20.5: CO2 uptake - C4 and CAM Pathways

The C4 Pathway

Photorespiration, caused by the oxygenase activity of RuBisCo/Oxygenase and the ensuing glycolate salvage (C2) pathway, significantly diminishes the efficiency of photosynthesis in C3 plants. Rest assured, some plants have found a way around the problem by adding a few steps before RuBisCo. The altered pathway is known as the C4 pathway, and plants that use it are called C4 plants. The initial steps involve the temporary capture of CO₂ as HCO₃^-into a 4C, not 3C, sugar. Plants that utilize the C4 pathway include maize, sorghum, sugarcane, and many tropical plants. An overview of the pathways emphasizing the steps that precede RuBisCo is shown in Figure \(\PageIndex{1}\).

This extra step (compared to C3 plants) concentrates CO₂ and effectively reduces the competing substrate O_2, thereby increasing Rubisco efficiency.

Experimental evidence shows that radiolabeled ¹⁴CO₂ is captured into the 4C molecule, oxaloacetate (OAA), a citric acid cycle and gluconeogenic intermediate, through the enzyme phosphoenolpyruvate carboxylase, which uses HCO₃^- as a substrate. The reaction takes place in mesophyll cells. OAA can be reduced to malate by NADPH via malate dehydrogenase or converted to aspartate via transamination (not shown in the figure). Malate moves into the bundle sheath cell.  The NADP-malic enzyme then decarboxylates it to pyruvate. Pyruvate can move back into the mesophyll cell, where it is converted to phosphoenolpyruvate (PEP) and then back to OAA by pyruvate phosphate dikinase and PEP carboxylase. CO₂ from malate decarboxylation is delivered to RuBisCo as a substrate.

Pyruvate dikinase is used in bacteria, protozoa, C4 plants, and another type, the Crassulacean type, discussed below. Its non-plant function is to produce ATP, similar to pyruvate kinase. The net reaction is:

ATP + phosphate + pyruvate = AMP + PP_i + H⁺ + phosphoenolpyruvate

Figure \(\PageIndex{2}\) shows a simplified mechanism for the reaction

The net reaction shows that in C4 plants, two molecules are phosphorylated by ATP. One is pyruvate, and the other is inorganic phosphate (Pi). Hence the name dikinase. The reverse reaction of ATP synthesis occurs in bacteria and protozoans. Two phosphorylated intermediates exist: Enz-His-P and Enz-His-PP_i. These are "activated" phosphate carriers in the phosphotransfer reactions to pyruvate and P_i, respectively. If PP_i is not hydrolyzed to 2P_i, as illustrated in the top left of Figure 2 above, the reaction is fully reversible.

In C4 plants, there are three reactions

1. Pyr + E-His-P ↔ PEP + E-His
2. E-His + ATP ↔ E-His-PP ^.AMP (^. indicates a noncovalent interaction)
3. E-His-PP ^.AMP + P_i ↔ E-His-P + AMP + PP_i

When PP_i is hydrolyzed, the net input of ATP to phosphorylated pyruvate is two ATP equivalents.

In the next step, PEP carboxylase carboxylates PEP in a carbon-capture reaction, using HCO₃^- as a substrate rather than CO₂ per se. PEP carboxylase also lacks competing oxidase activity. The product is malate, which releases locally high "saturating" concentrations of CO₂ in the bundle sheath cells, significantly suppressing the oxygenase activity of RuBisCo.

Pyruvate phosphate dikinase undergoes a very large conformational change in domain organization during the catalytic cycle. The two kinase activities are located at different sites in the enzyme. The phosphorylation of Pi occurs in the N-terminal domain, while the phosphorylation of pyruvate occurs in the C-terminal domain. The central domain, which links the N- and C-terminal domains via associated "tethers," contains the catalytic histidine involved in phosphotransfer. Domain swiveling occurs to allow sequential phosphotransfer reactions​​​​.

Figure \(\PageIndex{3}\) shows the Pfam domain structure for the protein.

