Biochemistry and Molecular Biology - Belyasova N.A. 2002

Metabolism. Energy-Yielding Processes
Capture of Light Energy by Biomolecules
Photosynthesis

Photosynthesis is a highly efficient process of capturing visible light energy and converting it into the chemical bond energy of biological molecules. This process takes place in the Cells of plants, Algae, and phototrophic Bacteria. In most of these organisms, photosynthesis is accompanied by The Use of Water as an electron donor and the release of molecular oxygen (Equation 13.1). Only in anoxygenic bacteria belonging to the Class Anoxyphotobacteria can certain other reduced compounds, such as hydrogen sulfide, serve as electron Donors instead of water, and in this case, no O₂ is formed (Equation 13.2).

CO₂ + H₂O → (CH₂O) + O₂     (13.1)

CO₂ + 2H₂S → (CH₂O) + H₂O + 2S     (13.2)

This observation belongs to Cornelis van Niel, who in 1931–1933, while still a graduate student, investigated the characteristics of photosynthesis in various bacteria and made a bold hypothesis that revolutionized researchers' views on the principles of photosynthesis. Based on the photosynthesis equation he derived for purple sulfur bacteria (13.2), C. van Niel suggested that it is water, rather than carbon dioxide, that splits during plant photosynthesis to form molecular oxygen, and he proposed a general equation for photosynthesis (13.3):

CO₂ + 2H₂A → (CH₂O) + H₂O + 2A     (13.3)

In the latter equation, H₂A represents an electron donor, which is water in oxygenic organisms and other substances (such as hydrogen sulfide, molecular hydrogen, or isopropanol) in anoxygenic bacteria. This reaction releases a dehydrated electron donor (A) and reduces carbon dioxide into an organic compound (CH₂O). These transformations constitute the core of the photosynthetic process.

It is interesting to note that water is a very poor electron donor, and none of the oxidizing agents available to Living organisms are powerful enough to strip hydrogen atoms from an H₂O molecule. This becomes possible only As a result of the photochemical transformations of special chlorophyll molecules, which thereby become strong oxidizing agents and acquire The ability to oxidize water.

The process of photosynthesis strictly requires the participation of membranes. In prokaryotes, this role is performed by invaginations of Cell/30.html">The Plasma Membrane, whereas in eukaryotes, it is carried out by thylakoid membranes located within METABOLISM/14.html">Chloroplasts. Thylakoids reside in the chloroplast stroma, forming stacked structures called grana. The internal space of the thylakoids is known as the lumen.

Thylakoid membranes house electron transport components grouped into Two Photosystems, as well as ATP synthase. The photosystems are used for Electron Transport and the coupled translocation of protons into the lumen, which generates a proton gradient across the thylakoid membrane. The energy of this proton gradient drives ATP synthesis, much like the process in Oxidative Phosphorylation. However, unlike the Respiratory Chain, electrons in photosystems move from a weak donor (water molecules) to a weak acceptor (NADP⁺), i.e., in the opposite direction to Respiration. Consequently, energy must be expended to drive this upward electron flow. Indeed, for the reduction of NADP⁺, electrons must be excited by light twice.

Characteristics of photosystem components. The components of photosystems are Structure/178.html">Protein Complexes containing photosensitive molecules (pigments) and Carriers of Reducing equivalents.

Pigments are mainly represented by chlorophylls, carotenoids, and phycobilins. The primary role in photosynthesis is played by chlorophyll a, a green, magnesium-containing tetrapyrrole pigment (Fig. 13.1). This chromophore is a magnesium porphyrin, structurally similar to heme (iron porphyrin). However, chlorophyll a has several key differences: a fifth, cyclopentane ring is fused to one of the pyrrole rings; one of the pyrrole rings is partially reduced; and one of the acidic side chains is esterified with phytol, a twenty-carbon, highly hydrophobic alcohol. The phytol tail imparts amphiphilic properties to the chlorophyll molecule and acts as an anchor that keeps the chromophore embedded in The Lipid Bilayer of the thylakoid membrane. Meanwhile, the hydrophilic HEAD (magnesium porphyrin) resides on the membrane surface facing the aqueous phase of the stroma, oriented parallel to the membrane plane. This achieves an optimal orientation of chlorophyll within the chloroplasts for the most efficient capture of light energy.

Fig. 13.1. STRUCTURE OF THE chlorophyll a molecule

Other chlorophylls of green plants (b, c, pheophytin), as well as those of certain algae and bacteria (Chlorobium chlorophyll, bacteriochlorophylls), differ from chlorophyll a in The structure of their pyrrole ring substituents, the absence of a magnesium ion (pheophytin), and their absorption spectra. The presence of chlorophylls with varying absorption spectra in The Cell broadens the range of captured radiation.

