Ördög Vince, Molnár Zoltán (2011)
Debreceni Egyetem, Nyugat-Magyarországi Egyetem, Pannon Egyetem
Table of Contents
Life on Earth depends on energy derived from the sun. Photosynthesis is the only process of biological importance that can harvest this energy. A large fraction of the planet’s energy resources results from photosynthetic activity in either recent or ancient times (fossil fuels). The term photosynthesis means literally “synthesis using light”. Photosynthetic organisms use solar energy to synthesize complex carbon compounds. The most active photosynthetic tissue in higher plants is the mesophyll of leaves. Mesophyll cells have many chloroplasts. In the chloroplasts, light energy is converted into chemical energy by two different functional units called photoystems. The absorbed light energy is used to power the transfer of electrons through a series of compounds to act as electron donors and electron acceptors. The majority of electrons ultimately reduce NADP+ to NADPH and oxidize H2O to O2. Light energy is also used to generate a proton motive force (PMF) across the thylakoid membrane. This PMF is used to synthesize ATP.
General concept of photosynthesis
Light has both particle and wave characteristics
Light has properties of both particles and waves (Figure 2.1). A wave is characterized by a wavelength. The light wave is a transverse (side-to-side) electromagnetic wave, in which both electric and magnetic fields oscillate perpendicularly to the direction of propagation of the wave and at 90o with respect to each other. Sunlight is like a rain of photons of different frequencies. Human eyes are sensitive to only a small range of frequencies – the visible-light region of the electromagnetic spectrum (Figure 2.2). The absorption spectrum of chlorophyll-a indicates approximately the portion of the solar output that is utilized by plants. An absorption spectrum provides information about the amount of light energy taken up or absorbed by a molecule or substance as a function of the wavelength of the light.
Chlorophyll appears green to our eyes because it absorbs light mainly in the red and blue parts of the spectrum, so only some of the light enriched in green wavelength is reflected into our eyes. Chlorophyll (Chl) in its lowest-energy, or ground state absorbs a photon and makes a transition to a higher-energy, or excited state (Chl*) (Figure 2.3). Absorption of blue light excites the chlorophyll to a higher-energy state than absorption of red light, because the energy of photons is higher when their wavelength is shorter. In the higher excited state, chlorophyll is extremely unstable, it very rapidly gives up some of its energy to the surrounding as heat, and enters the lowest excited state, where it can be stable for a maximum of several nanoseconds (10-9 s). Because of the inherent instability of the excited state, any process that captures its energy must be extremely rapid.
Figure 2.1 Light is a transverse electromagnetic wave, consisting of oscillating electric and magnetic fields (source: Taiz L., Zeiger E., 2010)
Figure 2.2 The electromagnetic spectrum (source: Taiz L., Zeiger E., 2010)
In the lowest excited state, the excited chlorophyll has four alternative pathways for disposing of its available energy:
Excited chlorophyll can re-emit a photon and thereby return to its ground-state – a process known as fluorescence.
The excited chlorophyll can return to its ground state by directly converting its excitation energy into heat, with no emission of a photon
Chlorophyll may participate in energy transfer, during which an excited chlorophyll transfers its energy to another molecule.
A fourth process is photochemistry, in which the energy of the excited state causes chemical reactions to occur. The photochemical reactions of photosynthesis are among the fastest known chemical reactions. This extreme speed is necessary for photochemistry to compete with the three other possible reactions of the excited state.
Figure 2.3 Light absorption and emission by chlorophyll (source: Taiz L., Zeiger E., 2010)
Photosynthetic pigments absorb the light that powers photosynthesis
The energy of sunlight is first absorbed by the pigments of the plant (Figure 2.4). All pigments active in photosynthesis are found in the chloroplast. The chlorophylls and bacteriochlorophylls are the typical pigments of photosynthetic organisms. Chlorophylls a and b are found in green plants, and c and d are found in some protists and cyanobacteria. All chlorophylls have a complex ring structure that is chemically related to the porphyrin-like groups found in haemoglobin and cytochromes.