Figure \(\PageIndex{4}\) shows an interactive iCn3D model of an AlphaFold-predicted model of chloroplast pyruvate, phosphate dikinase from Flaveria brownii (Brown's yellowtops) (Q39734)

Figure \(\PageIndex{4}\): AlphaFold-predicted model of chloroplast pyruvate, phosphate dikinase from Flaveria brownii (Brown's yellowtops) (Q39734). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VB6Aywuc2bLD38

The green represents the N-terminal NBD, the red is the central domain with the catalytic His (side chain shown as CPK-colored spheres and labeled), and the blue is the PEP/Pyr PBD.

Figure \(\PageIndex{5}\) illustrates the conformation changes that occur in the central domain (yellow in this figure) and the N-terminal nucleotide-binding domain (NBD, green) in various ligand-bound states.

Panel a–c show movement of the central domain (yellow). In (a), it swivels to face the PBD domain (PDB 5JVL/C), and in (c), it faces the NBD domain (TbPPDK (PDB 2X0S)). In (b), it is in an intermediate position.

Panels d-f show the movement of the NBD (three greens depict the three subdomains). Panel (d) is the state without a bound nucleotide (PDB 5JVJ/A), while panel (f) shows the fully closed, nucleotide-bound state (PDB 5JVL/A). Panel (e) shows a semi-closed, nucleotide-bound state (PDB 5JVL/C). Minges, A., Ciupka, D., Winkler, C. et al. Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase. Sci Rep 7, 45389 (2017). https://doi.org/10.1038/srep45389. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/

Figure \(\PageIndex{6}\) shows the interactions of bound substrates in different conformation states of PPDK.

Figure \(\PageIndex{6}\): Substrate binding sites of FtPPDK.

Panel (a) shows the semi-closed state of the PEP binding site (PDB 5JVL/A) with the catalytic H456 (yellow) pointing away from PEP.

Panel (b) shows the closed state of the PEP binding site (PDB 5JVL/C), showing interactions between PEP and surrounding residues, including the catalytic H456 (yellow).

Panel (c) shows the closed state of the nucleotide-binding site of 5JVL/D occupied with 2'-Br-dAppNHp, a nonhydrolyzable ATP analog. Interacting residues are highlighted. Minges, A. et al. ibid.

After RuBisCo delivers CO₂ from malate in the bundle sheath cell, the remaining reactions are the same as in the C3 pathway.

Once CO₂ is fixed into 3-phosphoglycerate in the bundle-sheath cells, the Calvin cycle's other reactions occur exactly as described earlier. Overall, the C4 pathways require more ATP. Each CO₂ fixed in the C4 pathway requires one PEP, which takes the equivalent of two ATPs. So, each CO₂ fixed in the C4 pathway takes five ATPs compared to three ATPs in the C3 pathway. As mentioned above, CO₂ affinity for RuBisCo (estimated from the K_M) decreases with increasing temperature, reducing the energetic efficiency of carbon capture. At higher temperatures (28-30 °C), the extra energy cost for the C4 pathway balances out the extra energy cost for the C3 pathway at higher temperatures.

Carbonic Anhydrases

We first encountered carbonic anhydrase when we discussed its mechanism in Chapter 6.1. We'll now discuss its function and activity in the C4 pathway in detail. Given the climate crisis driven by rising atmospheric CO₂ from burning fossil fuels, removing CO₂ from the atmosphere—a process called carbon sequestration for climate purposes—has become even more important. Understanding the role of carbonic anhydrase in CO₂ sequestration also becomes crucial.

Plants have three genes for carbonic anhydrase (α, β, and γ). Each can be differentially spliced, giving plants many different isoforms of this protein. They are most abundant in the chloroplast, cytoplasm, and mitochondria, and they have many additional roles beyond CO₂ fixation in C4 (and CAM) plants. Autotrophic (make their own "food") bacteria (such as cyanobacteria, also known as blue-green algae) have no internal organelles. However, there are carboxysomes, which are protein-bounded vesicles (much like a bacteriophage head), which contain not nucleic acid but RuBisCo and carbonic anhydrase in their internal compartment. The carbonic anhydrase converts HCO₃^- to CO₂ for reaction with RuBisCo. The carboxysome thus concentrates the CO₂-producing and fixing enzymes for photosynthesis.

