20.4: CO₂ uptake - Calvin Cycle and C3 organisms

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Learning Goals (ChatGPT o3-mini)

• Differentiate Between Light and Dark Reactions:
  • Explain the overall flow of energy from the light reactions (which produce ATP, NADPH, and O₂) to the dark reactions (which fix CO₂ into carbohydrates).
  • Describe why the term “dark reactions” is used despite these reactions occurring in the light.

• Outline the Calvin (C3) Cycle:
  • Describe the three phases of the Calvin cycle—carbon capture (CO₂ fixation), reduction (conversion of 3PG to G3P), and regeneration (reformation of RuBP)—and how they combine to produce triose phosphates for carbohydrate synthesis.
  • Explain the stoichiometry of the cycle, including the overall consumption of ATP and NADPH per CO₂ fixed.

• Examine the Role and Mechanism of RuBisCo:
  • Detail the dual enzymatic activities (carboxylase and oxygenase) of Rubisco and explain how it catalyzes the fixation of CO₂ into ribulose 1,5-bisphosphate (RuBP) to yield 3-phosphoglycerate.
  • Discuss the structural composition of Rubisco, including the large (chloroplast-encoded) and small (nucleus-encoded) subunits, and how multimer formation (e.g., the Rubiscosome) influences its catalytic efficiency.

• Understand Rubisco's Limitations and the Problem of Photorespiration:
  • Explain the competitive oxygenase activity of Rubisco that leads to the formation of 2-phosphoglycolate and 3-phosphoglycerate, outlining the factors (e.g., CO₂ and O₂ concentrations, temperature) that affect this competition.
  • Discuss the kinetic parameters (KM for CO₂ vs. O₂) of Rubisco and how these contribute to the enzyme’s reduced efficiency under current atmospheric conditions.

• Describe the Glycolate (Photorespiration) Pathway:
  • Outline the steps of the glycolate salvage pathway, including the conversion of 2-phosphoglycolate to glycine, the subsequent formation of serine, and the regeneration of 3-phosphoglycerate.
  • Discuss the cellular compartments involved in photorespiration (chloroplast, peroxisome, mitochondria) and the overall energetic cost of the process.

• Explore Plastid Diversity and Interconversion:
  • Identify different types of plastids (e.g., chloroplasts, amyloplasts, etioplasts, chromoplasts) and explain their roles in photosynthesis and storage.
  • Describe the process of “greening” (conversion of proplastids or etioplasts to chloroplasts) and “de-greening” (transition to leucoplasts or gerontoplasts) in response to developmental cues and environmental signals.

• Understand Metabolite Exchange and Carbon Partitioning:
  • Explain the function of the triose phosphate/phosphate translocator (TPT) in exchanging triose phosphates from the chloroplast with inorganic phosphate (Pi) from the cytosol, and its role in balancing carbohydrate synthesis and energy production.
  • Describe how the products of the Calvin cycle are used in the cytosol for the synthesis of sucrose and other metabolites.

• Connect CO₂ Fixation to Global Carbon Cycling and Climate Impacts:
  • Discuss the role of Rubisco in capturing atmospheric CO₂ and its significance for global primary production.
  • Evaluate how changes in atmospheric CO₂ concentrations and rising global temperatures might influence Rubisco’s carboxylase versus oxygenase activities, with implications for plant productivity and nutrient content.

• Analyze Experimental Approaches and Data Interpretation:
  • Interpret data from deuterium NMR studies that examine changes in isotopomer ratios (e.g., D6S/D6R) of photosynthetically generated glucose, linking these changes to shifts in Rubisco’s activity under different CO₂ levels.
  • Discuss how kinetic measurements and structural studies (including iCn3D models) contribute to our understanding of Rubisco’s function and regulation.

These learning goals aim to provide a comprehensive framework for understanding how the dark reactions of photosynthesis integrate with light reactions, the critical role of Rubisco in CO₂ fixation, and the challenges posed by photorespiration, all within the broader context of plant metabolism and environmental impact.

The source for the organization and some of the text derives from: Sindayigaya and Longhini. https://www.peoi.org/Courses/Courses...chem/biochem18 CC - https://creativecommons.org/licenses...sa/3.0/deed.en

Everything in the chapter is related to climate change.  For more general information on biochemistry and climate change, visit Chapter 32 Biochemistry and Climate Change. For more details on carbon fixation or capture, visit Chapter 32.16: Part 4 - Fixing Carbon Fixation.

Introductions 

We focused on the light reactions of photosynthesis. Now, let's turn our attention to the dark reactions, which fix CO₂ from the air and reduce it with NADPH produced, along with O₂, in the light reactions, to produce carbohydrates. The dark reactions don't just occur in the dark. The term differentiates them from the light-driven reactions using PSII and PSI. What is so interesting about plants is that they produce fuel from CO₂ using photons as a source of energy (autotrophs) and consume the fuels they make, using both anaerobic and aerobic respiration pathways. Their biosynthetic reactions occur mostly in the chloroplast, a type of plastid, subcellular organelles with specific functions such as photosynthesis or metabolite synthesis and storage. Plants also can not move to acquire fuel and nutrient molecules. They are subject to many growing conditions (differential light qualities and quantities, temperatures, and rainfall levels). Also, plant cells have cell walls in addition to a cell membrane. Figure \(\PageIndex{1}\) shows a simple cartoon showing the major photosynthesis motifs.

Figure \(\PageIndex{1}\): a Schematic depiction of the light and dark reactions in natural photosynthesis. Li, Y., Hui, D., Sun, Y. et al. Boosting thermo-photocatalytic CO₂ conversion activity by using photosynthesis-inspired electron-proton-transfer mediators. Nat Commun 12, 123 (2021). https://doi.org/10.1038/s41467-020-20444-1 Creative Commons Attribution 4.0 International License.

In this section, we will discuss how CO₂ from the atmosphere is "fixed" or "captured" in the formation of the simplest sugars (3 carbon molecules like glyceraldehyde-3-phosphate) in a process called the C3 or Calvin Cycle, which is also called the Calvin–Benson–Bassham (CBB) cycle, or the reductive pentose phosphate cycle (RPP cycle). Plants that use the C3 cycle are called C3 plants. There are two other major types of carbon capture pathways, the C4 and CAM pathways, which we discuss in the next section. All use a key enzyme, ribulose 1,5-bisphosphate carboxylase (RuBisCo), to covalently fix CO₂ into small carbohydrates, 3-phosphoglycerate. RuBisCo is the most abundant protein in the biosphere. Recent estimates suggest that there are about 0.7 gigatons (Gt = 10¹² tons) of it, with over 90% in the leaves (about 3% of their weight) of terrestrial plants. It captures about 120 Gt of atmospheric CO₂ each year. This enzyme has a second competing enzymatic activity. It is also an oxygenase, which fixes O₂ at the active site, decreasing its ability to fit O₂. That activity captures about 100 Gt of atmospheric O₂ each year.  Hence, the enzyme is often called ribulose 1,5-bisphosphate carboxylase/oxidase (but still abbreviated RuBisCo).

We will devote most of this chapter section to this most important enzyme. Along with RuBisCo, plants have pathways to convert the fixed CO₂ to 3C sugars and then through a unique pentose pathway, which runs in a reductive fashion, ultimately produce the sugar-containing molecules in plants we are most familiar with: sucrose and the glucose polymer starch.

