The light phase of photosynthesis. Photochemical processes in biological systems Photochemical processes of photosynthesis z scheme
Of all known photochemical processes in nature highest value It has photosynthesis. The founder of the theory of photosynthesis is K. A. Timiryazev. Photosynthesis is the basis for the existence of all life on earth. Photosynthesis of green plants is the only primary source of accumulation of organic matter on Earth, which serves to nourish humans and animals. All vegetation the globe creates annually about 120 billion tons of organic matter, of which about 10 billion tons are produced by humans, growing food and fodder plants on an area of about 2.5 billion hectares.
In the green leaf of a plant, under the influence of solar radiation, a whole complex of photochemical processes occurs, as a result of which starch, fiber, proteins, fats and other complex compounds are formed from water, carbon dioxide and mineral salts. organic matter. The process of photosynthesis is very complex. It is carried out with the direct participation of the most important natural photocatalyst, chlorophyll, and is accompanied by a whole cycle of chemical transformations that do not depend on solar radiation. These transformations involve big number various biocatalysts - enzymes. The overall photosynthesis equation is usually expressed as the reaction of converting carbon dioxide and water to hexose:
6CO 2 + 6H 2 O \u003d C 6 H 12 O 6 + 6O 2
However, this equation, like most of the total equations in biology, does not express the main features of the process.
The most important merit of K. A. Timiryazev is the materialistic scientific substantiation of photosynthesis. Timiryazev was the first to show that photosynthesis obeys the law of conservation and transformation of energy. Thus, the idealistic views on the process of photosynthesis, which explained it by the action of an immaterial "life force", were refuted.
An equally important merit of Timiryazev is the discovery of the role of chlorophyll as a sensitizer of photochemical reactions occurring during photosynthesis. He experimentally established that photosynthesis is carried out mainly in the red and blue rays of the visible spectrum. Timiryazev carried out the following experiment. A row of glass tubes, filled with a mixture of air and carbon dioxide, and containing one identical green leaf each, was exposed to sunlight spread out with a trihedral prism so that there was one tube in each part of the solar spectrum. The content of carbon dioxide in the tubes was determined every few hours. It turned out that the assimilation of CO 2 occurs only in those rays that are absorbed by chlorophyll, that is, in the red, orange and yellow parts of the spectrum.
Thus, Timiryazev showed that it is chlorophyll that is the absorber of light in green plants and that this pigment, by absorbing light quanta, has the ability to transfer them further to the molecules of substances that are the source of photosynthesis.
In these reactions, chlorophyll undergoes a reversible redox transformation. The structure of chlorophyll is based on a porphyrin core called chlorin. It consists of four CH-bridged pyrrole residues, which are linked by two main and two coordination bonds to the central magnesium atom. In addition, the chlorophyll molecule includes the remainder of the molecule of the high molecular weight unsaturated alcohol phytol. Currently, at least five types of chlorophyll are known, which differ from each other in the structure of the molecule.
In addition to chlorophyll, which is the main type of photosynthetic pigments, the green leaf (in the so-called chloroplasts, which are complex specialized biological structures) contains other pigments - carotenoids and phycobelins, which are usually called auxiliary. These pigments modern ideas, take a known part in photosynthesis, and also protect chlorophyll from photooxidation. In addition to pigments, the main components of chloroplasts, in which, in fact, the entire process of photosynthesis is carried out, are lipoid substances and proteins that contain a large number of enzymes necessary for the implementation of subsequent stages of photosynthesis that are not associated with exposure to solar radiation.
Many issues of photosynthesis, despite the rapid development of science, remain little studied to this day. As mentioned earlier, the process of photosynthesis consists of two stages - light and dark, and both of these stages are closely related.
Since the initial process of photosynthesis is the absorption of light by chlorophyll, photosynthesis can be approximately represented in the form of the following scheme.
In the light stage, chlorophyll, having absorbed a quantum of light, passes into an excited state and in this form, through a number of intermediate processes, causes the decomposition of a water molecule into a hydrogen atom H and an OH radical according to the scheme
where the symbol X conventionally denotes a chlorophyll molecule; X* - the same molecule in the active state.
Further, the chlorophyll molecule, by attaching a hydrogen atom, is restored. OH radicals, combining in pairs, form a hydrogen peroxide molecule H 2 O 2, which, as a fragile compound, decomposes into water and oxygen:
4OH \u003d 2H 2 O 2
2H 2 O 2 \u003d 2H 2 O + O 2
After the completion of these reactions, the dark stage of the photosynthesis process begins, the essence of which is the transfer of hydrogen by the reduced chlorophyll molecule to the molecule CO 2 with the formation of organic compounds such as carbohydrates. This process is carried out under the action of the corresponding enzymes according to the scheme: 4H + CO 2 \u003d CH 2 O + H 2 O
As a result, due to polymerization, the final product of photosynthesis is obtained - hexose C 6 H 12 O 6.
The fact that the oxygen released in the process of photosynthesis belongs to water, and not carbon dioxide, was proved by A.P. Vinogradov (1946) using the method of labeled atoms. So, when using water H 2 18 O, all its oxygen 18 O was found after photosynthesis in free molecular oxygen, and when working with C 18 O 2 and H 2 16 0, free oxygen 16 O is released, while oxygen 18 O was included in composition of organic compounds. The establishment of this fact was essential for the theory of photosynthesis, since earlier many scientists believed that molecular oxygen is formed by light decomposition or photolysis of CO 2 .
The above scheme of photosynthesis is only approximate and does not reflect all the details of this extremely complex phenomenon. IN last years it was found that the reduction of one CO 2 molecule to carbon takes not one, but 8-12 energy quanta. This indicates that during photosynthesis there are at least eight primary photochemical reactions that occur in a certain order with other (non-photochemical) reactions.