Figure \(\PageIndex{7}\):

F

In C3 plants, CO₂ (aq), which is dissolved CO2, is the actual substrate for RuBisCo, so available HCO3—is converted to CO2 by carbonic anhydrase. In C4 and CAM plants, CO2 (aq) is first converted to bicarbonate by carbonic anhydrase. HCO₃^- (aq) then serves as the substrate for the "carbon fixation" step. Hence, carbonic anhydrase has roles in C3, C4, and CAM plants.

All carbonic anhydrases have a Zn⁺² ion at the active site. The most prominent alpha form in plants was discovered in erythrocytes and is typically active as a monomer. It has a single large 10-strand beta sheet flanked by seven alpha helices. Three histidine side chains and water form a tetrahedral coordination around the Zn²⁺ ion. Gamma carbonic anhydrase is a trimer with three active sites at the interface between pairwise monomers, with the Zn-coordinating histidine side chains from two different subunits.

Beta carbonic anhydrase in plants is typically an octamer of identical subunits. Two cysteines, one histidine, and water coordinate the Zn ion. The monomer has four beta strands in a beta-sheet surrounded by alpha helices. An additional beta-strand is involved in monomer interactions. Because the active site lies at the interface of two subunits, the functional biological unit is the dimer; however, a tetramer and even an octamer are typically formed. The substrate-binding groups correspond one-to-one with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues closely superimposable by a mirror plane of symmetry. Therefore, despite differing folds, alpha- and beta-carbonic anhydrases have converged on a similar active-site design and likely share a common mechanism.

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of beta-carbonic anhydrase from Pisum sativum (pea) with bound acetate (1EKJ).

Figure \(\PageIndex{8}\): Beta-carbonic anhydrase from Pisum sativum (pea) with bound acetate (1EKJ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VZTEJPy1hPxK27

For the sake of simplicity, only two subunits (A - gray and B - magenta) of the octamer are shown. Acetate, a proxy for HCO₃^-2, is shown in spacefill binding between the two subunits in CPK-colored spheres. The side chains involved in Zn²⁺ binding, Cys 160, His 220, and Cys 223 (unlabeled), are on the A chain. The amino acids that bind acetate are distributed between the A chain (Asp 162, Gly 224, Val 184) and the B chain (Gln 151, Phe 169, and Tyr 205) and are labeled. The same groups are involved in substrate binding in alpha-carbonic anhydrase, but in a mirror-image orientation (with normal L-amino acids).

Beta carbonic anhydrase is highly expressed in leaves and is found in chloroplasts, mitochondria, and the cytoplasm. Many plants have it in the cytoplasm and chloroplasts.

• β-carbonic anhydrase (βCA) in C3 plants: Most of the βCA in leaves is in chloroplasts in mesophyll cells and may comprise up to 2% of leaf protein. Yet studies have shown that deleting the gene has minimal effect on the maximal rate of photosynthesis. However, by interference, the enzyme is probably most needed for HCO^3- production for biosynthesis.
• β-carbonic anhydrase (βCA) in C4 plants: Most of the βCA is found in the cytoplasm of mesophyll cells. There, it catalyzes the first reaction of the C4 pathway: CO₂(aq) to HCO₃- (aq). Mitochondrial βCA and γCA probably function to fix CO2 arising from oxidative respiration.

Crassulacean acid metabolism (CAM) pathway

Plants that encounter the chronic stress of low water availability have evolved another pathway to adapt to these conditions. Stomata in C3 plants open during the day to capture carbon from CO₂, but they can close when water is limited. This obviously will inhibit plant growth. In the CAM pathway, the stomata remain open at night, allowing for carbon capture when water loss through the stomata is lower. Carbonic anhydrase fixes the incoming CO₂, and a series of enzymes form malic acid, which is transported to vacuoles for storage and use during the light. This CAM pathway is described in Figure \(\PageIndex{9}\).