Carotenoids and phycobilins serve as accessory pigments. They further expand THE SPECTRUM OF energy absorbed by the Organism and, in addition, protect chlorophylls from excess light and oxidation by the oxygen released during photosynthesis. Carotenoids are yellow, orange, red, or brown pigments that strongly absorb in the violet region of the spectrum. Chloroplasts contain two groups of carotenoids: carotenes (Hydrocarbons, mostly tetraterpenes, discussed in detail in Chapter 17) and xanthophylls (chemically similar to carotenes but containing oxygen). Phycobilins are characteristic of cyanobacteria and the chloroplasts of red algae.

To capture light energy more thoroughly, pigments in the photosystems are assembled with Proteins into antenna complexes. Each antenna complex contains several hundred pigment molecules, and their primary function is to transfer the absorbed energy to chlorophyll a. Thus, an antenna complex can be likened to a funnel that "channels" energy toward the chlorophyll of the photosystem reaction center. Excitation energy (an exciton) is transferred from molecule to molecule via the electric field of an excited electron (specifically, an excited electron in a molecule that has absorbed a light quantum transfers its energy to a neighboring molecule in such a way that an electron in the neighbor becomes excited as well).

The electron-transporting Components of the photosystems include the cytochrome b₆/f complex (an integral membrane protein aggregate containing two Cytochromes: b₅₆₃ and f), pheophytin, membrane-bound plastoquinones (Q_A and Q_B), as well as mobile carriers such as plastoquinone Q_P, plastocyanin, and ferredoxin. Plastoquinone closely resembles ubiquinone in Structure and function (Fig. 12.1). Plastocyanin is a protein containing a single copper atom coordinated with amino acid side chains; during electron transfer, Cu alternates between the +1 and +2 oxidation states. Ferredoxin is an iron-sulfur protein of the 4Fe–4S^2- type that carries electrons. The chain of carriers terminates with an enzyme that transports electrons to NADP⁺.

It has been established that photosynthesis requires two photosystems: II and I. Photosystem II contains chlorophyll a with an absorption optimum at 680 nm (P₆₈₀) in its reaction center, whereas the reaction center of Photosystem I (P₇₀₀) contains chlorophyll a with an absorption optimum at 700 nm.

Light reactions of photosynthesis. The process of photosynthesis can be conventionally divided into two stages: light reactions and dark reactions. Light reactions require visible light and take place in the thylakoid membranes. Their ultimate outcomes are the reduction of NADP⁺ and the synthesis of ATP. Dark reactions can occur in the absence of visible light within the stroma, and they generally refer to the reduction of CO₂ involving ATP and NADPH.

In plants, photosynthetic electron transport begins with Photosystem II. The energy of absorbed light is transferred from the antenna complexes to the chlorophyll a of the P₆₈₀ reaction center, exciting one of its electrons. The excited electron is immediately transferred to the neighboring pheophytin, leaving a positively charged "hole" with a very high electron affinity in the chlorophyll molecule—essentially forming a positively charged radical, P₆₈₀^•+. This "hole" is very rapidly filled by an electron extracted from water by the water-splitting enzyme (H₂O → 2H⁺ + O₂ + 2e^-) with the participation of manganese ions. The excited electron migrates along the chain of carriers (Fig. 13.2) and reaches the second "hole" in P₇₀₀ of Photosystem I, which, in turn, was formed when a "hot" electron was transferred (the dashed arrow indicates the path of electrons entering the cyclic flow toward an electron acceptor, presumably plastoquinone). Here, the electron is excited a second time and is subsequently passed via Photosystem I carriers to NADP⁺ (Fig. 13.2). This movement of electrons along the carrier chain is called noncyclic flow, or the Z-scheme. An important distinguishing feature of this process is that the electron is excited twice in the reaction centers of the two photosystems, providing sufficient energy to reduce NADP⁺. The reaction producing NADPH is catalyzed by ferredoxin-NADP⁺ reductase, which contains FAD as a prosthetic group. It should be noted that the reduction of nicotinamide Cofactors requires hydride ions: two electrons from two molecules of reduced ferredoxin converge, and a proton is supplied from the stroma.

Fig. 13.2. Scheme of electron flows in the photosystems of thylakoid membranes. Zigzag arrows symbolize the photochemical excitation of electrons and their transition to a higher energy level; solid arrows indicate the path of electrons in the noncyclic flow; the dashed...

In addition to producing NADPH, the noncyclic electron flow drives the translocation of protons into the lumen, establishing an electrochemical gradient across the thylakoid membrane. This occurs during The transfer of hydrogen from reduced plastoquinone Q_P to the cytochrome b₆/f complex, which, as is known (Chapter 12), acts as an electron carrier. When protons flow out of the lumen into the stroma According to the principles of Facilitated Diffusion, they pass through ATP synthase channels, leading to the synthesis of ATP. This energy-storage mechanism is called Photophosphorylation, and its principle is similar to that of oxidative phosphorylation.