It is known that not every chlorophyll or other pigment molecule that has absorbed light and retained enough energy for a photochemical reaction is the center of such a reaction. In fact, photochemical activity, i.e., direct connection with a photochemical reaction, is carried out by only about one molecule out of 200-250 chlorophyll molecules. About this phenomenon A.G. Pasynsky writes: “... A misconception might arise that the bulk of chlorophyll is photochemically inactive and plays the role of a reserve substance in the leaf, as was sometimes assumed in the literature.
In fact, this situation is a necessary consequence of the quantum nature of the acting light. The absorption of light by a given chlorophyll molecule does not occur in a continuous stream; light quanta falling like raindrops are absorbed all the time by different chlorophyll molecules.
According to Rabinovich, even in direct sunlight, each chlorophyll molecule absorbs a quantum of light only once every 0.1 s, and under less favorable conditions, much less frequently. Meanwhile, the rate of subsequent enzymatic reactions is extremely high. If, under these conditions, each chlorophyll molecule were an independent center of a photochemical reaction associated with the necessary auxiliary enzymes, then such a device would be just as impractical as if each section of the roof, on which an individual drop of rain falls, was equipped with an independent drain. There simply wouldn't be enough space on the sheet for such a device, not to mention the fact that it could only be used for a small fraction of the time.
On the contrary, the combination of a large group (200-250) of chlorophyll molecules with one photochemical reaction center ensures its continuous operation, just as the attachment of one drain to a sufficiently large roof surface allows a continuous stream of water to be obtained from individual drops. It is clear that in this case the entire mass of chlorophyll molecules actively participates in the useful process, although it is associated with only one center for the conversion of absorbed radiant energy into chemical energy.
All this once again confirms the extreme complexity of the photosynthesis process, each stage of which requires not only certain environmental conditions, but also a very complex system of excipients, as well as a strictly defined internal structure of intracellular contents. The importance of structural factors is indicated by the fact that a green leaf subjected to mechanical stress (for example, if it is rolled on glass with a thick glass rod) loses its ability to photosynthesize.
The study of photosynthesis processes is very important not only from a purely theoretical point of view, but also from the point of view of obtaining high and stable yields. To get to know these processes, to learn how to control them - these are the tasks to be solved at the present time the efforts of a whole army of domestic and foreign scientists.
The effects of UV light are very important. dimerization reactions of nitrogenous bases in DNA and RNA. The main chromophores (a chromophore is a part of a molecule that absorbs light and determines the color of a substance) of DNA molecules are nitrogenous bases nucleotides. Absorption of UV light quanta by nitrogenous bases leads to the formation of electronically excited singlet states resulting from P → P* transitions.
In an electronically excited state, pyridine bases enter into a dimerization reaction, which consists in connecting two nitrogenous bases at a 5,6 double carbon bond to form a cyclobutane ring between the residues of nitrogenous base molecules. Thus, individual nucleotides are linked not only through phosphoric acid residues, but also through nitrogenous bases. For this reaction, the quantum yield is γ = 2 ∙10 -2 . This reaction causes so-called "point" mutations; 80% of all lethal mutations associated with UV exposure results from thymine dimerization.
At low irradiation intensity, useful point mutations occur. As a result of irradiation of parental forms with UV light and selection of useful traits, the wheat variety Erythrospermum-103 was created.
Z-scheme of photosynthesis
In higher plants, photosynthesis involves two photosystems I and II with their reaction centers, which include one P 700 molecule or two P 680 molecules, respectively, and the corresponding electron transport chains (Fig. 3.1 and 3.14). Photosynthesis reaction centers are large pigment-protein complexes embedded in photosynthetic membranes, with special packaging of pigments and electron carriers.
Photosystem I
Photosystem I transfers electrons to a small hydrophilic protein, ferredoxin, containing an (Fe-S) center. It is a mobile carrier capable of migrating across the membrane surface, like cytochrome c, and carrying electrons:
a) on NADP + -oxidoreductase - a protein complex built into the membrane that restores NADP:
NADP + + 2e - + H + ® NADPH.
The resulting NADPH is used in the Calvin cycle to synthesize glucose.
b) to plastoquinones, and then through the complex of cytochromes b 6 f - back to P 700. In this case, the complex of cytochromes b 6 f creates a proton gradient, which is used by H + -ATP synthase, built into the same membrane, for the synthesis of ATP. This cyclic photophosphorylation occurs because many more ATP molecules are required for carbon fixation and glucose synthesis than for NADP+.
Since some of the P 700 electrons are used in biosynthetic processes, the missing electrons are supplied by photosystem II. The flow of electrons from photosystem II through its electron transport chain also enters the cytochrome b 6 f complex, contributing to the generation of a proton gradient on the thylakoid membrane, and then through plastocyanin enters P 700 (Fig. 3.14). For its Z-like shape, this circuit is called the Z-circuit of photosynthesis.
Since the formation of one O 2 molecule requires the transfer of four electrons from two H 2 O molecules to two NADP +, and the transfer of one electron from water to NADP + requires the absorption of two photons (by two photosystems), the total equation for the processes of electron transfer in photosystems I and II can be written as:
2H 2 O + 2 NADP + + 8 photons > O 2 + 2 NADPH +2 H + .
During the destruction of chloroplasts by ultrasound, particles with a sedimentation constant of 38 S, 30 nm in diameter, retaining the ability to photosynthesize, were found. The electron micrographs of the membrane cleavages obtained by the freeze-etching method show large granules 10x15x18 nm in size and with a molecular weight of about 2 MDa. It is assumed that these are components of photosystem II located on the surface of the thylakoid membrane facing the intrathylakoid space. Smaller granules, which are supposed to lie on the stroma-facing surface of the thylakoid membrane, have not yet been identified.
The core complex of photosystem I includes large proteins RsaA and RsaB with a molecular mass of about 83 kDa, which carry PS I reaction centers, as well as about 90–100 chlorophyll a molecules and 12–16 carotenoid molecules.