The proteins and intermediates in the CAM pathways are ALMT9, aluminum-activated malate transporter; CA, carbonic anhydrase; MDH, malate dehydrogenase; OAA, oxaloacetate; ME, malic enzyme (NAD or NADP); P, phosphate; PEPC, phosphoenolpyruvate carboxylase; PEPCK, PEP carboxykinase; PPCK, PEPC kinase; PPDK, pyruvate, phosphate dikinase.

During the day, malic acid is transported back into the cytoplasm, where malic enzyme decarboxylates it, generating locally high CO₂ concentrations for use by RuBisCo in the C3 cycle. In CAM plants, other changes occur, including modifications to leaf structure and the activation of additional regulatory processes that coordinate metabolic gene expression.

Figure \(\PageIndex{10}\)s shows more details of the CAM cycle.

Thermodynamic comparison between the C3/C4 pathways and the Otto cycle

It is interesting to compare the thermodynamic efficiencies of plants with those of internal combustion engines, which operate according to the Otto cycle. In the cycle, chemical energy in the form of gasoline and O₂ is converted to thermal energy, which is then converted into mechanical energy. Both photosynthesis (the conversion of photon energy into chemical energy) and the Otto cycle have limited efficiencies.

• In internal combustion engines, power is partly limited by air intake and by different efficiencies at non-constant speeds (like stop-and-go).
• In the C3 pathway, efficiency is limited by the oxygenase activity of RuBisCo/Oxygenase (given the much higher concentration of atmospheric O₂ compared to CO₂) and the ensuing photorespiration pathway.
• Water availability also plays a role, as the net reaction of photosynthesis and glucose production, in simplified form, is 6CO₂ + 6H₂O → 6C(H₂O₆), so in high heat and low humidity, the process efficiency decreases.

Figure \(\PageIndex{11}\) shows a comparison of three stages: the storage component, the basic cycle, and a concentration mechanism, in photosynthesis and the internal combustion engine (ICE).

A concentrating mechanism in C4 plants and a turbocharger in turbocharged cars provide concentrated CO₂ and oxygen, respectively, to the core cycle (upper row). A storage mechanism in CAM plants allows carbon dioxide to be stored as malic acid at night and then passed to the Calvin cycle during the day, while a storage mechanism in hybrid electric vehicles (HEVs) allows energy to be stored in the battery during braking and then passed to the motor to power the drivetrain in parallel with the engine (bottom row).

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter describes the C4 and CAM photosynthetic pathways as evolutionary adaptations that overcome the photorespiratory inefficiency of the basic C3 Calvin cycle, presents the biochemistry of the key additional enzymes involved (particularly PEP carboxylase, pyruvate phosphate dikinase, and carbonic anhydrase), and situates these pathways within the context of climate change and food security.

The fundamental problem C4 plants address is RuBisCo's competing oxygenase activity, which becomes progressively more wasteful as temperature rises, as O₂ concentration greatly exceeds CO₂ in the mesophyll, and as active photosynthesis locally depletes CO₂. C4 plants — including the economically critical crops maize, sorghum, and sugarcane — solve this problem by spatially separating initial CO₂ capture from RuBisCo-catalyzed fixation, concentrating CO₂ in the bundle sheath cells where RuBisCo operates.

The C4 pathway begins in mesophyll cells, where carbonic anhydrase converts dissolved CO₂ to HCO₃⁻, which is then carboxylated by PEP carboxylase to form the four-carbon compound oxaloacetate (OAA). PEP carboxylase lacks competing oxygenase activity, a critical advantage over RuBisCo at this step. OAA is reduced to malate by NADP-malate dehydrogenase (or transaminated to aspartate in some C4 subtypes) and transported into the bundle sheath cells. There, NADP-malic enzyme decarboxylates malate to pyruvate, releasing CO₂ at locally high concentrations that saturate RuBisCo and suppress the oxygenase activity. The remaining Calvin cycle reactions proceed normally in the bundle sheath. Pyruvate returns to the mesophyll cell, where pyruvate phosphate dikinase (PPDK) converts it back to PEP, ready for another round of CO₂ capture.