Plastids

There are several types of these organelles. Photosynthesis occurs in chloroplasts, which have their own genome like mitochondria. Another common type is the amyloplasts, which lack pigmented molecules (i.e., they are colorless) and have no inner membrane. Rather, they are filled with starch. Chloroplasts and amyloplasts can interconvert. Chloroplasts are abundant in green leaves, while amyloplasts are predominately found in locations like potato tubers, where starch is stored. Light can drive the interconversion of plastids, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Transition pathways among various plastids. Choi et al. Frontiers in Plant Science. 12 (2021). https://www.frontiersin.org/article/...ls.2021.692024. DOI=10.3389/fpls.2021.692024 . Creative Commons Attribution License (CC BY)

Arrows show the characteristics and plastid interconversion pathways of the plastids. The transition to a chloroplast is called “Greening” and is identified with the number “1”. This transition is mainly triggered by light signals from proplastids, etioplasts, leucoplasts, and chromoplasts. Etioplasts can develop from proplastids in dark conditions, and this is identified by the number “2”. The number “3” indicates leucoplast development triggered by diverse development processes to generate starch, lipid, and protein-enriched sub-types called amyloplasts, elaioplasts, and proteinoplasts, respectively. Mainly during the ripening stage, diverse types of carotenoid crystals were generated within the plastids called chromoplasts from the proplastids, leucoplasts, and chloroplasts and this is identified with the number “4”. Together with etioplast and leucoplast development (2,3), chromoplast development (4) was identified as a “Non-greening” plastid transition. The loss of green color from the chloroplasts is called “De-greening”. It is identified with the number “5”, and these chloroplasts are then transited into leucoplast or gerontoplast by developmental regulation or during senescence, respectively.

CO₂ capture and the C3 Cycle

These processes are used in the synthesis of the simplest carbohydrates (3-carbon polyhydroxy aldehydes and ketones):

1. Carbon capture or fixation phase. We prefer the term carbon capture as this term is now used to describe how the world is seeking new ways (other than planting billions of trees) to "capture" excess CO₂ emitted through burning fossil fuels. In a reaction catalyzed by RuBisCo, atmospheric CO₂ ultimately reacts with a 5-carbon acceptor molecule, ribulose 1,5-bisphosphate (Ru1,5-BP, six carbons in total), to form two molecules of 3-phosphoglycerate (3PG), two 3C molecules.
2. Reduction phase: 3-phosphoglycerate is reduced to glyceraldehyde-3-phosphate (G3P). Three CO₂s are captured on reaction with 3 Ru1,5-BP to form 6 glyceraldehyde-3-phosphates (G3P). These can readily interconvert to the keto form, dihydroxyacetone phosphate (DHAP).
3. Regeneration phase: Five of the six G3Ps (15 Cs) react to form 3 three molecules of ribulose 1,5-bisphosphate (15 C2) to allow the catalytic C3 cycle to continue. The other G3P moves into the stroma as DHAP, which can be used in gluconeogenesis (reductive biosynthesis of glucose). This can be converted to polymer starch and the disaccharide sucrose (which we will discuss in a future session).

An overview of the Calvin or C3 cycle is shown below in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): C3 or Calvin Cycle. The three stages of the cycle in C3 plants and organisms are shown. Three CO₂s are captured for the net synthesis of one molecule of glyceraldehyde 3-phosphate.

The stoichiometry can be confusing until you count the number of carbon atoms and realize that the cycle must run three times to enable three carbon atoms from three CO₂ molecules to produce one net glyceraldehyde-3-phosphate (G3P). The G3P leaves the C3 cycle at the low left for glucose synthesis. The conversion of the 5 G3Ps that reform Ru1,5-BP requires ATP, as shown below:

\[\ce{5 glyceraldehyde-3P + 3 ATP → 3 ribulose-1,5-2P + 3 ADP + 2 P_i} \nonumber \]

with \(\ce{P_i}\) indicating inorganic phosphate. Hence, the net equation for three turns of the cycle, sufficient to produce one G3P is:

\[\ce{3 CO2 + 6 NADPH + 6 H^{+} + 9 ATP + 5 H2O → glyceraldehyde-3-phosphate (G3P) + 6 NADP^{+} + 9 ADP + 8 P_i } \nonumber \]

Even though glucose is not a product of the Calvin cycle, some texts use the following equation to show the stoichiometry to run the C3 cycle enough times (6) to fix 6 \(\ce{CO2}\) molecules, enough to make 1 glucose from a simple carbon atom counting perspective.

\[\ce{6 CO2 + 12 NADPH + 12 H^{+} + 18 ATP + 10 H2O → 2 glyceraldehyde-3-phosphate (G3P) + 12 NADP^{+} + 18 ADP + 16 P_i } \nonumber \]

Remember that NADPH and ATP are produced in the light reactions in about the same ratio used in the C3 cycle (2NADPH/3ATPs). The net 8 P_is made as products will react with 8 ADP to regenerate 8 ATP in the light reaction. The 9th Pi is incorporated in a triose-phosphate in the light reaction, so one Pi must be imported from the cytoplasm by an inner membrane triose-phosphate/phosphate translocator, which we will discuss below. In the dark, when ATP and NADPH are not produced, CO₂ capture is also inhibited.

A more detailed diagram showing the detailed reactions to regenerate ribulose 1,5-bisphosphate (Ru1,5BP) is shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Detailed reaction diagram for the Calvin (C3) cycle (after Hashunuma et al. Journal of Experimental Botany, 61 (2010). doi:10.1093/jxb/erp374. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/2.5)

Abbreviated reactions for synthesizing sucrose, glucogenic amino acids, and fatty acids are also shown.

You should note the reactions for the conversion of the 6C sugar molecule fructose-6-P (F6P) (glycolytic and gluconeogenic intermediate) to the 5C molecule Ru5P and Ru1-5BP are analogous to the reactions of the nonoxidative part of the pentose phosphate pathway (PPP), which generates 5C sugars for the synthesis of nucleotides, nucleic acids, and some amino acids. Hence, we won't discuss them further.

Carbon capture of CO₂ into 3-Phosphoglycerate - RuBisCo

This key enzyme requires a Mg²⁺ ion and proceeds through a carbamoylated lysine side chain, which acts as an "activator CO₂". The Mg²⁺ ion orients key side chains. The resulting 6C molecule cleaves into two molecules of 3PG.  

Rubisco is composed of two proteins: a large chain (around 55 K) whose gene is found in the chloroplast, and a small chain (around 12K) whose genes are in the nucleus. The large chain, which dimerizes, binds the ribulose-1,5-bisphosphate substrate between monomers in the dimer.  Four dimers of the large chain (8 subunits) interact with eight small chains to form a 16-mer in cyanobacteria, red and brown algae, and higher plants.  Multimeric units other than 16 are also found, with the large chain dimer being the smallest active unit.

 Rubiscos exist in 4 forms:

• Form I:  The 16-mer described above
• Form II:  Multimers of the large chain dimer, (L₂)_n, where n = 1-4
• Form III: Archaeal L₂ or (L₂)₅
• Form IV: Rubisco-like protein with no CO₂ or O₂ fixing activities

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (8RUC).  The eight large subunits are colored in varying shades of magenta/purple.  The eight small subunits are in varying shades of cyan.

Figure \(\PageIndex{5}\): Spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (8RUC). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...X2s7BDc8xQ5r19. (Long load time)

A possible mechanism of RuBisCo from Synechococcus elongatus, a unicellular cyanobacterium, is shown in Figure \(\PageIndex{6}\). 

Figure \(\PageIndex{6}\): Mechanism of ribulose-bisphosphate carboxylase (type I) (after https://www.ebi.ac.uk/thornton-srv/m-csa/entry/907/. Creative Commons Attribution 4.0 International (CC BY 4.0) License)

The green shows the carbamoylated lysine side chain ("activator CO₂"). Ribulose 1,5-bisphosphate is converted to an enediolate, which engages in a nucleophilic attack on the CO₂ to form a 6C sugar. Hydroxylation at C-3 of this sugar is followed by aldol cleavage. Ultimately, two 3PGs are produced, one containing the carbon atom from CO₂ (red).  The "activator CO2" (the carboxylated lysine) is hydrolyzed and removed at night, inactivating the enzyme. 

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of a single heavy and light chain of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from Synechococcus PCC6301 (1RBL). (long load time)

Figure \(\PageIndex{7}\): Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from Synechococcus PCC6301 (1RBL). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...BkwkrBmYVwRJ6A

The light chain is shown in cyan, and key residues in the heavy chain are shown in CPK-colored sticks and labeled. Bound to the heavy chain is a substrate analog/inhibitor, 2-carboxyarabinitol-1,5-diphosphate. It is produced in plants, and in the dark inhibits the enzyme. With increasing light, its concentration decreases.