The primary electron donor in photosystem I has not yet been isolated in its pure form, but it has been well characterized using spectral methods. Its light absorption spectrum contains 2 main bands at 700 and 430 nm. In plant chloroplasts, the ratio of P 700 and other chlorophylls is approximately 1:400. It is believed that the reaction center P 700 is formed by the dimer of chlorophyll a. Near P 700 in the reaction center are the primary and secondary electron acceptors A 0 and A 1 . The role of A 0 is played by a chlorophyll molecule, and with an absorption band in the region of 693-695 nm, and the secondary electron acceptor A 1 is phylloquinone (vitamin K 1). Recall that quinones are intramembrane hydrophobic molecules.
Laser spectroscopy methods have shown that the electron transfer from P 700 to A 0 occurs in less than 10 ps. Then, in 20–50 ps (according to various sources), the electron passes to A 1 . From it, an electron in 200 ps (according to other sources, in 20-50 ns) passes to the 4Fe-4S cluster (F X) located inside the photosynthetic membrane. Then, over the next 170 ns - on a dimer of two 4Fe-4S clusters (F A and F B), located on the surface of the thylakoid membrane facing the stroma, and, finally, in 0.5-100 μs - on ferredoxin (Fd). These components of PS I are retained in the photosynthetic membrane by a complex of PsaA, Psa B … PsaL proteins. It should be noted that iron-sulfur proteins have not yet been isolated and characterized, because they are easily denatured when trying to isolate. The hydrophilic protein ferredoxin weighing about 10.7 kDa contains a 2Fe-2S cluster. It is retained on the stromal surface of the thylakoid membrane due to electrostatic interaction with the PsaD protein (Rubin, 2000). Ferredoxin is the main switch that directs electrons either to Fd: NADP + -oxidoreductase for NADPH synthesis, or to the cytochrome b 6 f complex, from which they return one by one to P 700 through the water-soluble protein plastocyanin. The transfer of electrons to and from ferredoxin to other molecules takes much longer, microseconds, due to the required diffusion of this protein.
The mechanism of electron transfer in photosynthetic chains of electron transport, as in mitochondria, is twofold - tunneling in pigment-protein supramolecular complexes and transfer between complexes by a pool of mobile carriers.
The sequence of arrangement of redox agents in the ETC of chloroplasts in accordance with their redox potential and data on their localization in complexes reflects Z-scheme of photosynthesis(See Figure 3.32). According to the Z-scheme, there is a sequential transfer of electrons from PSII to PS1, and two photosystems are combined into a single chain of electron transfer from water to NADP + . The idea of the existence of two photosystems in chloroplasts and their joint sequential work first arose in the 1940s. based on the experiments of the laboratory of R. Emerson, who discovered the effect of the red drop in the quantum yield of photosynthesis when chloroplasts are illuminated with monochromatic far red light
(λ > 680 nm), which excites only PS1, and the effect of enhancing the quantum yield of photosynthesis when added to the far red light illumination with a wavelength of about 650 nm (excitatory PSII). Later, it was shown that electron transport in chloroplasts is possible not only from water to NADP + with the participation of two photosystems, but also other alternative pathways with the participation of only one of the two photosystems. In addition, molecular oxygen can act as the final electron acceptor in the ETC of photosynthesis instead of NADP+. Currently distinguished non-cyclic, cyclic and pseudo-cyclic electron transport in chloroplasts.
Non-cyclic electron transport- this is the transfer of electrons from water to NADP +, carried out with the participation of two photosystems, cytochrome b 6 f-complex and intermediate low-molecular carriers - a pool of plastoquinones (PQ), plastocyanin (Pc), ferredoxin (PD) and ferredoxin-NADP-oxidoreductase (FNR). It is accompanied by the release of oxygen and the reduction of NADP + . Associated with ATP synthesis (non-cyclic photophosphorylation).
Cyclic electron flow carry out separately PS1 or PSII.
Cyclic transport involving FS1(see Fig. 3.33) includes the transfer of electrons from reduced ferredoxin (PD RRRR) back to oxidized P. This involves a pool of plastoquinones, cytochrome b 6 f-complex and, probably, a special enzyme PD-quinone oxidoreductase (FQR). Cyclic electron flow involving PS1 is associated with ATP synthesis (cyclic photophosphorylation) and can provide additional ATP synthesis necessary for carbon assimilation processes. It can be an alternative way to use the energy of light at its high intensities.
Rice. 3.34. Structural and functional organization of the FSI complex (according to Wei-Zhong He,
1996, with changes):
(Mn) 4 - Mn-containing cluster; Tyr z - tyrosine-161 of protein D1 - primary electron donor for P; Tyr D - tyrosine-161 of protein D2; cit. b 559 cytochrome - b559; Chl Z, Chl D - accompanying chlorophylls a; Kar - β-carotene; Q A , Q B - plastoquinones; I - protein; α, β - subunits. The dotted line indicates a possible cyclic electron flow
Cyclic flow of electrons in PSII is associated with the reverse transfer of electrons from the reduced primary quinones Q A and (or) Q B to the oxidized pigment of the P reaction center - This involves cytochrome b 559 ,β-carotene and accompanying chlorophyll molecules A(Chl Z or Chl D) of the PSII reaction center (Fig. 3.34).
The cyclical flow of electrons in PSII is an alternative way of harnessing the energy of light. It is activated under conditions when the light intensity exceeds the ability of the ETC to utilize its energy or when the water-oxidizing system of chloroplasts is damaged.
Pseudocyclic electron flow- electron transfer from water to oxygen - was first studied by A. Mehler (Mehler, 1951) and named after him - Mehler reaction.
Oxygen reduction can occur as in PS1 , as well as in FSII. In this case, the absorption of oxygen can compensate for its release during the oxidation of H 2 O. As a result, the only product of this process, as in the case of a cyclic electron flow, will be ATP synthesized during pseudocyclic photophosphorylation.
