How do the light reactions communicate with the dark reactions to regulate the crucial process of fixing CO₂ into biomolecules? The principal means of regulation is alteration of the stromal environment by the light reactions. The light reactions lead to an increase in pH and stromal concentrations of Mg²⁺, NADPH, and reduced ferredoxin—all of which contribute to the activation of certain Calvin-cycle enzymes (Figure 23.11).

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As stated earlier, the rate-limiting step in the Calvin cycle is the carboxylation of ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate. This step is catalyzed by rubisco, a very slowly acting enzyme. The activity of rubisco increases markedly on exposure to light. How do the light reactions modify the activity of rubisco? Recall that CO₂ must be added to lysine 201 of rubisco to form the carbamate that is essential for catalytic activity, a process facilitated by rubisco activase. Carbamate formation is favored by alkaline pH and high concentrations of Mg²⁺ ion in the stroma, both of which are consequences of the light-driven pumping of protons from the stroma into the thylakoid space. Magnesium ion concentration rises because Mg²⁺ ions from the thylakoid space are released into the stroma to compensate for the influx of protons. Thus, light leads to the generation of regulatory signals as well as ATP and NADPH.

Light-driven reactions lead to electron transfer from water to ferredoxin and, eventually, to NADPH. Both reduced ferredoxin and NADPH regulate enzymes from the Calvin cycle. An important protein in these regulatory processes is thioredoxin, a 12-kDa protein containing neighboring cysteine residues. Thioredoxin cycles between an oxidized form with a disulfide bond between the two cysteines and a reduced sulfhydryl form (Figure 23.12). In chloroplasts, oxidized thioredoxin is reduced by ferredoxin in a reaction catalyzed by ferredoxin–thioredoxin reductase. The reduced form of thioredoxin activates rubisco and other Calvincycle enzymes, as well as many enzymes in other metabolic pathways in the cell by reducing disulfide bridges that control their activity including the chloroplast ATP synthase (Table 23.1). Thus, the activities of the light and dark reactions of photosynthesis are coordinated through electron transfer from reduced ferredoxin to thioredoxin and then to component enzymes containing regulatory disulfide bonds (Figure 23.13).

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Rubisco is among the most important enzymes in life because it provides organic carbon for the biosphere. However, this enzyme has a wasteful side. Rubisco sometimes reacts with O₂ instead of CO₂, catalyzing a useless oxygenase reaction. The products of this reaction are phosphoglycolate and 3-phosphoglycerate (Figure 23.14). Phosphoglycolate is not a versatile metabolite. A salvage pathway recovers part of its carbon skeleton, recycling three of the four carbon atoms of two molecules of glycolate. However, one carbon atom is lost as CO₂. This process is called photorespiration because, like cellular respiration, O₂ is consumed and CO₂ is released. However, photorespiration is wasteful because organic carbon is converted into CO₂ without the production of ATP, NADPH, or another energy-rich metabolite. This wastefulness raises the question, what is the biochemical basis of this inefficiency? The results of structural studies show that, when the reactive enediolate intermediate is formed, loops close over the active site to protect the enediolate. A channel to the environment is maintained to allow access by CO₂. However, like CO₂, O₂ is a linear molecule that also fits the channel. In essence, the problem lies not with the enzyme but in the unremarkable structure of CO₂. Carbon dioxide lacks chemical features that would allow discrimination between it and other gases such as O₂, and thus the oxygenase activity is an inevitable failing of the enzyme. Although evolutionary processes have enhanced the preference of rubisco for carboxylation—for instance, the rubisco of higher plants is eightfold as specific for carboxylation as that of photosynthetic bacteria—photorespiration still accounts for the loss of up to 25% of the carbon fixed. Another possibility exists, however. The oxygenase activity may not be an imperfection of the enzyme, but rather an imperfection in our understanding. Perhaps the oxygenase activity performs a biochemically important role that we have not yet discovered.

The oxygenase activity increases more rapidly with temperature than the carboxylase activity, presenting a problem for tropical plants. How do plants, such as sugarcane and corn that grow in hot climates, prevent very high rates of photorespiration? The solution to the problem illustrates one means by which photosynthesis responds to environmental conditions.