Figure \(\PageIndex{8}\) shows one active site of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol bisphosphate (8RUC). showing the "activator CO₂" bound in a Schiff base link to a lysine side chain (KCX201). The bound activated substrate analog, 2-carboxyarabinitol bisphosphate, contains a -CO₂^- group that coordinates with the Mg²⁺ ion (green sphere) instead of the actual CO₂ (O=C=O) substrate, which would be fixed.

Figure \(\PageIndex{8}\): Active site of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol bisphosphate (1RUC). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structure/icn3d/share.html?PTo2P4jRp3HJrHb99

 

Rubisco Reacts with both CO₂ and O₂ 

Rubisco is a slow carboxylase with a k_cat of around 2-10 CO₂/sec. In addition, it can bind another substrate, O₂, and engage in a competing reaction of photorespiration (oxidation) of ribulose 1,5-bisphosphate to form one molecule of 3-phosphoglycerate (3PG) and one molecule of 2-phosphoglycolate (2PG), as shown in Figure \(\PageIndex{9}\) below. (Note that 2-phosphoglycolate is not 2-phosphoglycerate!) In contrast to most oxygenases, no cofactor is required for the RuBisCo/Oxygenase activity.  It takes just a simple Mg²⁺ ion. A more detailed mechanism is shown in Figure 20.

Figure \(\PageIndex{9}\): RuBP conversion by Rubisco through the carboxylase (a) and the oxygenase (b) reactions. Tommasi, I.C. The Mechanism of Rubisco Catalyzed Carboxylation Reaction: Chemical Aspects Involving Acid-Base Chemistry and Functioning of the Molecular Machine. Catalysts 2021, 11, 813. https://doi.org/10.3390/catal11070813.  CC BY) license (https://creativecommons.org/licenses/by/4.0/

Following RuBP (1) enolization, the 2,3-enol(ate) intermediate (2) may react with CO2(a) or O2(b) co-substrates. The carboxylase reaction produces the 2-carboxy-3-keto-arabinitol 1,5-bisphosphate intermediate (3), undergoing protonation to the 2-carboxylic acid before hydration. The C2-C3-scission reaction in C3-gemdiolate (5) occurs in a concerted mechanism upon P1 protonation, producing two molecules of 3-phospho-D-glycerate (3PGA, 6). The oxygenase reaction produces 3-phospho-D-glycerate (3PGA,6) and 2-phosphoglycolate (2PG,7).  

The competing reaction with O₂ was only a potential problem after widespread photosynthesis increased the O₂ concentration in the air to 20%.  Species evolved to help minimize this problem.  Before we describe those adaptations, let's first look at how Rubisco can differentiate the two nonpolar substrates, CO₂ and O₂.  Given the symmetric arrangement of δ⁺ and δ^-charges in CO₂, it has a net zero dipole, as does O₂, so it would not align/orient in a field generated by two poles (+ and -). However, CO₂, but not O₂, would align in a field generated by four charged poles, so it has a quadrupole moment, as shown below in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\):  CO₂ aligning in a quadrupole field. (field lines from https://commons.wikimedia.org/wiki/F...quadrupole.svg)

The dipole unit is the debye, and the quadrupole unit is the debye^.Angstrom. Table \(\PageIndex{1}\) below shows some dipole and quadrupole moments values for simple gases.  CO₂ has the highest quadrupole moment of all these simple gases.

Molecule	Dipole moment (D)	Quadrupole moment (D Å)
CO2	0.000	4.30
CH4	0.000	0.02
H2	0.000	0.66
O2	0.000	0.39
CO	0.112	2.5
N2	0.000	1.52

Table \(\PageIndex{1}\): Dipole and Quadrupole moments for some simple gases.  Castro-Muñoz, R., Fíla, V., 2018. Progress on Incorporating Zeolites in Matrimid®5218 Mixed Matrix Membranes towards Gas Separation. Membranes 8, 30. https://doi.org/10.3390/membranes8020030

The active site of Rubisco has a high electrostatic field gradient in the dimeric form of the enzyme.  Figure \(\PageIndex{11}\) shows an interactive iCn3D model showing the electrostatic potential surface in the active site between two heavy chains of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) (8RUC).  It is complex. 

Figure \(\PageIndex{11}\): Electrostatic potential surface in the active site between two heavy chains of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) (8RUC). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...2JG3x4csVnEq67

Two heavy chains are shown in light pink and cyan, and one light chain is shown in gray.  The active site regions are shown as an electrostatic surface potential with blue positive and red negative.  The model has an activated substrate analog, 2-carboxyarabinitol bisphosphate, shown spacefill (hard to see given the electrostatic surface potential).  The residue labeled 201KCX is the carbamate of Lys201.  

Presumably, the electrostatic field in the active site facilitates through the subtle interaction of the quadrupole CO₂ compared to O₂.

The mechanisms that differentiate the binding of CO₂ and O₂ must also overcome the high intracellular concentration of O₂ (around 250 μM) compared to CO₂ (7–8 μM in C3 plants and 80 μM in C4 plants). The enhanced affinity for CO₂ (about 30-fold) compared to O₂ helps overcome these concentration barriers. No classic "binding pocket" exists for CO₂ and O₂, so diffusion and binding are likely guided by the electrostatic potential gradients along the diffusion surface. Figure \(\PageIndex{12}\) below the electrostatic potential molecular surface of O₂ and CO₂ (top), calculated from electron density measurement using quantum mechanics, and the electrostatic surface of their binding pockets

Figure \(\PageIndex{12}\): Electrostatic potential molecular surface of O₂ and CO₂ (top) and the electrostatic surface of their binding pockets.  Tommasi, I.C. et al., ibid.

(top) Computed electrostatic potential molecular surfaces of CO₂ (left) and O₂ (right). The color scheme follows commonly accepted conventions: blue, positive; red, negative. The value of the Qzz component of the quadrupole moment, as calculated by Stec, is −3.239 e a₀² for CO₂ and −0.232 e a₀² for O₂.  Note that the ratio of these values is about 14, about equal to the earlier quadrupole moments discussed above with units of debeye^.Å)

(bottom) a ribbon representation of the catalytic domain with bound gaseous ligands and surfaces colored by the electrostatic potential. O₂ (in red) and CO₂ (in purple) lie in a positively charged cavity (blue) of the TIM barrel. (Figure from ref. [15], used by permission of PNAS (copyright © 2012)).

Both CO₂ and O₂ are situated in a tiny "cavity" that is blue (positive potential, C-terminal domain) and red (negative potential, N-terminal domain) just above it.  The quadrupole moment of CO₂ is 10-15x that of O₂ which helps explain its higher affinity in the localized electrostatic potential gradients around the gas molecules.

Molecular dynamic (MD) simulations show an interaction preference for CO₂.  There are many subunit-subunit interfaces, and all appear in MD to preferentially interact with CO₂, which probably moves from the solvent through large:small subunit interface to the active site.  The CO₂ does not localize long at any residues but seems to occupy areas instead.  Since CO₂ has no dipole, it moves more closely to the small hydrophobic side chain (Ala, Val, Leu, Ile) and the main chain.  CO₂ has more interactions in every active site and the large:large subunit interface (whose electrostatic potential in the active site is shown above) and in the large:small subunit interface. 

In efforts to quantitate the preference of CO₂ over O₂ using MD, investigators found that over many different species of Rubisco, the relative distribution of CO₂ and O₂ to the small and large subunits was on average 1.8 for CO₂ and 1.4 for O₂, with the number of oxygen bound to either subunit lower. This is true even though CO₂ has a lower solvation energy than O₂ in water, so additional energy must be spent to desolvate CO₂ differentially.  The hydrophobic interactions likely promote the movement of CO₂ to the active site where electrostatic-based potentials favor CO₂ binding.  These results suggest that the small subunit acts like a "mini reservoir" for O2, which then diffuses to the large subunit region of the active site.  From a simple thermodynamic perspective, CO₂ would be favored to move along the surface and through spaces in the protein guided by transient interactions that be water.  The active site in Rubisco is not in a deep pocket but in shallow grooves near the surface.   Although the enzyme is slow (2-10 CO₂/s), it's not much slower than the average enzyme.  The median turnover number k_cat (under saturating conditions) of enzymes is about 10 s^-1, with most following between 1-100.   Its concentration is very high in chloroplasts, which helps increase the fixing of CO₂.  