The pseudocyclic electron flow leads to the formation of reactive oxygen species (superoxidanion radical O , hydrogen peroxide H 2 O 2), so the activation of the process can cause disturbances in the photosynthetic apparatus. This alternative electron transport is activated at high light intensities under conditions of deficiency of oxidized NADP+ in chloroplasts.
To transfer electrons from water to NAD, plants have developed a mechanism that uses two quanta in series for a non-cyclic electron flow. Two independent photochemical events occur one after the other, and for each of them in a certain area of the cell there is a special photosynthesis apparatus. Of course, these two sites must be appropriately connected.
Graphs of this dual process in its options are called schemas. It is believed that first there is a "lift" of the electron caused by the energy of light into photosystem II, where it is accepted by a specific acceptor. Then the electron undergoes a series of spontaneous (dark) reactions. At the same time, it is sequentially transmitted along a long chain of redox compounds with ever-decreasing negative potentials, i.e.
to weaker reducing agents. In the end, the electron fills the hole in the photosystem, this hole appeared earlier when one electron was removed from the system. Only after that, the second quantum of light energy is applied to the electron, now in system I, and the electron is accepted by the acceptor, which in this case becomes a much stronger reducing agent than system II acceptor.
Rice. 12.1. Simplified -uchem for non-cyclic electron flow. The y-axis indicates the standard potentials of redox compounds. Their numerical values are approximate.
(Less simplifying and closer to reality, we can say that in any this moment in each of the two photosystems there is a certain number of holes distributed randomly.) As a result, the final product is reduced ferredoxin, which, through an enzyme containing flavin, transfers electrons to
The first scheme was proposed by Hill and Bendall. Other authors have also expressed or accepted the idea that photosynthesis is based on a two-step process; for the detailed development of the circuit, great efforts were made by numerous researchers. Then it was necessary to identify the members of the electron transport chain and arrange them in the appropriate order. Among the most effective methods we can mention the study of action spectra, reaction kinetics by pulsed spectrometry and the study of defective mutations. On fig. 12.1 shows a simplified new form-schemes (as amended).
As far as is known, the scheme is applicable to all plants
Although different groups of plants vary greatly in photosensitive accessory compounds. One problem is the order of the compounds: cytochrome - plastodianin - photosystem - the order shown here is based on the results of Knuff and Arnon, as well as Zidov et al.
Rice. 12.2. Action spectrum of chlorella for spectrally pure light. The y-axis shows the amount of oxygen released per quantum:
The starting point that served as the basis for the creation of the hypothesis of two quanta was the observation that in plants, in cases where suitable light quanta of different wavelengths acted on them together, an increase in the yield of photosynthesis was observed. The action spectrum for monochromatic light is shown in Fig. 12.2, but in the long wavelength range, two different quanta, acting synergistically, give a greater output than the calculated sum of the two separate outputs (Fig. 12.3). For example, with a mixture of "red" and "far red" quanta, an increase in photosynthesis by 30% was found. The enhancement is due to differences between the action spectra of the two systems. As Myers writes: “The following thought experiment gives the best idea of the amplification effect: when a plant is irradiated with light of a wavelength and another light with a correctly selected wavelength, the intensity of photosynthesis is higher than the sum of the intensities obtained by separate irradiation. Gain can be even more clearly described as an increase in quantum yield, measured at wavelength , when a second (non-measurable) beam with the right wavelength is added
In plants in photosystem I, the photosensitive substance of the active center is . In photosystem II, the active center absorbs at a shorter wavelength - about. Apparently, both active centers consist of modified chlorophylls.
Rice. 12.3. The amplifying effect of chlorella. The action of far red light of a certain wavelength is complemented by (near) short wavelength red light. I - with additional light; II - without additional light.
Duysens called the light active in photosystem I "light I", and in photosystem ". Photosystems I and II can be partially separated by preparative methods. In this case, either mechanical forces or detergents are used.
In photosynthetic bacteria, no amplification effect was found.
2 solar energy the primary source of all biological energy. Photosynthetic cells use energy sunlight for the formation of glucose and other organic products. These organic products serve as a source of energy and carbon for heterotrophic cells.
3 general characteristics photosynthesis Photosynthesis is a complex multistage system of photophysical, photochemical and dark biochemical processes in which the energy of sunlight is transformed by photosynthetic organisms (bacteria and plants) into forms of energy available to them: chemical (NADPH, ATP) or electrochemical (H). Using this energy, the cell, using the photosynthetic system, synthesizes from simple inorganic substances carbon dioxide and water, glucose and other complex organic substances. The product of these reactions is also molecular oxygen released from water molecules.
4 Photosynthesis is one of the most important mechanisms of life on Earth, because, due to it, both complex highly ordered organic substances and oxygen are formed, which are consumed by other organisms (bacteria, plants and animals) as sources of building material and energy. The main total reaction of photosynthesis: 6CO 2 + 6H 2 O + hν C 6 H 12 O 6 + 6O kcal / mol
5 The essence of photosynthesis is to obtain high-energy electrons due to the energy of light quanta absorbed by chlorophyll. In order to prevent the return of the electron to its original state and the waste of the received energy for thermal processes, the excited electron is very quickly transferred along the electron transport chain to NADP+, which is then used in biosynthetic reactions. The initial source of electrons is water, which then splits and releases oxygen. This oxygen, which is a by-product of photosynthesis, is breathed by all aerobic organisms.
7 1) Light-harvesting complexes CCKI and CCKII belonging to photosystems I and II. They contain pigments that absorb light quanta with different wavelengths. The energy absorbed by them migrates from pigment to pigment, dissipating slightly at each step, until it reaches the reaction center with a minimum energy level. There, it causes the excitation of photosynthetic pigments - chlorophyll P 700 or P 680 molecules in one of two photosystems - PS I or PS II, respectively.
