorylation makes it active at night and insensitive to inhibition by malate. Dephosphorylation by a protein phosphatase in the day brings the enzyme to low activity.

Regulation of PEP

• Pyruvate–phosphate dikinase is regulated by a bifunctional kinase-phosphatase enzyme. In the dark, ADP-dependent phosphorylation deactivates the enzyme, while in the light, Pi-dependent dephosphorylation restores its activity.