Regulation of Rubisco by Multimer Formation and Phase Separation

As mentioned above, Rubisco can exist as multimers, including dimers, tetramers, hexamers, octamers, and 16-mers.  Different multimers don't seem to significantly differentiate per se between the competing substrates, CO₂ and O₂. Higher multimers can be induced in the presence of small ligands. The functional complex is often called the Rubiscosome, and its aggregation state varies across evolution. Figure \(\PageIndex{13}\) shows structures of the different multimers of Rubisco.

Figure \(\PageIndex{13}\): Crystal structure of a tetrameric RuBisCO.  Comparison of RuBisCO oligomeric states illustrating dimer positioning within a multimer. Form II dimer, tetramer, and hexamer are shown alongside form I′ octamer and form I hexadecamer. Protein Data Bank (PDB) codes (left to right): 5RUB, 7T1C, 5C2C, 6URA, and 1RBL.  Albert K. Liu et al. Structural plasticity enables evolution and innovation of RuBisCO assemblies. Sci. Adv.8, eadc9440(2022). DOI:10.1126/sciadv.adc9440. Creative Commons Attribution License 4.0 (CC BY). https://creativecommons.org/licenses/by/4.0/

These different multimeric structures have evolved to meet their respective organisms' environmental and biochemical needs.  These examples illustrate how, surprisingly, different aggregation states can evolve without great changes in active site requirements. Indeed, the active site is formed between two catalytic large chain monomers.  A large chain dimer has two active sites per dimer.

Genomics and phylogenetic analyses show that Rubisco originated about 3 billion years ago when the competing substrate O₂ in the atmosphere was barely present and CO₂ concentrations were 10K% higher than now.  Hence, the enzyme evolved when O₂  increased and CO₂ decreased.  Key events in the evolution of different forms of Rubisco are shown in Figure \(\PageIndex{14}\) below. 

Figure \(\PageIndex{14}\): The evolutionary history of rubisco in the context of atmospheric CO₂ (%) and O₂ (%) following divergence from the ancestral rubisco-like protein (RLP).  Bouvier JW, Emms DM, Kelly S. Rubisco is evolving for improved catalytic efficiency and CO₂ assimilation in plants. Proc Natl Acad Sci U S A. 2024 Mar 12;121(11):e2321050121. doi: 10.1073/pnas.2321050121. Epub 2024 Mar 5. PMID: 38442173; PMCID: PMC10945770.  Creative Commons Attribution License 4.0 (CC BY).

Gray vertical bars indicate important branch points in the phylogeny at which Rubisco diverged into different evolutionary lineages. To provide additional context, the time period at which the First and Second Great Oxidation events occurred along this evolutionary trajectory is also labeled and referenced as gray vertical bars. Graphics of atmospheric CO₂ and O₂ levels were adapted from the TimeTree resource [http://www.timetree.org].

Although the small subunit has no catalytic activity, its presence improves enzymatic activity and facilitates the formation of multimers.  The multiple forms of Rubisco and evolutionary changes in the enzyme haven't had a huge effect on the specificity of the enzyme for CO₂ compared to O_{2. However,} evidence suggests there have been slow increases in the activity and specificity of the enzyme for CO₂.  The enzyme has evolved slower than over 98% of enzymes throughout life, so other strategies are deployed to increase carbon capture.  These include increasing Rubisco production in cells, localizing Rubisco near the site of CO₂ import/formation, and phase separation of Rubisco into aggregates.

Rubisco kinetics in extinct and living angiosperms show increases in CO₂/O₂ specificity (S_C/O), carboxylase turnover rates (k_catC), and carboxylation efficiencies (k_catC/K_C) in angiosperms, as shown in Table \(\PageIndex{2}\) below.

Rubisco	SC/O (mol mol^−1)	kcatC (s^−1)	kcatC/KC (s^−1 µM^−1)	KC (µM)	KC^air (µM)	KO (µM)	KC/KO (µM µM^−1)
Last common angiosperm ancestor	81.1 ± 1.9	2.6 ± 0.3	0.16 ± 0.02	16.3 ± 2.1	24.8 ± 2.8	484.1 ± 56.4	0.034 ± 0.004
Extant angiosperms	87.1 ± 0.5	3.4 ± 0.1	0.20 ± 0.01	17.6 ± 0.5	26.4 ± 0.7	517.2 ± 14.7	0.035 ± 0.001

Table \(\PageIndex{2}\):  Rubisco kinetics in extinct and extant angiosperms.  Bouvier JW et al., ibid.

Another mechanism helps Rubisco differentiate between CO₂ and O₂ as competing substrates in some photosynthetic organisms such as the unicellular algae Chlamydomonas. High local concentrations of Rubisco can lead to its phase separation.  Such regulation would have the goal of increasing the localized concentration of CO₂, allowing the enzyme to act closer to its maximal velocity.  This would occur if the phase-separated condensate were close to the source of CO₂ or included in a heterogeneous condensate.  One example is the carboxysome in prokaryotes, which contains both Rubisco and carbonic anhydrase, which converts CO₂ (g) into bicarbonate, HCO₃^-. These proteins are encapsulated in an icosahedral protein shell consisting of hexamers of a protein called CcmK2.  Click this link for an iCn3D model showing part of the shell from the marine cyanobacterium Prochlorococcus (8WXB).  CO₂ is converted to HCO₃^- using carbonic anhydrase in the cytosol, which traps the now anionic carbon in the cell.  Bicarbonate can then be transported into the carboxysome and reconverted to CO₂ by encapsulated carbonic anhydrase.  The CO₂ is hence delivered to Rubisco at a high concentration and with minimal O₂ as a competitor for Rubisco.

Figure \(\PageIndex{15}\) below shows a model representing the selective permeability of the carboxysome shell to confine metabolite flux for driving the CBB cycle

Figure \(\PageIndex{15}\): Schematic model representing the selective permeability of the carboxysome shell to confine metabolite flux for driving the CBB cycle.  Faulkner, M., Szabó, I., Weetman, S.L. et al. Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein. Sci Rep 10, 17501 (2020). https://doi.org/10.1038/s41598-020-74536-5Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

CcmK2 hexamers have a concave side facing outwards to the cytoplasm and a convex side facing inwards to the lumen. Based on the positive electrostatic charge of the central pore, CcmK2 acts as a tunnel for HCO₃⁻ influx and a barrier to O₂ and CO₂, precluding O₂ influx and leakage of CO₂ from the carboxysome lumen to the cytoplasm. Larger molecules RuBP and 3-PGA can likely pass through CcmK2 but require a conformational change in the CcmK2 pore involving a flip of the Ser39 side chain.

Microalgae (eukaryotic) contain pyrenoids, condensates containing Rubisco surrounded by a covering of starch. They are found in the stroma of chloroplasts. They can be connected by tubes containing carbonic anhydrase.  Figure \(\PageIndex{16}\) shows the CO₂ concentrating function of pyrenoids in Chlamydomonas and Hornwort, a land plant.  In Chlamydomonas, EPCY1 (similar to LCI5 with Uniprot ID Q94ET8), a linker protein that binds to the small subunit of Rubisco, phase-separates with Rubisco.  The pyrenoid accounts for about 30% of global CO₂ fixation.

Figure \(\PageIndex{16}\):  CO₂ concentrating function of pyrenoids.  Mary Williams.  Characterization of pyrenoid-based CO2-concentrating mechanism in hornworts.  July 26, 2024.  https://plantae.org/characterization...-in-hornworts/.  Creative Commons A-NC 2.0 License.

There is no listing for EPYC1 in Uniprot.  Another protein (LCI5, Uniprot ID: Q94ET8) from Chlamydomonas reinhardtii with a close sequence is listed.  An analysis of the amino acid sequence of LCI5 with PhaSePro, a database of proteins driving liquid-liquid phase separation (LLPS) in living cells, confirms that the protein has 4 mostly identical 60 amino acid tandem repeats between amino acids 52 and 291 that can drive LLPS.  It also states that the common name for LCI5 is EPYC1.  An analysis of the amino acid sequence using PSIPRED Workbench shows at least 5 regions of disorder in the protein as illustrated in Figure \(\PageIndex{17}\) below.