PPDK is a mechanistically fascinating enzyme with a domain-swiveling architecture. Its catalytic histidine, located in the central domain, must physically reposition between two spatially separated active sites: the N-terminal nucleotide-binding domain (NBD), where the histidine is phosphorylated by ATP (forming the E-His-PP·AMP intermediate), and the C-terminal PEP/pyruvate-binding domain (PBD), where the phosphate is transferred to pyruvate to form PEP. This sequential phosphotransfer mechanism — proceeding through E-His-P and E-His-PP·AMP intermediates — means that each CO₂ captured in the C4 pathway costs the equivalent of two ATP (one phosphate goes to pyruvate to form PEP, and pyrophosphate from the ATP reaction is hydrolyzed to drive the reaction forward). Combined with the three ATPs for the Calvin cycle, each CO₂ fixed in C4 costs five ATP versus three in C3. However, at temperatures above approximately 28–30°C, the extra energetic cost of C4 is offset by the elimination of photorespiratory carbon and energy loss, making C4 more efficient under warm conditions — precisely the conditions where C4 crops like maize are most productive.

Carbonic anhydrase plays distinct but complementary roles in all three photosynthetic strategies. In C3 plants, it converts HCO₃⁻ to CO₂ to supply RuBisCo in the chloroplast. In C4 and CAM plants, it operates in the opposite direction in mesophyll cells, converting CO₂ to HCO₃⁻ for use by PEP carboxylase. In prokaryotic carboxysomes, carbonic anhydrase regenerates CO₂ from stored bicarbonate to supply the encapsulated RuBisCo at high local concentrations. Plants possess three genetically distinct carbonic anhydrase families (α, β, and γ) with different quaternary structures: α-carbonic anhydrase is monomeric with a ten-strand β-sheet core; β-carbonic anhydrase (most abundant in plant leaves) is an octamer of dimeric units with an active site at the dimer interface where Cys-Cys-His tetrahedral coordination of Zn²⁺ mirrors the His-His-His coordination of the α form; γ-carbonic anhydrase is a trimer with active sites at pairwise subunit interfaces. Despite entirely different protein folds, the active-site geometry of α and β carbonic anhydrases is related by a mirror-image arrangement — a striking case of convergent evolution of a chemically identical active site.

The CAM pathway temporally, rather than spatially, separates CO₂ capture from RuBisCo fixation, enabling plants in chronically water-limited environments (such as succulents, cacti, and bromeliads) to minimize water loss. In CAM plants, stomata open at night when transpiration is low, allowing CO₂ to enter. Carbonic anhydrase converts it to HCO₃⁻ for PEP carboxylase, which carboxylates PEP to oxaloacetate. NADP-malate dehydrogenase reduces OAA to malate, and the ALMT9 transporter transports malate into vacuoles for overnight storage as malic acid. During the day, stomata close to prevent water loss; stored malic acid is released from vacuoles, decarboxylated by malic enzyme or PEPCK to release CO₂ at locally high concentrations, and RuBisCo fixes the CO₂ through the normal Calvin cycle. PPDK regenerates pyruvate or PEP for the next night's carbon capture.

The chapter concludes with an illuminating analogy between these three photosynthetic strategies and internal combustion engine systems: the C3 Calvin cycle is the basic engine; the C4 CO₂-concentrating mechanism is equivalent to a turbocharger that pre-concentrates the key substrate; and the CAM pathway is analogous to a hybrid battery that stores energy (in this case, carbon) temporally for later use when conditions are optimal. This framing reinforces a central theme: both engineering and evolution deploy analogous strategies to overcome thermodynamic and kinetic limitations in energy-conversion systems. From a climate perspective, the mechanistic differences between C3 and C4 photosynthesis explain why increasing atmospheric CO₂ does not equally benefit all plants — C4 species gain little from elevated CO₂ because they already concentrate it internally — and why understanding RuBisCo's oxygenase competition, exacerbated by warming temperatures, is directly relevant to global food security.