The different type of carotenoids found in photosynthetic organisms are all linear molecules with multiple conjugated double bonds. Absorption bands in the 400 to 500 nm region give carotenoids their characteristic orange colour. Carotenoids are found in all photosynthetic organisms. The light energy absorbed by the carotenoids is transferred to chlorophyll for photosynthesis; because of this role they are called accessory pigments. Carotenoids also help to protect the organism from damage caused by light.
Figure 2.4 Molecular structure of some photosynthetic pigments (source: Taiz L., Zeiger E., 2010)
Phycobilisomes serve as the primary light-harvesting antennae for photosystem II in cyanobacteria and red algae. These supramolecular complexes are primarily composed of phycobiliproteins, brilliantly coloured family of water soluble proteins bearing covalently attached, open-chain tetrapyrroles known as phycobilins. Absorbed light energy is transferred by very rapid, radiation-less downhill energy transfer from phycoerythrin or phycoerythrocyanin (if present) to C-phycocyanin and then to allophycocyanin species that act as the final energy transmitters from the phycobilisome to the photosystem II or photosystem I reaction centers.
An action spectrum depicts the magnitude of a response of a biological system to light as a function of wavelength. For example, an action spectrum for photosynthesis can be constructed from measurements of oxygen evolution at different wavelength. Action spectra were very important for the discovery of two distinct photosystems operating in O2-evolving photosynthetic organisms.
Light-harvesting antennas and photochemical reaction centers
The absorption of the light energy is a cooperation between many chlorophylls and carotenoid molecules (Figure 2.5). The majority of the pigments serve as an antenna complex, collecting light and transferring the energy to the reaction center complex, where the chemical oxidation and reduction reactions leading to long-term energy storage take place. Even in bright sunlight, a single chlorophyll molecule absorbs only a few photons each second. If there were a reaction center associated with each chlorophyll molecule, the reaction center enzymes would be idle most of the time, only occasionally being activated by photon absorption. However, if a reaction center receives energy from many pigments at once, the system is kept active a large fraction of time. Several hundred pigments are associated with each reaction center, and each reaction center must operate four times to produce one molecule of oxygen – hence the value of 2500 chlorophylls per O2. The reaction centers and most of the antenna complexes are integral components of the photosynthetic membrane. In eukaryotic photosynthetic organisms, these membranes are found within the chloroplast; in photosynthetic prokaryotes, the site of photosynthesis is the plasma membrane or membranes derived from it.
Figure 2.5 Basic concept of energy transfer during photosynthesis (source: Taiz L., Zeiger E., 2010)
Oxygen-evolving organisms have two photosystems
The quantum yield of photochemistry is nearly 1.0, the actions of about ten photons are required to produce each molecule of O2, so the overall maximum quantum yield of O2 production is about 0.1. Any photon absorbed by chlorophyll or other pigments is as effective as any other photon in driving photosynthesis. However, the yield drops dramatically in the far-red region of chlorophyll absorption (greater than 680 nm). Emerson discovered the enhancement effect. He measured the rate of photosynthesis separately with light of two different wavelength and then used the two beams simultaneously (Figure 2.6). When red and far-red light were given together, the rate of photosynthesis was greater than the sum of the individual rates. These and others observations were eventually explained by experiments performed in 1960 that led to the discovery that two photochemical complexes, now known as photosystem I and II (PSI and PSII), operate in series to carry out the early energy storage reactions of photosynthesis. Photosystem I absorbs far-red light, photosystem II absorbs red light. Another difference between the photosystems is that:
Photosystem I produces a strong reductant, capable of reducing NADP+, and a weak oxidant.
Photosystem II produces a very strong oxidant, capable of oxidizing water, and a weaker reductant than the one produced by photosystem I.