8 2) The excited chlorophyll P 700 molecule, which is part of photosystem I (PS I), is able to ionize: P e The electron torn off from it is transferred along the electron transport chain to NADP +, restoring it: NADP + + e + H + NADPH. NADPH is further used in the Calvin cycle to synthesize glucose. The vacancy on formed after the departure of an electron is filled either by its own electron returning along the CET as a result of cyclic transfer, or by an electron coming from PS II.
9 3) In photosystem II, due to the excitation energy, an electron is detached from the chlorophyll P 680 molecule: P e and transferred along the electron transport chain to chlorophyll P 700, filling the vacancy formed there.
10 4) A vacancy formed on chlorophyll is filled with an electron, which is split off from a water molecule using the so-called water-splitting system, which is part of photosystem II. In addition to an electron, it splits off protons from a water molecule: 2H 2 O 4 H e + O 2 At the same time, hydrogen ions H +, which are involved in creating a proton gradient, are also split off from water, and molecular oxygen is also released, which is a by-product of this process.
11 5) The transfer of electrons along the ETC leads to the simultaneous transfer of H + protons through the membranes of thylakoids of chloroplasts into the stroma by a complex of cytochromes b 6 f, similar to the mitochondrial complex bc 1. The processes of proton release during water splitting and their transfer through the membrane create an electrochemical proton gradient, which serves as energy source for ATP synthesis.
14 Chloroplasts Photosynthesis takes place in chloroplasts, the organelles of plant cells. They usually have a biconvex shape like saucers folded together with a diameter of 3-10 microns and a thickness of 1.5-3 microns. In a plant cell, there are from 1 to 1000 chloroplasts.
15 A cross section of a pea (Pisum sativum) leaf cell has passed through two chloroplasts. The chloroplast is delimited from the cytoplasm by two membranes - outer and inner. The outer membrane is smooth, the inner one forms outgrowths - lamellae. The thylakoids are stacked on the lamellae. The picture clearly shows stacks of thylakoids - grana. Chlorophyll molecules are concentrated in the thylakoids of the gran between the layers of proteins and lipids. It is able to capture the energy of sunlight, with the help of which carbohydrates are formed from water and carbon dioxide. Accumulations of carbohydrates are visible in the picture as dark spots. Transmission microscope, x
18 Plant chloroplasts are similar to mitochondria in that they also use the electron transport chain and the chemiosmotic principle to convert energy. They also have a double-membrane envelope comprising a highly permeable outer membrane and an inner low-permeable membrane with embedded carrier proteins. The inner region, analogous to the mitochondrial matrix, is called the stroma. But chloroplasts do not have cristae, but have a special system of internal membranes - thylakoids - that form the third internal compartment.
19 Thylakoids are flat membranous sacs with a diameter of about 0.5 µm. They are packed in stacks - grains. Inside them is the thylakoid space. Separate grana are interconnected by lamellae - more extended layers. Thylakoid membranes contain, in addition to lipids and proteins, photoreceptive pigments - various modifications of chlorophyll and a number of other pigments that form a light-harvesting antenna. They contain two photosystems, PS I and PS II, which carry out photosynthesis and H-ATP synthetase (F 0 F 1), which synthesizes ATP, as in mitochondria.
20 Ultrastructure of thylakoids. A. Thylakoid grana. B. The surface of the thylakoid, revealed by the method of freezing-etching. Areas with large granules (B) are visible, representing the intrathylakoid surface of the thylakoid membrane, and areas with small granules (C), representing the surface of the thylakoid membrane facing the stroma. Large granules are presumably components of photosystem II; the nature of small granules has not yet been established.
21 The stroma contains its own DNA of chloroplasts, a protein-synthesizing apparatus, enzymes of the Calvin cycle and other metabolic processes, sugars, lipids, organic acids and other compounds. Just as in mitochondria, chloroplast DNA encodes only a part of the necessary proteins, while other proteins or their subunits are encoded in the cell nucleus. It is assumed that chloroplasts, like mitochondria, arose in the early stages of plant evolution as a result of a symbiosis of plant cells and some prokaryotic cells.
22 Photosynthetic pigments The main pigment of photosynthesis is chlorophyll a (Chl a). It consists of a porphyrin head approximately 1.5x1.5 nm in size, which has hydrophilic properties, and a long hydrophobic tail - phytol - about 2.0 nm long. In the center of the porphyrin ring of chlorophylls, there is an Mg 2+ ion bound by coordination bonds with nitrogen atoms. Other forms of chlorophyll - b, c, d, as well as bacteriochlorophyll - differ in side substituents and distribution double bonds in the porphyrin ring.
23
26 The absorption spectra of chlorophylls have two intense bands: the Soret band characteristic of porphyrins in the blue or near ultraviolet part of the spectrum and the "red" light absorption band in the nm region (in bacteriochlorophylls it is shifted to the near infrared nm region). The absorption of light in the red region of the spectrum is provided by the interaction of the magnesium ion with the conjugated p-electron system of the porphyrin ring. (For comparison, heme containing in the porphyrin ring not Mg 2+, but Fe 2+ absorbs light not in the red, but in the green-yellow part of the spectrum).
27 In the cell, the "red bands" of chlorophyll absorption are shifted to the long-wavelength region compared to the spectra obtained in organic solvents, which indicates a significant role of pigment-pigment, pigment-protein, and pigment-lipid interactions due to the dense packing of chlorophyll molecules in the photosynthetic membrane.
28 Methods of low-temperature and differential spectrophotometry revealed up to 10 modifications of chlorophyll a, which differ in absorption and fluorescence spectra (i.e., in the arrangement of energy levels), in extractability from the membrane (i.e., in hydrophilicity/hydrophobicity), etc. They are called "native forms" of chlorophyll a. So, P 670 (absorbing light nm), P 680, P 700, etc. have been isolated. The main factor that determines the properties of these native forms is pigment-pigment interactions, i.e. the formation of dimers and larger aggregates. In pigment aggregates, due to the interaction of p-electron systems, the electron cloud is "smeared" over two or more porphyrin rings. Accordingly, the upper energy level decreases and the ability to absorb longer wavelength red light increases.