Besides the noncyclic flow described above, cyclic electron flows can also occur in thylakoid membranes. In these pathways, an electron is excited only once—in the reaction center of Photosystem I—and no NADPH is produced. The flow becomes cyclic when electrons from ferredoxin are transferred not to NADP⁺, but back to plastoquinone Q_P (Fig. 13.2, 13.3, dashed arrows). This movement of electrons provides the cell with ATP only, but not NADPH. Cyclic electron flow predominates under low NADP⁺ concentrations, which result from the intracellular accumulation of NADPH.

Figure 13.3 illustrates the arrangement of photosystem components, their substrates, and products. As can be seen, the main products of the light reactions of photosynthesis—NADPH and ATP—accumulate in the stroma, precisely where they are consumed in the dark reactions.

Dark reactions of photosynthesis. The dark reactions of photosynthesis encompass the fixation of CO₂, which involves its reduction and incorporation into Organic compounds. In most photosynthetic organisms, this process takes place via The Calvin Cycle (named after Melvin Calvin, who was awarded the Nobel Prize for its discovery).

The Calvin cycle can be conditionally divided into three stages: 1) carboxylation of ribulose diphosphate; 2) reduction of 3-phosphoglycerate to an aldehyde; 3) regeneration of ribulose diphosphate, the CO₂ acceptor.

Carbon dioxide reduced in the Calvin cycle enters the stroma of green plant chloroplasts through Stomata in leaves and green stems, whereas in algal and cyanobacterial cells, it enters in dissolved form. The key reaction of CO₂ reduction is the carboxylation of ribulose diphosphate. This reaction is catalyzed by an unusual enzyme—ribulose diphosphate carboxylase/oxygenase—which exhibits both carboxylase and oxygenase activities (Fig. 13.4).

Fig. 13.4. Conversion reactions of ribulose diphosphate catalyzed by ribulose diphosphate carboxylase/oxygenase

The oxygenase activity is manifested in the absence of CO₂ and in the presence of O₂, with the products of this reaction participating in Photorespiration. Ribulose diphosphate carboxylase/oxygenase operates very slowly: its catalytic rate is hundreds of times lower than that of most Other Enzymes. Consequently, this enzyme can constitute up to half of the total protein in chloroplasts. It is considered to be the most abundant protein on Earth, quantitatively prevailing over all other proteins. Regulation of the Calvin cycle rate is implemented at the level of this enzyme.

The reduction of the carboxylase reaction products (two molecules of 3-phosphoglycerate) proceeds with the participation of phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. The reverse Reactions Catalyzed by these enzymes also occur in Glycolysis (Fig. 9.3). This process consumes ATP energy and reducing equivalents supplied by NADPH.

The regeneration of ribulose diphosphate is accomplished through intermolecular rearrangements involving transaldolases and transketolases (which also participate in the Pentose Phosphate Pathways described in Chapter 9). Three compounds enter these transformations: glyceraldehyde-3-phosphate, dihydroxyacetone phosphate formed from it via isomerization, and fructose-1,6-diphosphate, which is formed in an aldol Condensation reaction from two triose phosphates (see Glycolysis, Chapter 9).

As a result of the described transformations, 6 molecules of CO₂ yield 1 molecule of hexose through 6 turns of the Calvin cycle. This process consumes 18 molecules of ATP and 12 molecules of NADPH (Fig. 13.5). Thus, carbon dioxide fixation is energetically very costly for the cell: incorporating just a single molecule of CO₂ into an organic compound requires 3 molecules of ATP and 2 molecules of NADPH.

The Calvin cycle is depicted as closed; however, much like the TCA cycle, many of its intermediates serve as precursors for The Biosynthesis of cellular components. For instance, 3-phosphoglycerate can be converted into Pyruvate (glycolysis reactions); erythrose-4-phosphate into aromatic Amino Acids; ribose-5-phosphate into NUCLEOTIDES; and hexose phosphates into Polysaccharides (the latter three intermediates are formed during the intermolecular rearrangement stage). Just as in other cycles, anaplerotic reactions exist that prevent the cycle from stalling.