Figure 2.6 The rate of photosynthesis when red and far-red light are given together is greater than the sum of the rates when they are given apart (source: Taiz L., Zeiger E., 2010)
Organization of the photosynthetic apparatus
The chloroplast is the site of photosynthesis
In photosynthetic eukaryotes, photosynthesis takes place in the subcellular organelle known as the chloroplast (Figure 2.7). The most striking aspect of the structure of the chloroplast is the extensive system of internal membranes known as thylakoids, which are the site of the light reactions of photosynthesis. The carbon reduction reactions, which are catalyzed by water-soluble enzymes, take place in the stroma, the region of the chloroplast outside the thylakoids. Thylakoid membranes closely associated with each other are known as grana lamellae, and the exposed membranes in which stacking is absent are known as stroma lamellae. Two separate membranes, each composed of a lipid bilayer and together known as the envelope, surround most types of chloroplasts. The chloroplast also contains its own DNA, RNA, and ribosomes.
Figure 2.7 Schematic picture of the overall organization of the membranes in the chloroplast (source: Taiz L., Zeiger E., 2010)
Thylakoids contain integral membrane proteins
A wide variety of proteins essential to photosynthesis are embedded in the thylakoid membranes. The reaction centers, the antenna pigment-protein complexes, and most of the electron carrier proteins are all integral membrane proteins. Thylakoid membrane proteins have one region pointing toward the stromal side of the membrane and the other oriented toward the interior space of thylakoid, known as lumen. The chlorophylls and accessory light-gathering pigments are always pigment-protein complexes. Antenna and reaction center chlorophylls are organized within the membrane so as to optimize energy transfer in antenna complexes and electron transfer in reaction centers.
Photosystem I and II are spatially separated in the thylakoid membrane
The PSII reaction center, along with its antenna chlorophylls and associated electron transport proteins, is located predominantly in the grana lamellae (Figure 2.8). The PSI reaction center and its associated antenna pigments and electron transfer proteins, as well as the ATP synthase enzyme that catalyzes the formation of ATP, are found almost exclusively in the stroma lamellae and at the edges of the grana lamellae. The cytochrome b6f complex of the electron transport chain that connects the two photosystems is evenly distributed between stroma and granum lamellae. Thus the two photochemical events that take place in O2-evolving photosynthesis are spatially separated. This separation implies that one or more of the electron carriers that function between the photosystems diffuses from the grana region of the membrane to the stroma region, where electrons are delivered to photosystem I. A strict one-to-one stochiometry between the two photosystems is not required. The ratio of PSII to PSI is about 1.5:1, but it can change when plants are grown in different light conditions.
Figure 2.8 Organization of the protein complexes of the thylakoid membrane (source: Taiz L., Zeiger E., 2010)
Organization of light-absorbing antenna systems
The antenna systems of different classes of photosynthetic organisms are remarkably varied, in contrast to the reaction centers, which appear to be similar in even distantly related organisms. The variety of antenna complexes reflects evolutionary adaptation to the diverse environments in which different organisms live.
Antenna systems contain chlorophyll and are membrane associated
The size of antenna system varies considerably in different organisms, ranging from 200 to 300 chlorophylls per reaction center in higher plants, to a few thousand pigments per reaction center in some types of algae and bacteria. In almost all cases, the antenna pigments are associated with proteins to form pigment-protein complexes. The physical mechanism by which excitation energy is converted from the chlorophyll that absorbs light to the reaction center is fluorescence resonance energy transfer, often abbreviated as FRET. By this mechanism the excitation energy is transferred from one molecule to another by a nonradiative process. Approximately 95 to 99% of the photons absorbed by the antenna pigments have their energy transferred to the reaction center, where it can be used for photochemistry. The energy transfer among antenna pigments in the reaction center is a purely physical phenomenon, electron transfer involves chemical (redox) reactions.
