20.4: CO₂ uptake - Calvin Cycle and C3 organisms

Search Fundamentals of Biochemistry

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

Introduction

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 is simply used to differentiate them from the light-driven reactions using PSII and PSI. What is so interesting about plants is they produce fuel from CO₂ using photons as a source of energy (they are autotrophs) and also consume the fuels they make, using both anaerobic and aerobic respiration pathways. Their biosynthetic reactions take place mostly in the chloroplast, a type of plastid, which are 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 a large range of growing conditions (differential light qualities and quantities, temperatures, and rainfall levels). Also, plant cells have cell walls in addition to a cell membrane. A simple cartoon showing the major motifs of photosynthesis is shown in Figure \(\PageIndex{1}\).

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 logically 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, to 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 chloroplast which has its own genome, like the mitochondria. Another common type is the amyloplasts, which lack pigmented molecules (i.e. they are colorless) and have no inner membrane. In plants, 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)

The characteristics and plastid interconversion pathways of the plastids are shown by arrows. The transition to a chloroplast is called “Greening” and is identified with the number “1”. This 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 that is 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” and 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 the use of 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, 6 carbons in total), to form two molecules of 3-phosphoglycerate (3PG). (2, 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, in the form of DHAP where it can be used in gluconeogenesis (reductive biosynthesis) of glucose. This can be converted to polymer starch and also 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 actual number of carbon atoms and realize that the cycle has to run 3 times to enable 3 carbon atoms from 3 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 3 turns of the cycle, sufficient to produce 1, 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 as they are 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 also is 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 the synthesis of 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) pathway 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 2 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.  4 dimers of the large chain (8 subunits) interact with 8 small chains to form a 16-mer in cyanobacteria, red and brown algae, and higher plants.  Other 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 8 large subunits are colored in varying shades of magenta/purple.  The 8 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 carbamoylated lysine side chain ("activator CO_2") is shown in green. 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 of which contains the carbon atom from CO₂ (red).  The "activator CO2" (the carboxylated lysine) is hydrolyzed and removed at night, which inactivates 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, it inhibits the enzyme. With increasing lights, 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 (labeled KCX201). The bound activated substrate analog, 2-carboxyarabinitol bisphosphate, contains a -CO₂^- group that coordinates with the Mg²⁺ ion (green sphere) in place 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) is described as occurring 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 0 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 in Figure \(\PageIndex{10}\) below.

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 values for dipole and quadrupole moments 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 shows that 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 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 is 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 just above it red (negative potential, N-terminal domain).  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 small hydrophobic side chain (Ala, Val, Leu, Ile) and the main chain.  CO₂ has more interaction in every active site as well as 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 differentially desolvate CO₂.  The hydrophobic interactions likely promote the movement of CO₂ to the active site where electrostatic-based potentials likely 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 rather in shallow groves 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 have evolved to meet the environmental and biochemical needs of their respective organisms.  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 2 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 not present and CO₂ concentrations were 10K% higher than now.  Hence the enzyme evolved over time 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).

Important branch points in the phylogeny at which Rubisco diverged into different evolutionary lineages are indicated by gray vertical bars. To provide additional context, the time-period at which the First and Second Great Oxidation events occurred along this evolutionary trajectory are 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 O₂, although 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 was close to the source of CO₂ or is included in a heterogeneous condensate itself.  One such 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 high concentration and with minimal O₂ as a competitor for Rubisco.

Figure \(\PageIndex{15}\) below shows a model epresenting 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, which are 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 in 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 that there are 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 4 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 4 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.

A program called 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 in 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 less dependent on their charge distribution on the protein. Low κ values are found in 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 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 is 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 phase separation of Rubisco 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 as well as 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 of it.

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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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 remaining G3P not used to resynthesize ribulose 1,5BP can be used for the synthesis of 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 for the synthesis of sucrose, which can be transported around the plant as a source of carbon. 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, which would decrease ATP and also 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 shown in gray with the 3PG in colored sticks and conserved residues (T188, K204, F263, Y339, K362, R363) that interact with both Pi and 3PG. There would presumably be an outward- and inward-open conformation that is triggered on ligand binding.

Activity Regulation by Light

Given their role in photosynthesis, you would expect even the dark reaction enzymes would 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 4 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 which activate them. In the absence of light, the process reverses, 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 so, also by the cleavage of a critical disulfide bond, but in this case, it results in enzyme inactivation.

We saw the role of thioredoxin in the previous chapter section when we discussed the regulation of photosynthesis as well as the ATP synthase of the chloroplast.

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 itself, 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 and 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 and 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 not present, 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). The higher K_M for O₂ is nearly offset by O₂s greater solubility 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 wasteful oxygenase activity effect. Finally, as CO₂ is captured by the enzyme, the ratio of the local concentrations of O₂/CO₂ also goes up. All of these make the efficiency of RuBisCo 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 reaction 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.