Figure \(\PageIndex{17}\): Disorder Plot for  LCI5 (Uniprot ID Q94ET8).  PSIPRED Workbench.  

Most analyses for tandem disordered repeats in EPYC1 in the literature show 4-5 such repeats.  However, in those plots, the sequence for amino acids 259 to the end is NOT the same as in the Uniprot sequence for LCI5 (aka EPYC1). Two different ways to align the sequence of the literature amino acid sequence and the Uniprot sequences are shown in Figure \(\PageIndex{18}\) below.  

Top Panel

Bottom Panel

Figure \(\PageIndex{18}\): sequence of the tandem repeats in LCI5 (aka EPYC1) for literature and Uniprot amino acid sequences. Top panel fromhttps://pmc.ncbi.nlm.nih.gov/articles/PMC7736253/ ,  bottom panel from https://pmc.ncbi.nlm.nih.gov/articles/PMC7736253/  and https://pmc.ncbi.nlm.nih.gov/articles/PMC6793452/.

In either case, there are at least four tandem repeats in which there is a clear alignment of hydrophobic, polar, and charged (Red for D, E and Blue for R, K)

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of the AlphaFold structure of LCI5 with tandem repeats from Chlamydomonas reinhardtii (Q94ET8).  The four internal repeats from the bottom panel above are shown and labeled in Red, Orange, Cyan, and Brown.

Figure \(\PageIndex{19}\): AlphaFold structure of LCI5 (EPYC1) with tandem repeats from Chlamydomonas reinhardtii (Q94ET8). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structure/icn3d/share.html?7m6GBaZBbJWV9USw6

Color code the basic sidechain (R and K) blue and the acidic sidechains (D and E) in red sticks in iCn3D as follows:

• Click on the external link
• Select, advanced
• Input this expression to select all the Lysines:  $Q94ET8.A:K
• Name the selection All_Lysines
• Repeat for the other amino acids (R, D, and E) and name them appropriately
• Ctrl-click the selections you just made in the Selected Sets window, then choose the following:  Style, Side Chains, Stick
• Color each as follows:  Choose the named selection and then choose the following:  Color, Unicolor, then blue for Ks and Rs, and red for Ds and Es.

8 EPYC1 proteins bind to 8 sites on the small subunits of Rubisco in Form I Rubisco (L₈S₈) multimer.   Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the EPYC1(106-135) peptide-bound large-small chain Rubisco dimer (7JSX)

Figure \(\PageIndex{20}\): EPYC1(106-135) peptide-bound Rubisco (7JSX). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structu...VWZB2C5pyZrLw6

The small chain is shown in light cyan and the large chain is in light magenta. The EPYC1 peptide binds largely through ion..ion interactions mostly to the small unit. 

Indeed, if EPYC1 is added to Rubisco in vitro, it forms liquid droplets.  EPYC1 doesn't act as a scaffold per se to which Rubisco attaches and phase separates.  Rather, it is through multiple low-affinity interactions that the interactions of EPYC1 and Rubisco lead to phase separation. It occurs even at low concentrations of both. The linker protein EPYC1, with multiple arginines and lysines, is positively charged.  They interact with negative side chains in the small rubisco subunit.   Accordingly, their interactions and phase separation are salt-dependent.

CIDER (Classification of Intrinsically Disordered Ensemble Regions) was used to calculate the charge distribution of side chains in the large and small subunits of Rubisco and EPYC1.  These parameters were calculated.

• κ (kappa, 0-1): A measure of the extent of charge segregation in a sequence.  All three proteins are weak polyampholytes with small fractions of + and - side chains. As such, they all form globular-like structures that are less dependent on the protein's charge distribution. Low κ values are found when charges are mixed along the chain and not highly segregated.  This minimizes self-aggregation and the formation of hairpins in EPYC1 with itself, for example.  Even strong polyampholytes with low κ values form more random coil structures. 
• FCR: The fraction of charged residues in a sequence
• NCPR: The net charge per residue (can be positive or negative)
• Hydropathy: The mean hydropathy of a sequence, calculated as the average of a 0-9 using the Kyte-Doolittle hydrophobicity scale. 
• Fraction disorder promoting: The fraction of residues that promote disorder.

The results are shown in Table \(\PageIndex{3}\):below

seq	length	κ	FCR	NCPR	Hydropathy	Fraction disorder promoting
LCI5 (aka EPYC1)	321	0.137	0.184	0.109	3.883	0.841
small Rubisco	185	0.169	0.189	0.038	4.464	0.589
large Rubisco	475	0.138	0.240	-0.013	4.274	0.623

Table \(\PageIndex{3}\): Charge Distribution in Rubisco and LCI5 (EPYC1)

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the surface of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (8RUC) colored code by charge.  

Figure \(\PageIndex{21}\): Surface of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (8RUC) colored code by charge. Red indicates negative charge and blue positive. (Copyright; author via source). Click the image for a popup or use this external link:  https://www.ncbi.nlm.nih.gov/Structu...7QgFy3UMqyLPy5 

The full-length linker protein LCI5 (aka EPYC1), which has highly disordered links, forms a multitude of low-affinity, polyvalent electrostatic interactions with the Rubisco 16-mer, promoting aggregation and phase separation. 

Here is a potential model showing these interactions: (Figure 6E from https://pmc.ncbi.nlm.nih.gov/articles/PMC7736253/#F7) - Awaiting permission for use of the actual image instead of this link: https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d65f/7736253/6c90be375842/nihms-1640018-f0006.jpg

It should be noted that Rubisco in the carboxysome also interacts with an intrinsically disordered linker protein CsoS2 (Carboxysome assembly protein CsoS2B), which leads to Rubisco's phase separation together with carboxysomal carbonic anhydrase (CsoSCA ) in α-cyanobacteria.

Figure \(\PageIndex{22}\) shows an interactive iCn3D model of the carboxysomal mini-shell icosahedral assembly from CsoS1A and CsoS2  from Halothiobacillus neapolitanus (8B12).  The major carboxysome shell protein CsoS1A is in the gray cartoon, the carboxysome shell vertex protein CsoS4A in the purple spacefill, and the CsoS2 is in the cyan cartoon.

Figure \(\PageIndex{22}\): Carboxysomal mini-shell icosahedral assembly from CsoS1A and CsoS2  from Halothiobacillus neapolitanus (8B12). (Copyright; author via source). Click the image for a popup or use this external link: https://www.ncbi.nlm.nih.gov/Structure/icn3d/share.html?mE2g19pAtcMaAoiy6.

Carboxysomes self-assemble in a process guided by the intrinsically disordered linker protein CsoS2, which connects multiple shell proteins, carbonic anhydrase, and Rubisco to form the ultimate phase-separated carboxysome.  Interactions are mediated in part by a repetitive Ile(Val)-Thr-Gly ([IV]TG) motif in CsoS2 and the β-strand in CsoS1A through His79 and main chain hydrogen bonds in the main chain.  The domain and tandem repeat structures of CsoS2 and its interactions with CsoS1A are illustrated in Figure \(\PageIndex{23}\) below.

Figure \(\PageIndex{23}\): CsoS2 binds to the shell through multivalent interactions with shell proteins and highly conserved interfaces via novel [IV]TG repeats.  Ni, T., Jiang, Q., Ng, P.C. et al. Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly. Nat Commun 14, 5512 (2023). https://doi.org/10.1038/s41467-023-41211-y.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

Panel a shows the domain arrangement of CsoS2. The N-terminal, Middle and C-terminal domains are colored in pink, green and red, respectively. Three dashed boxes indicate the structured fragments resolved in T = 9 shell. 

Panel b shows the CryoEM densities of F1-F3 with atomic models. c CsoS2 interactions with shell components, viewed from inside. Three structured fragments in the C-terminal domain, F1, F2 and F3, are identified and labelled. 

Panels d–f: Interaction interfaces between CsoS2 F1 (d), F2 (e) and F3 (f) fragments with shell components, CsoS1A (blue/green) and CsoS4A (purple). 

Panel g shows the alignment of CsoS2-CsoS1A interacting motifs, showing the CsoS2 [IV]TG motif (green) in contact with CsoS1A His79. 