The antenna funnels energy to the reaction center
The sequence of pigments within the antenna that funnel absorbed energy toward the reaction center has absorption maxima that are progressively shifted toward longer red wavelength. This red shift in absorption maximum means that energy of the excited state is somewhat lower nearer the reaction center than in the more peripheral portions of the antenna systems. In all eukaryotic photosynthetic organisms that contain both chlorophyll a and chlorophyll b, the most abundant antenna proteins are members of a large family of structurally related proteins. Some of these proteins are associated primarily with photosystem II and are called light-harvesting complex II (LHCII) proteins; others are associated with photosystem I and are called LHCI proteins. These antenna complexes are also known as chlorophyll a/b antenna proteins. The structure of the LHCI proteins is generally similar to that of the LHCII proteins. All of these proteins have significant sequence similarity.
Mechanisms of electron transport
Electrons from chlorophyll travel through the carriers organized in the “Z scheme”
In the “Z scheme” of the O2-evolving photosynthetic organisms all the electron carriers known to function in electron flow from H2O to NADP+ are arranged vertically at their midpoint redox potentials (Figure 2.9). Components known to react with each other are connected by arrows, so the Z scheme is really a synthesis of both kinetic and thermodynamic information. The large vertical arrows represent the input of light energy into the system.
Figure 2.9 “Z scheme” of photosynthesis (source: Taiz L., Zeiger E., 2010)
Photons excite the specialized chlorophyll of the reaction centers (P680 for PSII; P700 for PSI), and an electron is ejected. The electron than passes through a series of electron carriers and eventually reduces P700 (for electrons from PSII) or NADP+ (for electrons from PSI). Almost all the chemical processes that make up the light reactions of photosynthesis are carried out by four major protein complexes: photosystem II, the cytochrome b6f complex, photosystem I, and the ATP synthase. These four integral membrane complexes are vectorially oriented in the thylakoid membrane to function as follows (Figure 2.10):
Photosystem II oxidizes water to O2 in the thylakoid lumen and in the process releases photons into the lumen
Cytochrome b6f oxidizes plastohidroquinone (PQH2) molecules that were reduced by PSII and delivers electrons to PSI. The oxidation of plastohidroquinone is coupled to proton transfer into the lumen from the stroma, generating a proton motive force.
Photosystem I reduces NADP+ to NADPH in the stroma by the action of ferredoxin (Fd) and the flavoprotein ferredoxin-NADP reductase (FNR).
ATP synthase produces ATP as protons diffuse back through it from the lumen into the stroma.
Figure 2.10 The transfer of electrons and protons in the thylakoid membrane is carried out vectorially by four protein complexes (source: Taiz L., Zeiger E., 2010)
The photosystem II
PSI and PSII have distinct absorption characteristics. The reaction center chlorophyll of photosystem I absorbs maximally at 700 nm in its reduced state. Accordingly, this chlorophyll is named P700. The analogous optical transient of photosystem II is at 680 nm, so its reaction center chlorophyll is known as P680.
Photosystem II is contained in a multisubunit protein supercomplex. The core of the reaction center consists of two membrane proteins known as D1 and D2, as well as other proteins. The primary donor chlorophyll, additional chlorophylls, carotenoids, phaeophytins, and plastoquinones are bound to the membrane proteins D1 and D2. Water is oxidized to oxygen by photosystem II. Four electrons are removed from two water molecules, generating an oxygen molecule and four hydrogen ions. The protons are released into the lumen of the thylakoid. These protons are eventually transferred from the lumen to the stroma by translocation through ATP synthase. In this way, the protons released during water oxidation contribute to the electrochemical potential driving ATP formation. Manganese (Mn) is an essential cofactor in the water-oxidizing process. A classic hypothesis in photosynthesis research postulates that Mn ions undergo a series of oxidations – known as S states, and labelled S0, S1, S2, S3, and S4 – that are linked to H2O oxidation and the generation of O2.