30 In addition to chlorophyll, photosynthetic cells also contain other pigments: pheophytins, carotenoids, phycoerythrins, phycobilins, etc. All of them contain long chains of conjugated bonds, in which the π-electron cloud is “smeared” over the entire length, which also leads to a decrease in excited energy levels and allows the absorption of visible light quanta. Carotenoids have a chain length of approximately 40 carbon atoms. They absorb light in the nm region and are yellow or orange in color. Phycobilins absorb light in the nm region. All of them complement the absorption spectrum of chlorophyll, absorbing not only red light, but also light from other parts of the spectrum, allowing plants to effectively capture solar radiation.
31 Light-harvesting complexes To increase the efficiency of light utilization, photons are captured not only by chlorophyll molecules in the reaction centers, but also by auxiliary pigments that transfer the energy of electronic excitation to the reaction center. Indeed, the absorption cross section of a chlorophyll molecule is cm 2, therefore, at the intensity of daytime sunlight quantum / (cm 2 sec), the intensity of absorption of quanta by one molecule is not more than 1-10 quanta per second. But the duration of the cycle of using the energy of a photon from its absorption to the release of an O 2 molecule is about 0.01-0.02 sec. Consequently, most of the time between the absorption of individual quanta, chlorophyll must "idle". Therefore, the combination of dozens of pigment molecules that supply energy to one reaction center increases the efficiency of photosynthesis.
33 In addition to filling time intervals, the efficiency of light collection is increased by “spectral filling”, i.e. absorption of quanta in the blue-green-yellow region, in which chlorophylls practically do not absorb light. This role is played by a variety of pigments - chlorophylls, carotenoids, phycoerythrins, phycobilins, etc., which have light absorption bands in this spectral region. Dozens of light-harvesting pigment molecules surround photosystems I and II. The energy of absorbed photons is converted into the energy of electronic excitation of pigment molecules. It is transferred from molecule to molecule with some dissipation and, after several random walks, gets to the reaction centers of photosystems I and II - P 680 or P 700, which are "energy traps", since the process of energy migration is interrupted in them. Energy is not transferred further, but is used to generate high-energy electrons and photosynthesis itself.
34 Previously, when a definite spatial organization of pigment molecules was not established, their random arrangement in the photosynthetic membrane was assumed, and the rather good term "light-harvesting antenna" was used. But in recent decades, when it became known that pigment molecules are associated with proteins that organize their arrangement, the term “light-harvesting pigment-protein complexes” (LSPBC or simply LSC) began to be used. IN international literature the abbreviation LHC (Light Harvesting Complexes) is used to designate them.
35 Usually, SSPBCs surround the functional core, the central core complex, in which the reaction centers of PS I or PS II are located. Trimers of Lhcb1/Lhcb2 proteins are located on the periphery of PS I and PS II, while the central, core part is surrounded by monomeric antenna proteins. In PSII, these are the Lhca1, Lhca2, Lhca3, and Lhca4 proteins, and in PS II, the Lhcb3, Lhcb4, Lhcb5, and Lhcb6 proteins (or CP 25, CP29, CP26, and CP24).
36 Each Lhc trimmer monomer in the PSII photosystem is a protein of three α-helices 4.3 long; 5.1 and 3.2 nm. These proteins bind 7 molecules of chlorophyll a, 5 molecules of chlorophyll b and 2 molecules of lutein. The porphyrin rings of chlorophylls are arranged in two layers perpendicular to the plane of the thylakoid membrane near its outer and inner planes.
37 Scheme of the structure of PS I (a) and PS II (b). (a) shows the arrangement of trimeric (Lhcb1/Lhcb2) and monomeric light harvesting protein complexes (Lhca1, Lhca2, Lhca3 and Lhca4). The core complex in the center contains approximately 90 chlorophyll a molecules. (b) shows the approximate positions of trimeric (Lhcb1/Lhcb2) and monomeric light harvesting protein complexes (Lhcb3, Lhcb4, Lhcb5 and Lhcb6). The center shows a dimer of two core complexes, each of which consists of the reaction center proteins D1/D2 and cytochrome b559, surrounded by antenna proteins CP43 and CP47 containing chlorophyll a molecules (according to S. Jansson, 1994).
38 Spatial structure of one of the monomers in the trimeric complex Lhc. Its three α-helices contain 7 molecules of chlorophyll a (in the center, lighter), 5 of chlorophyll b (on the periphery, darker), and 2 of lutein (black) (according to Nelson and Cox, 2005).
40 Energy Migration Mechanisms The most important mechanism of electronic excitation energy migration in a photosynthetic membrane is the Förster inductive resonance mechanism. It is carried out with the participation of singlet excited levels (S 1) subject to the obligatory rules: 1) the donor must fluoresce; 2) the acceptor must have an absorption band corresponding to the emission band of the donor; 3) the distance between them should not exceed 4-6 nm.
41 Energy transfer occurs due to the fact that during the existence of the excited state of the donor (c) the oscillating excited electron generates an alternating electromagnetic field. If its frequency coincides with one of the resonant frequencies of the excited acceptor molecule, then nonradiative resonant energy transfer occurs: electron oscillations are excited in the acceptor, and the donor molecule nonradiatively returns to its original state. Typical distances of inductive-resonant energy transfer are 2-5 nm, and the characteristic time is sec. In this way, energy migration between different pigment-protein complexes can be carried out.