Panel h shows the consensus sequences of CsoS2 C-terminal F1, F2 and F3 fragments from 100 CsoS2 sequences, plotted with Weblog. Asterisks indicate the conserved repeating [IV]TG motif present in each fragment.

Figure \(\PageIndex{24}\) below shows a summary representation of phase-separated Rubisco, which preferentially interacts with CO₂ if localized near high concentrations.

Figure \(\PageIndex{24}\): Phase Separation of Rubisco.  PHASE SEPARATION 101. Animation Lab. Margot Riggi, Janet Iwasa, et al. Creative Commons Licensing CC BY 4.0 - https://creativecommons.org/licenses/by/4.0/

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Recent Updates:  2/27/25

The Skeptics - But isn't the CO₂ we emit into the atmosphere just plant food?  

This argument is used by many who say that we shouldn't worry about dumping CO₂ from burning fossil fuels into the atmosphere.  As with some arguments, there is partial truth in the statement. Of course, CO₂ is plant "food," as it is fixed by Rubisco in photosynthesis.  This incomplete and misleading description is an example of a logical fallacy.  Several applicable ones are described below: 

• Half-truth (Cherry-picking Fallacy) – The argument selectively presents only one fact (that CO₂ benefits plants) while ignoring other relevant facts, such as how excessive CO₂ contributes to global warming, ocean acidification, and extreme weather, which can harm ecosystems, including plants.

• Hasty Generalization – Just because CO₂ benefits plants in some cases doesn't mean that increasing CO₂ indefinitely is beneficial. It overlooks the complex ecological and climatic consequences.

• Oversimplification – The argument reduces a complex scientific issue to a single, misleading cause-and-effect relationship for climate change.

• Straw Man Fallacy (in some contexts) – If this argument is used to dismiss concerns about climate change without addressing the broader scientific consensus, it misrepresents the issue and creates a weaker version of the opponent’s position

Recent studies have shown that increasing CO_{2 atm} increases plant carbon capture. Of course, it would since it is a substrate for Rubisco, which does not run at V_max given CO_{2 atm} concentrations.  The carboxylation rate increases compared to the enzyme's oxidase rate.  Studies documenting this effect use deuterium NMR.

As you learned in Organic Chemistry, protons (¹H) have characteristic NMR shifts from 1-12 ppm.  The proton resonates at a frequency of 100 - 800 MHz.  Deuterium (²H or just D, natural abundance 0.015%) has a broader signal since it has a small magnetic dipole moment.  It has an optimal resonant frequency of 61 MHz. Samples for ¹H NMR experiments are typically dissolved in D₂O or CD₃OD since the Ds are mostly invisible at the frequencies used.  Nevertheless, D-NMR is useful, for instance, to differentiate different stereoisomers of D-substituted molecules.  These isomers are called isotopomers.  Figure \(\PageIndex{25}\) illustrates two isotopomers of deuterium-labeled glucose, an end product of photosynthesis.

Figure \(\PageIndex{25}\): Two C6 deuterium isomers of D-glucose (straight chain).

The two C6 Hs on glucose in the top panel are identical substituents, so the C6 carbon center does not exist as stereoisomers.  However, if each was separately substituted with deuterium, two stereoisomers, called isotopomers, exist. The original unsubstituted glucose is prochiral at C6, with one of the Hs being proR and the other proS.

Rubisco has both carboxylase and oxygenase activities, and the ratio of D6^S/D6^R isotopomers can give insight into the relative rates of both activities.  As CO_{2 atm} increases, the products from the carboxylase activity should increase as those from the oxygenase decrease or remain unchanged.  The D6S/D6R isotopomer ratio change is seen experimentally in glucose form sunflower leaf starch grown in different concentrations of CO_{2 atm}, as shown in Figure \(\PageIndex{26}\) below.  The individual D-NMR peaks from glucose obtained from plants grown at higher CO_{2 atm} have lower NMR shifts (ppm). In addition, the D6^R peak for glucose from leaves exposed to high CO_{2 atm} is higher in amplitude peak, indicating increased concentrations of the D6^R isotopomer. As CO_{2 atm} increases, D6^S/D6^R decreases.

Figure \(\PageIndex{26}\): Effect of [CO₂] on the D6^S/D6^R isotopomer ratio of photosynthetically generated glucose moieties in sunflower (H. annuus).  I. Ehlers, A. Augusti, T.R. Betson, M.B. Nilsson, J.D. Marshall, & J. Schleucher, Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C3 plants over the 20^th century, Proc. Natl. Acad. Sci. U.S.A. 112 (51) 15585-15590, https://doi.org/10.1073/pnas.1504493112 (2015). This article is freely available online through the PNAS open access option, for which there is permission for the general reuse of figures for non-commercial and educational purposes without requesting permission.  

Panel (A): Deuterium NMR spectrum of a glucose derivative displaying one signal for each of the seven glucose isotopomers. The signals’ integrals are proportional to the isotopomer abundances.

Panel (B): Excerpts of deuterium NMR spectra of glucose prepared from sunflower leaf starch, showing signals from the D6^Sand D6^R isotopomers of the C6H₂ glucose group. The solid and dashed spectra were acquired from glucose formed at CO_{2 atm} levels of 280 ppm and 1,500 ppm CO₂, respectively. The dashed spectrum has been shifted sideways to ease comparison.

Panel (C): Note:  This plot has been linearized by plotting 1/[CO_{2 atm}] on the bottom x-axis.  The Dependence of the D6^S/D6^R ratio of glucose from sunflower leaf starch on 1/[CO₂] (in units of 10⁻³ ppm⁻¹) during growth [r² = 0.88, slope 0.057 ±0.006 (SEM), P < 10⁻⁷, n = 17, individual plants except for 180 and 280 ppm, where material from two to four plants had to be pooled for each sample].

A similar relationship between D6^S/D6^R and 1/[CO₂] was seen in beet sucrose samples obtained from 1890 to 2012.  

Mechanistically, this decrease in the D6^S/D6^R in leaves grown at high CO_{2 atm}, arising from the increase in D6^R peak, can be explained by the metabolic pathways for the carboxylase and oxygenase reactions in C₃ metabolism. In photooxidation (photorespiration), one G3P is produced along with one 2-phosphoglycolate, a 2-carbon molecule, as CO₂ was not fixed (see The Glycolate Pathway at the end of chapter).  This can be converted to G3P by a different set of reactions in the chloroplast and in the peroxisome.  Given the complexity of the isotope effect in enzyme reactions, it has always been observed that different sets of reactions lead to a common reactant displaying different isotopomer distributions. The carbon group added to 2-phosphoglycolate to form 3-phosphoglycerate comes from serine using peroxisomal serine hydroxymethyl transferase. Serine purchased commercially uses that enzyme for its production and has a unique and strong isotopomer signal at its carbon C3.

Changes in the D6^S/D6^R ratio in tree rings in tropical Toona cilita trees in Asia and Australia over ≈ 100 years have been observed with increasing CO_{2 atm} and tree diameter, a measure of irradiance at the tree's crown. The photosynthetic efficiency (increased carboxylation decreased photorespiration) increased with CO_{2 atm} but decreased with tree diameter, probably reflecting limitation on water movement to the canopy and reduced leaf CO₂ with high irradiance.  (Note: the D6S/D6R saturates with tree height and, at some point, no longer increases.) This also correlates with the biosphere primary production increase (carbon fixation into glucose) between 1981-2020.  

Global warming caused by increased CO_{2 atm} from burning fossil fuels will have very bad effects on plants.  Higher temperatures lead to more water in the atmosphere and on the surface, which can be good since plants need rain for growth.  However, extreme weather caused by higher global temperatures leads to floods or droughts, which harm plants. Of course, plants will not easily adapt to higher temperatures in the future.