Fig. 13.5. The Calvin cycle

The rate of the Calvin cycle is strictly regulated, as excessive cellular energy should not be squandered on CO₂ fixation. The core principle of regulation is to match the rate of CO₂ fixation to the rate of the light reactions of photosynthesis, which supply the ATP and NADPH required for fixation. Therefore, despite the Calvin cycle not directly requiring visible light, it proceeds most intensively under illumination. The primary rate-limiting step of carbon dioxide fixation is the carboxylation of ribulose diphosphate. Carboxylase activity increases significantly upon illumination due to the following factors:

1) carboxylase is allosterically activated by fructose-6-phosphate and inhibited by fructose-1,6-diphosphate. In turn, the levels of these products are controlled by the enzyme fructose-1,6-diphosphatase, which is activated by light and catalyzes The breakdown of fructose diphosphate;

2) another allosteric activator of carboxylase is NADPH, The amount of which increases in the light under a high non-cyclic electron flow;

3) the rate of this enzymatic reaction increases as the pH rises from 7 to 9. The enzyme Functions in the stroma, and its alkalinization results from the acidification of the lumen during the generation of a proton gradient across the thylakoid membrane;

4) carboxylase is activated by manganese ions released into the stroma when protons are transferred into the lumen during electron transport.

Additionally, light activates another Calvin cycle enzyme—glyceraldehyde-3-phosphate dehydrogenase—which apparently alters its Specificity (from NADH to NADPH) under the action of light.

The C₄ pathway of CO₂ fixation. The aforementioned process of CO₂ incorporation into 3-phosphoglycerate (resulting from the carboxylase reaction of ribulose diphosphate carboxylase/oxygenase) is termed the C₃ pathway, and plants utilizing it are called C₃ plants. Concurrently, an alternative C₄ pathway (the Hatch–Slack cycle) exists, distinguished by an additional initial stage: the fixation of CO₂ into a four-carbon compound (oxaloacetate). This reaction is catalyzed by phosphoenolpyruvate carboxylase, an enzyme that operates much faster than ribulose diphosphate carboxylase/oxygenase. Consequently, C₄ plants incorporate carbon dioxide into oxaloacetate far more efficiently than C₃ plants incorporate it into 3-phosphoglycerate. However, the C₄ pathway requires extra stages and Energy Expenditure: fixing one molecule of CO₂ consumes 5 molecules of ATP instead of 3 in the C₃ pathway (Fig. 13.6). Nevertheless, C₄ plants (such as corn, sugarcane, sorghum, and many temperate weeds) grow much faster than C₃ plants (such as wheat, rye, oats, and rice). This discrepancy is driven by photorespiration—a wasteful process characteristic of all C₃ plants that is virtually absent in C₄ plants.

Fig. 13.6. The Hatch–Slack cycle (the C₄ pathway of CO₂ fixation)

Photorespiration. This process stems from the oxygenase activity of ribulose diphosphate carboxylase/oxygenase. Molecular oxygen competes with CO₂ for the Active Site of this enzyme, causing a fraction of the ribulose diphosphate to be converted into phosphoglycolate (Fig. 13.4). This does not occur in C₄ plants, most of which originate from tropical regions where intense moisture evaporation forced plants to evolve a mechanism for closing stomata—through which gas exchange occurs—during the hottest hours of the day (under maximum solar irradiation). To prevent this mechanism from reducing the rate of CO₂ fixation, these plants store carbon dioxide within malate or aspartate (formed from oxaloacetate via the Hatch–Slack cycle) and subsequently utilize it as needed via decarboxylation reactions. In this scenario, the concentration of CO₂ in green plant Tissues remains consistently high, eliminating competition by O₂ for the enzyme's active site, as molecular oxygen enters the cells in limited amounts when the stomata are closed.

The substrate for photorespiration is glycolate, produced by the dephosphorylation of phosphoglycolate. Unlike mitochondrial respiration (which also occurs in plants in the dark), photorespiration's consumption of O₂ and release of CO₂ are not coupled with energy conservation; rather, they consume reducing equivalents and ATP, while carbon fixed in the Calvin cycle is futilely lost as CO₂. Research shows that photorespiration can lead to the re-oxidation and release of up to 50% of the carbon fixed in the Calvin cycle. The adaptive significance of this energetically wasteful process, which constrains plant growth efficiency, remains an unresolved puzzle.

The sequence of events in photorespiration is illustrated in Fig. 13.7. As can be seen, individual stages of this process take place across different Organelles. The net outcome of photorespiration is The formation of one phosphoglycerate molecule (3 "C" atoms) from two glycolate molecules (4 "C" atoms), with oxygen participation leading to The oxidation of the fourth carbon atom into carbon dioxide. Evidently, the purpose of this complex sequence of transformations in photorespiration is to recover at least a portion of the carbon from accumulated excess glycolate back into the cycle.

Fig. 13.7. Photorespiration

During photorespiration, one carbon atom is lost for every four, and it should be taken into account that energy has already been expended on the fixation of this carbon atom in the Calvin cycle. In addition, the oxidation of Glycine and deamination of Serine release ammonia, the incorporation of which into amino acids will again require energy expenditure. As a result, photorespiration reduces the potential yield of C₃ plants by 30–40%.