One means of overcoming the inherent oxygenase activity of rubisco is generating a high local concentration of CO₂ at the site of the Calvin cycle in the photosynthetic cells. The key to accomplishing this is the synthesis of four-carbon (C₄) compounds such as oxaloacetate that carry CO₂ from mesophyll cells on the surfaces of leaves to interior bundle-sheath cells, which are the major sites of photosynthesis (Figure 23.15). Decarboxylation of the four-carbon compound in a bundle-sheath cell maintains a high concentration of CO₂ at the site of the Calvin cycle. The resulting three-carbon compound, pyruvate, returns to the mesophyll cell for another round of carboxylation.

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The C₄ pathway for the transport of CO₂, also called the Hatch–Slack pathway after its discoverers Marshall Davidson Hatch and Charles Roger Slack, starts in a mesophyll cell with the condensation of CO₂ and phosphoenolpyruvate to form oxaloacetate, in a reaction catalyzed by phosphoenolpyruvate carboxylase. Oxaloacetate is then converted into malate by an NADP⁺-linked malate dehydrogenase. Malate enters the bundle-sheath cell and is decarboxylated within the chloroplasts by a different isozymic form of NADP⁺-linked malate dehydrogenase. The released CO₂ enters the Calvin cycle in the usual way by condensing with ribulose 1,5-bisphosphate. Pyruvate formed in this decarboxylation reaction returns to the mesophyll cell. Finally, phosphoenolpyruvate is formed from pyruvate by pyruvate-P_i dikinase. The net reaction of this C₄ pathway is

Thus, the energetic equivalent of two ATP molecules is consumed in transporting CO₂ to the chloroplasts of the bundle-sheath cells. In essence, this process is active transport: the pumping of CO₂ into the bundle-sheath cell is driven by the hydrolysis of one molecule of ATP to one molecule of AMP and two molecules of orthophosphate. The CO₂ concentration can be 20-fold greater in the bundle-sheath cells than in the mesophyll cells as a result of this transport.

When the C₄ pathway and the Calvin cycle operate together, the net reaction is

Note that 30 molecules of ATP are consumed per hexose molecule formed when the C₄ pathway delivers CO₂ to the Calvin cycle, in contrast with 18 molecules of ATP per hexose molecule in the absence of the C₄ pathway. The elevated CO₂ concentration resulting from the expenditure of an extra molecule of ATP enhances the rate of photosynthesis in tropical plants where light is abundant and minimizes the energy loss caused by photorespiration. Plants that rely on the C₄ pathway are called C₄ plants.

Tropical plants with a C₄ pathway do little photorespiration because the high concentration of CO₂ in their bundle-sheath cells accelerates the carboxylase reaction relative to the oxygenase reaction. The geographical distribution of plants having this pathway (C₄ plants) and those lacking it (C₃ plants) can now be understood in molecular terms. C₄ plants have the advantage in a hot environment and under high illumination, which accounts for their prevalence in the tropics. C₃ plants, which consume only 18 molecules of ATP per hexose molecule formed in the absence of photorespiration (compared with 30 molecules of ATP for C₄ plants), are more efficient than C₄ plants in temperate environments (Figure 23.16).

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Water is an important player in photosynthesis; if there is no water to provide electrons, oxygenic photosynthesis stops. Water availability is not a problem in temperate areas or hot, wet tropics, but it is not abundant in deserts (Figure 23.17). A crucial adaptation for such plants, and many non-desert plants as well, is the controlled opening and closing of the stomata (singular, stoma), microscopic pores located on the underside of plant leaves that are used for gas exchange. Such plants keep the stomata of their leaves closed in the heat of the day to prevent water loss (Figure 23.18). As a consequence, CO₂ cannot be absorbed during the daylight hours when it is needed for hexose synthesis. Rather, CO₂ enters the leaf when the stomata open at the cooler temperatures of night. To store the CO₂ until it can be used during the day, such plants make use of an adaptation called crassulacean acid metabolism (CAM), named after the family Crassulaceae (including many succulents). Carbon dioxide is fixed by the C₄ pathway into malate, which is stored in vacuoles. During the day, malate is decarboxylated and the CO₂ becomes available to the Calvin cycle. In contrast with C₄ plants, CAM plants separate CO₂ accumulation from CO₂ utilization temporally rather than spatially.

Although CAM plants do prevent water loss, the use of malate as the sole source of carbon dioxide comes at a metabolic cost. Because the malate storage is limited, CAM plants cannot generate CO₂ as rapidly as it can be imported by C₃ and C₄ plants. Consequently, the growth rate of CAM plants is slower that that of C₃ and C₄ plants. The saguaros cactus, which can live up to 200 years and reach a height of 60 feet, can take 15 years to grow to only one foot in height.

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