Also of great concern is the change in the nutritional composition of the plants growing at higher CO_{2 atm}, a problem that will likely affect food insecurity.  Carbon stocks (sugars and starches) may increase, but nutrients derived from soil decrease.  Significant protein and micronutrient reduction occurs, such as iron, magnesium, and zinc.  These changes are especially observed in rice, wheat, potatoes, and barley.  This problem affects humans and herbivores, which have greater concentrations of these nutrients than the plants they eat.  Figure \(\PageIndex{27}\) shows the reduction in plant protein and micronutrients at elevated CO_{2 atm} levels (568 to 590 ppm compared to present levels of 427 on 2/27/25). The data was obtained through a multiyear study at free-air CO₂ enrichment (FACE) facilities under field conditions in China and Japan. The names to the right in the bar graph are particular rice lines (cultivars).  The decreased availability of nutrients for herbivores will affect the entire food chain.

Figure \(\PageIndex{27}\): Reduction in plant protein and micronutrients relative to CO_{2 atm}. Chunwu Zhu et al. Carbon dioxide (CO₂) levels this century will alter the protein, micronutrients, and vitamin content of rice grains, with potential health consequences for the poorest rice-dependent countries.Sci. Adv. 4, eaaq1012(2018).DOI:10.1126/sciadv.aaq1012.   Distributed under a Creative Commons Attribution-NonCommercial License 4.0 (CC BY-NC).

Left panel:  Average reduction in grain protein at elevated relative to ambient [CO₂] for 18 cultivated rice lines of contrasting genetic backgrounds grown in China and Japan using free-air CO₂ enrichment (FACE) technology. Bars are ±SE. *P < 0.05 and **P < 0.01 (see Methods for additional details).

Right Panel: Average reduction in grain micronutrients, iron (Fe), and zinc (Zn) concentration at elevated relative to ambient [CO₂] for 18 cultivated rice lines of contrasting genetic backgrounds grown in China and Japan using FACE technology.  Bars are ±SE. *P < 0.05 and **P < 0.01 for a given cultivar. CO₂; **P < 0.01 is based on all cultivars (see Methods for additional details).

Similar effects were seen for B vitamin content.

The next step: 3-Phosphoglycerate to Glyceraldehyde 3-Phosphate and dihydroxyacetone phosphate

The 3PG produced by RuBisCo is converted to the triose glyceraldehyde-3-P (G3P) (which can readily isomerize to dihydroxyacetone phosphate) using typical glycolytic enzymes run in reverse, except that NADPH is used as a reductant instead of NADH. In addition, the stromal and cytosolic enzymes derive from different genes. The G3P not used to resynthesize ribulose 1,5BP can be used to synthesize starch, sucrose, etc, as illustrated in Figure 4 above.

Exchange of trioses and phosphate across the inner membrane

The inner chloroplast membrane has a triose-phosphate/phosphate translocator (TPT), an antiporter that brings into the stroma Pi in exchange for a triose phosphate, either dihydroxyacetone phosphate or 3-phosphoglycerate. The importance of this was discussed above. The exported triose can be used to synthesize sucrose, which can be transported around the plant as a carbon source. Trioses within the chloroplast can also be converted to glucose and onto glycogen as the organelle becomes an amyloplast. If the translocator is inhibited, Pi would decrease in the chloroplast, decreasing ATP and starch synthesis.

The structure of TPT has been determined with the bound ligands, 3-phosphoglycerate and inorganic phosphate, in an occluded conformation from Galdieria sulphuraria, an extremophilic unicellular species of red algae. Figure \(\PageIndex{25}\) shows an interactive iCn3D model of a triose-phosphate/phosphate translocator from the red algae (5Y78).

Figure \(\PageIndex{25}\): Triose-phosphate/phosphate translocator from red algae (5Y78). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...mVNeFkXibxXr39

The model shows two monomers, one with red cylindrical alpha helices and spacefill 3-phosphoglycerate. The other subunit is gray, with the 3PG in colored sticks and conserved residues (T188, K204, F263, Y339, K362, R363) that interact with both Pi and 3PG. An outward- and inward-open conformation would presumably be triggered on ligand binding.

Activity Regulation by Light

Given their role in photosynthesis, you would expect even the dark reaction enzymes to be regulated by light. Indeed, four C3 cycle enzymes are. They are ribulose 5-phosphate kinase, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase. The regulation is affected by photon-induced disulfide bond formation between two cysteine side chains in the enzymes. When oxidized (disulfide bond form), the enzymes are inactive. Under light conditions, PSII, cyto b6f, and PSI work in electron transport to move electrons from H₂O to ferredoxin and onto a small soluble protein, thioredoxin, which has a disulfide. The enzyme catalyzing this last step is ferredoxin-thioredoxin reductase. On reduction, the disulfide in thioredoxin is cleaved, and the now free sulfhydryls in thioredoxin are used to cleave the disulfide in the four enzymes mentioned above in a similar fashion to how β-mercaptoethanol in excess can cleave disulfides in proteins. This leads to conformational changes in the four enzymes that activate them. The process reverses without light, and the enzymes are inhibited. For fuel at night, plants mobilize starch for energy.

A simple mechanism to show how thioredoxin catalyzes disulfide bond reduction in target proteins is shown in Figure \(\PageIndex{26}\).

Figure \(\PageIndex{26}\): Mechanism of thioredoxin reduction of target proteins. https://en.Wikipedia.org/wiki/Thiore...le:FigMech.png

The first enzyme in the oxidative branch of the pentose pathway, glucose 6-phosphate dehydrogenase, uses NADP⁺ as an oxidizing agent, producing NADPH. In the light, there is lots of NADPH produced from the light reactions of photosynthesis, so it makes biological sense that under these conditions, glucose 6-phosphate dehydrogenase activity is inhibited. It is also regulated by the cleavage of a critical disulfide bond, but it results in enzyme inactivation in this case.

We saw the role of thioredoxin in the previous chapter section when we discussed the regulation of photosynthesis and the chloroplast's ATP synthase.

Figure \(\PageIndex{27}\) shows an interactive iCn3D model showing a comparison of the structures of oxidized (1ERU) and reduced (1ERT) human thioredoxin.

Figure \(\PageIndex{27}\): Comparison of the structures of oxidized (1ERU) and reduced (1ERT) human thioredoxin. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...XPVsErV2JWxdA6.

The two subunits of thioredoxin, linked by a disulfide, are shown in gray. Press the "a" key to toggle between the oxidized form, with Cys32-Cys35 disulfide shown as a yellow stick, and the reduced form with the reduced and separated Cys 32 and Cys 35 shown in colored spheres. Not that the hydrogen covalently attached to the free cysteine side chain does not show in a crystal PDB structure.

SUMMARY

Photosynthesis in vascular plants takes place in chloroplasts. In the CO₂-assimilating reactions (the Calvin cycle), ATP and NADPH are used to reduce CO₂ to triose phosphates. These reactions occur in three stages: the fixation reaction, catalyzed by Rubisco; reduction of the resulting 3-phosphoglycerate to glyceraldehyde 3-phosphate; and regeneration of ribulose 1,5-bisphosphate from triose phosphates. Rubisco condenses CO₂ with ribulose 1,5-bisphosphate, forming an unstable hexose bisphosphate that splits into two molecules of 3-phosphoglycerate. Rubisco is activated by covalent modification (carbamoylation of Lys201) catalyzed by Rubisco activase. It is inhibited by a natural transition-state analog, whose concentration rises in the dark and falls during daylight. Stromal isozymes of the glycolytic enzymes catalyze the reduction of 3-phosphoglycerate to glyceraldehyde 3-phosphate; each molecule reduced requires one ATP and one NADPH. The cost of fixing three CO₂ into one triose phosphate is nine ATP and six NADPH, which are provided by the light-dependent reactions of photosynthesis. An antiporter in the inner chloroplast membrane exchanges Pi in the cytosol for 3-phosphoglycerate or dihydroxyacetone phosphate produced by CO₂ assimilation in the stroma. Oxidation of dihydroxyacetone phosphate in the cytosol generates ATP and NADH, thus moving ATP and reducing equivalents from the chloroplast to the cytosol. Four enzymes of the Calvin cycle are activated indirectly by light. They are inactive in the dark so that hexose synthesis does not compete with glycolysis—which is required to provide energy in the dark.