43 Within a single local light-harvesting complex adjacent to PSII or PSII, where pigment molecules are densely packed into a quasi-crystalline structure, their electronic levels can be socialized into zones. In this case, an exciton mechanism of energy migration is possible, in which the absorption of a light quantum leads to the appearance of an exciton - a collective excitation delocalized over the entire system of molecules. The excitation of the donor molecule propagates through the upper vibrational levels to the entire system of acceptors in a time sec, which is less than the relaxation time of the vibrational states. This is the fastest way to transfer energy. At the end of this time interval, the exciton energy can lead to the excitation of a particular acceptor molecule, which is located at a distance of up to 1–1.5 nm from the donor.
44 Specific mechanisms of energy transfer in FSPBCs depend on their structure and the packing density of pigments. The transfer of excitation energy between neighboring pigment molecules occurs in 0.2-5 ps, and between different pigment-protein complexes that are farther away, it is slower. Thus, the methods of laser spectroscopy revealed characteristic times of energy transfer of the order of 0.15-0.3 ps, 2-6 ps and ps. They may correspond to energy migration between adjacent chlorophyll molecules over a distance of about 1 nm, between individual pigment clusters, and between monomers in a trimer. In the first two cases, the exciton mechanism of energy migration is possible, and in the third, inductive-resonant transfer is more probable. The time between the absorption of a photon by antenna pigments and the capture of energy by the reaction center of photosystem I is estimated as ps, while that of photosystem II is about 300 ps.
45 So, in a light-harvesting antenna, the energy absorbed by short-wavelength forms of pigments migrates to longer-wavelength ones. Along the way, some of the energy dissipates, but the main part at the end "flows" onto the P 680 or P 700 pigments of the reaction centers, where charge separation and further photochemical processes occur due to it.
46 Z-scheme of photosynthesis In higher plants, photosynthesis involves two photosystems I and II with their reaction centers, which include one P 700 molecule or two P 680 molecules, respectively, and the corresponding electron transport chains. The reaction centers of photosynthesis are large pigment-protein complexes embedded in photosynthetic membranes, with special packaging of pigments and electron carriers.
47 Photosystem I Photosystem I transfers electrons to the small hydrophilic protein ferredoxin containing the (Fe-S) center. This is a mobile carrier capable of migrating over the surface of the membrane, like cytochrome c, and transferring electrons: a) to NADP + oxidoreductase - a protein complex built into the membrane that reduces NADP: NADP + + 2e + H + NADPH. The resulting NADPH is used in the Calvin cycle to synthesize glucose.
48 b) to plastoquinones, and then through the cytochrome b 6 f complex back to P 700. In this case, the cytochrome b 6 f complex creates a proton gradient that is used by H + - ATP synthase built into the same membrane for ATP synthesis. This cyclic photophosphorylation occurs because many more ATP molecules are required for carbon fixation and glucose synthesis than for NADP+.
49 Since some of the P 700 electrons are used in biosynthetic processes, the missing electrons are supplied by photosystem II. The flow of electrons from photosystem II through its electron transport chain also enters the cytochrome b 6 f complex, thereby contributing to the generation of a proton gradient on the thylakoid membrane, and then through plastocyanin enters P 700. For its Z-like form, this scheme is named Z scheme of photosynthesis.
50 Since the formation of one O 2 molecule requires the transfer of four electrons from two H 2 O molecules to two NADP +, and the transfer of one electron from water to NADP + requires the absorption of two photons (by two photosystems), the total equation for the processes of electron transfer in photosystems I and II can be written as: 2H 2 O + 2NADP + + 8 photons O 2 + 2NADPH + 2H +
52 The core complex of photosystem I includes large proteins RsaA and RsaB with a molecular weight of about 83 kDa, which carry PS I reaction centers, as well as around chlorophyll a molecules and carotenoid molecules. The primary electron donor in photosystem I has not yet been isolated in its pure form, but it has been well characterized using spectral methods. Its light absorption spectrum contains 2 main bands at 700 and 430 nm. In plant chloroplasts, the ratio of P 700 and other chlorophylls is approximately 1:400. It is believed that the reaction center P 700 is formed by the dimer of chlorophyll a. Near P 700 in the reaction center are the primary and secondary electron acceptors A 0 and A 1. The role of A 0 is played by the chlorophyll a molecule with an absorption band in the nm region, and the secondary electron acceptor A 1 is phylloquinone (vitamin K 1). Recall that quinones are intramembrane hydrophobic molecules.
54 Photosystem II Photosystem II is more complex than photosystem I, since it includes a water-oxidizing system that supplies electrons to the reaction center. The PSII core complex is a dimer of two subunits, which include two almost identical proteins D1 and D2, which are the structural basis for the organization of reaction centers, cytochrome b 559, as well as antenna proteins CP43 and CP47. The composition of the reaction center of photosystem II, in addition to 6 molecules of chlorophyll a, includes two molecules of pheophytin, two molecules of β-carotene and one molecule of cytochrome b 559. They are retained and placed in the photosynthetic membrane by a pair of almost identical integral proteins D 1 and D 2 and several smaller proteins.
55 In photosystem II, chlorophyll a, P 680, serves as the primary electron donor. It is assumed that the role of the P 680 reaction center in PSII is played by the chlorophyll dimer. But spectral properties indicate that P 680 may contain six more chlorophyll molecules located at some distance. The study of P 680 is difficult because it is a very strong oxidizing agent and can oxidize neighboring molecules. Indeed, for the oxidation of a water molecule, its redox potential must exceed +810 mV.
56 The separation of charges and the transfer of electrons in the reaction center of photosystem II occur approximately in the same way as in photosystem I. Laser spectroscopy methods have shown that after absorption of a light quantum, an electron is transferred from excited chlorophyll P 680 * to the primary acceptor pheophytin in 3 ps with an efficiency of % (FF) is a pigment of the porphyrin series, which does not contain a coordinated metal atom, - with the formation of a radical pair (R FF). Over the next 150 ps, the electron is transferred to the plastoquinone PQA (or simply QA), which is strongly bound to proteins, and from it to the weakly bound plastoquinone PQB (or QB), located outside the reaction center. The latter, having received two electrons and adding two protons from water, turns into a completely reduced form PQ B H 2, which, after diffusion inside the membrane, transfers electrons to the complex of cytochromes b 6 f.