Phaeophytin and two quinones accept electrons from photosystem II
In the electron acceptor complex phaephytin acts as an early acceptor in photosystem II. Phaeophytin passes electrons to a complex of two plastoquinones in close proximity to an iron atom. The two plastoquinones, PQA and PQB, are bound to the reaction center and receive electrons from phaeophytin in a sequential fashion. Transfer of the two electrons to PQB reduces it to PQB2-, and the reduced PQB2- takes two protons from the stroma side of the medium, yielding a fully reduced plastohydroquinone (PGH2). The plastohydroquinone transfers its electrons to the cytochrome b6f complex.
Electron flow through the cytochrome b6f complex also transports protons
The cytochrome b6f complex is a large multisubunit protein with several prosthetic groups. It is distributed equally between the grana and the stroma regions of the membranes. The precise way by which electrons and protons flow through the cytochrome b6f complex is not yet fully understood, but a mechanism known as the Q cycle accounts for most of the observations. In this mechanism, plastohydroquinone (PQH2) is oxidized, and one of the two electrons is passed along a linear electron transport chain toward photosystem I, while the other electron goes through a cyclic process that increases the number of protons pumped across the membrane. In the linear transport chain, the oxidized Rieske protein (FeSR) accepts an electron from PQH2 and transfers it to cytochrome f. Cytochrome f then transfers an electron to the blue-coloured copper protein plastocyanin (PC), which in turn reduces oxidized P700 of PSI. The plastocyanin (PC) is a small, water soluble, copper-containing protein that transfers electrons between the cytochrome b6f complex and P700. This protein is found in the lumenal space.
The photosystem I reaction center reduces NADP+
The PSI reaction center complex is a large multisubunit complex. In contrast to PSII, in which the antenna chlorophylls are associated with the reaction center, but present on separate pigment-proteins, a core antenna consisting of about 100 chlorophylls is an integral part of the PSI reaction center. The core antenna and P700 are bound to two proteins, PsaA and PsaB. Electrons from PSI reaction center are transferred to ferredoxin (Fd), a small, water-soluble iron-sulfur protein. The membrane-associated flavoprotein ferredoxin-NADP-reductase (FNR) reduces NADP+ to NADPH, thus completing the sequence of noncyclic electron transport that begins with the oxidation of water.
Some of the cytochrome b6f complexes are found in the stroma region of the membrane, where photosystem I is located. Under certain conditions, cyclic electron flow is known to occur from the reducing side of photosystem I via plastohydroquinone and the b6f complex and back to P700. This cyclic electron flow is coupled to proton pumping into the lumen, which can be utilized for ATP synthesis but does not oxidize water or reduce NADP+.
Proton transport and ATP synthesis in the chloroplast
A fraction of the captured light energy is used for light-dependent ATP-synthesis, which is known as photophosphorilation. It is widely accepted that photophosphorilation works via the chemiosmotic mechanism, which was first proposed in the 1960s by Peter Mitchell. Chemiosmosis appears to be a unifying aspect of membrane processes in all forms of life. The basic principle of chemiosmosis is that ion concentration differences and electric-potential differences across membranes are sources of free energy that can be utilized by the cell. Electron flow is accompanied with the proton flow from one side of the membrane to the other. The direction of proton translocation is such that the stroma becomes more alkaline (fewer H+ ions) and the lumen becomes more acidic (more H+ ions) as a results of electron transport. Mitchell proposed that the total energy available for ATP synthesis, which he called the proton motive force, is the sum of a proton chemical potential and a transmembrane electric potential. Transmembrane pH difference of one pH unit is equivalent to a membrane potential of 59 mV.
The ATP is synthesized by an enzyme complex known by several names: ATP synthase, ATPase, and CF0-CF1. This enzyme consists of two parts: a hydrophobic membrane-bound portion called CF0 and a portion that sticks out into the stroma called CF1. Remarkable aspect of the mechanism of he ATP synthase is that the internal stalk and probable much of the CF0 of the enzyme rotate during catalysis. The enzyme is actually a tiny molecular motor. Three molecules of ATP are synthesized for each rotation of the enzyme. The stoichiometry of protons translocated to ATP formed is 14/3, or 4.67.