Photorespiration - RuBisCo/Oxygenase and the Glycolate Cycle - Another Look

As autotrophs, plants make their fuels. They use that fuel to make ATP to power endergonic reactions like protein synthesis, cell division, etc. As eukaryotic cells, they have mitochondria and can use both aerobic and anaerobic respiration to produce ATP. In the dark, when photons are absent, they carry out mitochondrial aerobic respiration as they break down carbohydrates to CO₂ and water, the reverse of photosynthesis.

They also use O₂ in another process that is driven by light. As we detailed above, the enzyme that captures carbon, RuBisCo, has oxygenase activity. RuBisCo uses O₂ in a process called photorespiration, which produces CO₂ in a competing reaction. The final products of the reaction with CO₂ using RuBisCo are two 3C molecules, 3-phosphoglycerate (3PG). Using O₂ as a substrate produces 1 molecule of the 3C 3PG and 1 molecule of a 2C analog, 2-phosphoglycolate (not 2-phosphoglycerate). 2-phosphoglycolate is also named carboxymethylphosphate. About one out of every four turnovers of the enzyme produced this metabolic dead product. Given this non-trivial side reaction, the enzyme should be called ribulose 1,5-bisphosphate carboxylase/oxygenase.

Compare the K_M values (9 μM for CO₂ and 350 μM for O₂ or 39x higher for O₂) for the enzyme and the equilibrium concentrations of the gases in aqueous solution (11 μM for CO₂ and 250 μM for O₂ or 23x higher for O₂). Oxygen's greater solubility nearly offsets the higher K_M for O₂, so modern conditions lead to a significant waste of the CO₂ capture efficiency of RuBisCo/Oxy. At higher temperatures in a warming world, the equilibrium ratio of solution concentrations of O₂/CO₂ increases, as does the affinity (based crudely on K_M values) of CO₂. Both of these exacerbate the effect of wasteful oxygenase activity. Finally, as the enzyme captures CO₂ is captured by the enzyme, the ratio of the local concentrations of O₂/CO₂ also goes up. All of these factors make RuBisCo's efficiency worse.

Figure \(\PageIndex{28}\) shows another mechanism for the reaction of both CO₂ and O₂ with RuBisCo/Oxygenase.

Figure \(\PageIndex{28}\): Mechanism for the reaction of both CO₂ and O₂ with RuBisCo/Oxygenase. (after Kannappan et al. J. Phys. Chem. B 2019, 123, 2833−2843)

Note again that in contrast to most oxygenases, no cofactor is required for the RuBisCo/Oxygenase.

The glycolate pathway

The 2-phosphoglycolate (carboxylmethyl phosphate) "waste" product of the oxygenase activity of RuBisCo/Oxygenase is recycled through a complex pathway that is called "photorespiration". It occurs in three different organelles, the chloroplast, the peroxisome, and the mitochondria. Part of the generalized pathway is shown in Figure \(\PageIndex{29}\).

Figure \(\PageIndex{29}\): The glycolate photorespiration salvage pathway (adapted from Hu et al. Plant Cell. 2012;24.doi:10.1105/tpc.112.096586)

Multiplying the stoichiometry represented in the figure by 2 shows that 2 molecules of 2-phosphoglycolate produce 2 molecules of glycine. These get converted to two molecules of serine. We will see the mechanisms for some of these reactions in the chapter on amino acid metabolism. The net reaction is:

\[\ce{2 Glycine + NAD^{+}+ H2O → serine + CO2 + NH3 + NADH} \nonumber \]

The serine is eventually converted to 3-phosphoglycerate, which can be used again in the C3 cycle. Note that CO₂ is produced in the glycolate pathways that started with the use of O₂ as a substrate by RuBisCo/Oxygenase. Hence, the whole system uses O2 and produces one CO₂, so the combined reactions are usually called photorespiration. It's not an ideal term since it is wasteful compared to mitochondrial respiration. Some prefer to call the combined pathway of Rubisco oxygenase and the glycolate pathways the C2 cycle.

Summary

This chapter shifts focus from the light-dependent reactions of photosynthesis to the “dark reactions,” a series of biochemical pathways that fix CO₂ into organic compounds, primarily through the Calvin (C3) cycle. Although termed “dark reactions,” these processes occur during the day and are distinguished from the light reactions by their independence from direct photon absorption.

Calvin Cycle Overview:

• CO₂ Capture and Fixation:
  Atmospheric CO₂ is fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) into ribulose 1,5-bisphosphate (RuBP), producing an unstable 6-carbon intermediate that splits into two molecules of 3-phosphoglycerate (3PG).
• Reduction and Regeneration:
  3PG is then reduced to glyceraldehyde-3-phosphate (G3P) using ATP and NADPH generated during the light reactions. Most G3P molecules are recycled to regenerate RuBP, while a fraction is used for the synthesis of carbohydrates such as starch and sucrose.

Rubisco: Structure, Function, and Limitations:

• Dual Activity:
  Rubisco catalyzes both the carboxylase and oxygenase reactions. Its primary role is to fix CO₂; however, it also reacts with O₂, leading to photorespiration—a process that diverts energy and fixed carbon away from carbohydrate synthesis.
• Structural Composition and Evolution:
  The enzyme is composed of large subunits (encoded by the chloroplast genome) and small subunits (nuclear-encoded), forming multimeric complexes (often a 16-mer in higher plants). Rubisco’s slow catalytic rate and competing oxygenase activity are attributed to its evolutionary origin in a high-CO₂, low-O₂ environment, which has become suboptimal under modern atmospheric conditions.
• Mechanistic Insights:
  Detailed structural studies reveal how the enzyme’s active site, including a carbamoylated lysine and a bound Mg²⁺ ion, facilitates CO₂ fixation. Despite its inefficiency, high cellular concentrations of Rubisco ensure sufficient carbon fixation.

Photorespiration and the Glycolate Pathway:

• Competing Reaction with O₂:
  When Rubisco acts as an oxygenase, it produces one molecule of 3PG and one molecule of 2-phosphoglycolate. The latter is a “waste” product that enters the glycolate salvage pathway (photorespiration), a multi-organellar process spanning the chloroplast, peroxisome, and mitochondrion.
• Energetic and Metabolic Costs:
  Photorespiration consumes energy and releases CO₂, thus reducing the overall efficiency of photosynthetic carbon fixation, especially under conditions of high O₂ and elevated temperatures.

Plastid Dynamics and Metabolite Transport:

• Plastid Diversity and Interconversion:
  The chapter discusses various plastids—chloroplasts for photosynthesis, amyloplasts for starch storage, and others—and how they interconvert based on developmental and environmental signals.
• Triose Phosphate/Phosphate Translocator (TPT):
  The TPT exchanges triose phosphates from the chloroplast with inorganic phosphate from the cytosol, linking the dark reactions with cytosolic carbohydrate metabolism.

Regulation by Light and Redox Control:

• Enzymatic Regulation:
  Key Calvin cycle enzymes are modulated by light through the ferredoxin–thioredoxin system. In the light, reduced thioredoxin activates these enzymes by cleaving regulatory disulfide bonds, ensuring coordination between the light and dark reactions.
• Balancing Carbon Fixation and Photorespiration:
  The enzyme’s competing activities are influenced by substrate concentrations and environmental conditions, with plants employing mechanisms (e.g., phase separation of Rubisco in carboxysomes or pyrenoids) to increase local CO₂ concentrations and enhance fixation efficiency.

Environmental and Global Implications:

• Impact of Elevated CO₂ and Climate Change:
  Changes in atmospheric CO₂ levels affect Rubisco kinetics and plant nutrient composition. Although higher CO₂ can boost carbon fixation, it may also lead to imbalances in nutrient content (e.g., reduced protein and micronutrient levels) in crops, with broader implications for food security.
• Photorespiratory Flux and Plant Productivity:
  The efficiency of the dark reactions is a critical determinant of overall photosynthetic performance, influencing how plants adapt to changing environmental conditions and how global carbon cycles are maintained.

In summary, this chapter integrates the biochemical mechanisms underlying CO₂ fixation, the regulation and limitations of Rubisco, and the metabolic consequences of photorespiration. It highlights the complexity of plant metabolism, emphasizing how dark reactions, though indirectly driven by light, are finely tuned to optimize carbon assimilation and respond to environmental challenges.