58 A. Spatial organization of the reaction center of photosystem II (according to X-ray diffraction analysis with a resolution of 3.8) The arrows show the path of electrons from the donor, Mn ions in the Mn 4 cluster of the water-oxidizing system, to the secondary quinone QB. B. Scheme of the primary charge separation in photosystem II, showing that the initial electron donor for pheophytin (Phe) is chlorophyll associated with D1, and the resulting “hole” migrates to P680 (after Barber, 2003)
59 Complex of cytochromes b6f The complex of cytochromes b 6 f is similar to the mitochondrial complex of cytochromes bc 1. It is involved in the transfer of an electron from photosystem I (from ferredoxin) or from photosystem II (from plastoquinone PQ B H 2) through plastocyanin to photosystem I. This complex is dimer of two identical monomers. It contains two cytochrome b 6 with high and low potential hemes (denoted as b H and b L, respectively), cytochrome f with heme f, another heme x with an unknown function, and β-carotene, whose functions are also unknown. It also includes the iron-sulfur protein Riske with a center (Fe 2 -S 2).
60 Plastoquinone PQ B probably transports protons across the photosynthetic membrane by a mechanism similar to the Q-cycle in the mitochondrial complex of cytochromes bc 1. In any case, it is assumed that there is a plastoquinone binding site in the area of contact of each monomer with another monomer, so that between them, a pair of plastoquinones PQ and PQH 2 can be placed, which can carry out the Q-cycle. But unlike mitochondria, where hydrogen ions are transferred from the matrix to the intermembrane space, in chloroplasts, protons are transferred inside the thylakoids. This results in the creation of an electrochemical proton gradient used by H+-ATP- synthase (F 0 F 1 ) to produce ATP.
61 The joint work of the complex of cytochromes b 6 f, NADP + -oxidoreductase and the water-oxidizing system creates a 1000-fold proton concentration gradient. As a result, the intrathylakoid volume acquires a pH of 5, while the pH of the stroma is about 8. This creates a proton motive force of about 200 mV on the thylakoid membrane (one pH unit is approximately equal to 60 mV), almost entirely due to the difference in H + concentrations, and not to the membrane potential (thylakoid membrane permeable to Mg 2+ and Cl - ions, and they level the potential difference). This proton driving force is used by H + - ATP synthetase, the same as in mitochondria, to synthesize ATP.
63 The transfer of protons by the complex of cytochromes b 6 f in thylakoids occurs according to the Q-cycle mechanism, as in the mitochondrial complex of cytochromes bc 1. One of the PQH 2 electrons is transferred to the low-potential heme b L, and the other to the Fe 2 -S further through cytochrome f to plastocyanin. At the same time, protons are absorbed from the stroma, attaching to plastoquinone (Q + 2H + QH 2), which are then released into the thylakoid.
64 Water-oxidizing system The initial source of electrons for the electron-transport systems of photosynthesis is water. It also supplies hydrogen ions to create the proton gradient. But to obtain them, it is necessary to split the water molecule: 2H 2 O 4 H e + O 2
65 It takes at least four photons to split two water molecules. But the photosynthetic machinery can only manipulate one electron at a time. To solve this problem, photosynthetic cells have a special water-oxidizing (or water-splitting) system closely, structurally and functionally, associated with photosystem II. It transfers the electron taken from the water to the P 680 to compensate for the loss of an electron in the process of charge separation. That is, it is a direct electron donor for P 680. The molecular nature of this electron donor (it was designated as Z) remained unknown for a long time, but then it was found that one of the tyrosines of the D1 polypeptide (YZ) plays its role. But, since P 680 can accept only one e each, and four of them should be released during the splitting of water, then there must be a catalytic center capable of binding two water molecules, accumulating the split off electrons and directing them one at a time to P 680.
66 It has been suggested that the water-oxidizing system operates as a 4-stroke cyclic mechanism. Indeed, in experiments with PSII illumination with short flashes, the relative oxygen yield was maximum after the 1st and 5th flashes. It is assumed that the complex producing O 2 exists in five intermediate oxidation states: S 0, S 1, S 2, S 3 and S 4, the transition between which occurs when illuminated by successive flashes of light. This sequence is called an S-cycle.
68 But what substance can accumulate 4 electrons, changing its valency by 4 units? They turned out to be manganese ions. Manganese was discovered long ago in the composition of PSII, and it was known that if it is less than 4 atoms per P 680 molecule, then O 2 is not released. Manganese can be in several stable states from Mn +2 to Mn +7. For a protein-bound ion, this is referred to as Mn(II), Mn(III), etc. It is shown that the transition S 0 S 1 S 2 changes the oxidation state of manganese: Mn(II) Mn(III) Mn(IV).
69 Using X-ray spectroscopy, four-manganese clusters were found in a photosynthetic membrane and their structure was studied. They consist of two dimers in which manganese atoms are connected by oxygen bridges. It is assumed that after the removal of four electrons, the oxidized manganese cluster in the S 4 state is able to oxidize two water molecules. This happens at the final stage of the S 4 S 0 cycle.
75 At the first stage of the Calvin cycle, carbon is fixed (assimilated) from carbon dioxide, i.e. the attachment of a CO 2 molecule to a five-carbon molecule of ribulose-1,5-bisphosphate with its splitting into two three-carbon molecules of 3-phosphoglycerate. Then, with the participation of NADPH and ATP, it is reduced to glyceraldehyde-3-phosphate, which is used for the synthesis of sugars and starch, as well as for the production of ATP in the glycolytic process. And finally, with the participation of ATP, the regeneration of the carbon acceptor, ribulose-1,5-bisphosphate, can occur, thus closing the cycle.
