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Photophosphorylation Secondary article Article Contents Andrew N Webber, Arizona State University, Tempe, Arizona, USA . Introduction Photophosphorylation is the process through which photosynthetic organisms convert light energy to adenosine triphosphate. . Physical Organization of the Process in Chloroplasts . Chlorophylls and Accessory Pigments . Light Absorption by Antennas Introduction . Photosystems I and II: Structures, Organization, Light Absorption, Mechanisms Photosynthetic organisms are able to convert light energy into a chemically useful form (adenosine triphosphate; ATP) by a process termed photophosphorylation. Photophosphorylation is performed by membrane-associated protein complexes that serve to capture the light energy (the reaction centres) and subsequently use the captured energy to make ATP (the ATP synthase complex). The unifying principle in all photosynthetic organisms (prokaryotic or eukaryotic) is that they use the captured light energy to initiate electron transfer, and produce a charge separation across a membrane. Following the charge separation process, secondary electron transfer reactions occur energetically downhill, with some of the energy being conserved as a proton electrochemical gradient across the membrane. It is this proton electrochemical gradient that is the driving force for ATP production. In oxygenic photosynthetic organisms, two different reaction centres, termed photosystem II and photosystem I, act in series to transfer electrons from the ultimate electron donor, water, to the terminal electron acceptor, NADP 1 . Photosystem II generates a powerful oxidant capable of extracting electrons from water, generating oxygen and protons. Electrons are transferred from photosystem II to an organic molecule termed plastoquinone to generate the reduced form, plastoquinol. Electrons from plastoquinol are then transferred to the cytochrome b6/f complex. Cytochrome f then reduces plastocyanin, which then transfers its electron to photosystem I. A second charge separation generates a powerful reductant that eventually transfers electrons to ferredoxin, and ultimately to NADP 1 . The proton gradient is generated by water oxidation and electron transfer between photosystem II and photosystem I. Physical Organization of the Process in Chloroplasts In eukaryotic organisms, photosynthesis occurs inside a specialized cellular organelle known as the chloroplast (Figure 1). The chloroplast is delimited by a double membrane, called the outer and inner envelope membrane. The outer membrane is porous to most molecules, whereas the inner membrane serves as a barrier, controlling import . Establishment of a Proton Electrochemical Potential and Use for ATP Synthesis . Production of Oxygen . Anoxygenic Photosynthetic Organisms . Reaction Centre of Purple Bacteria and export of sugars, phosphate and other molecules, using specialized transport proteins. The inside of the chloroplast is called the stroma. The stroma contains the soluble enzymes involved in the reduction of carbon dioxide to sugars. The chloroplast is a semiautonomous organelle and contains its own DNA. The chloroplast genome is very small and contains information for the biosynthesis of only a limited number of chloroplast components. The remainder of the genes required for chloroplast biogenesis are located in the nucleus (Webber and Baker, 1996). Inside the chloroplast is an extensive highly structured membrane, termed the thylakoid, that contains the protein complexes required for light harvesting, electron transfer, water oxidation and ATP synthesis. The thylakoid is a single membrane that forms a large flattened sac enclosing the lumen. The membrane is highly folded, forming large stacks (grana) interconnected by regions of unstacked, nonappressed membrane regions, termed the stromal lamellae (Figure 1). The physiological reason for the extensive membrane stacking is still unclear. The highly structured nature of the thylakoid, however, is thought to reflect a lateral organization of protein complexes in the membrane. Photosystem II and associated light-harvesting complexes are primarily associated with the stacked regions, whereas photosystem I and the ATP synthase complex are located in the stromal lamellae. Electron transfer between photosystem II and photosystem I, via the cytochrome b6/f complex, is facilitated by lateral diffusion of plastoquinone and plastocyanin. Chlorophylls and Accessory Pigments Light energy is captured by specialized pigments that are mostly found associated with proteins. In plants and algae, the major pigments are chlorophyll a, chlorophyll b and carotenoids. Chlorophylls contain a porphyrin ring attached to a hydrophobic phytol side-chain. Chlorophylls ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Photophosphorylation Figure 1 (a) Electron micrograph of an isolated chloroplast. The chloroplast is delimited by a double envelope (CE) membrane. Inside is the stroma (S) containing the thylakoid membrane. The thylakoid membrane is highly folded, forming granal stack regions (GS) interconnected by stromal exposed membrane regions. (b) Schematic of the thylakoid membrane showing the major protein complexes and associated cofactors. absorb blue and red light, and so appear green to the eye. In chlorophyll b a formyl group replaces a methyl group on the porphyrin ring, causing a slight red shift in the absorption maxima, thus increasing the range of wavelengths of light absorbed. Chlorophylls serve both as lightharvesting antenna pigments and as components of the electron transfer chain (described below). 2 Carotenoids absorb blue-green light with absorption maxima between 420 nm and 480 nm. Carotenoids serve as accessory light-harvesting pigments, and also fulfil a photoprotective role in the antenna. Excess light conditions can be potentially damaging to the photosynthetic apparatus if the energy is not dissipated in a harmless form. When excited, chlorophyll can undergo a process called ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Photophosphorylation intersystem conversion, where the excited singlet state forms a triplet state. The triplet state of chlorophyll is long lived and may transfer its energy to oxygen. Carotenoids are able to quench the energy from potentially damaging excited molecules and dissipate the energy in a harmless form. Certain carotenoids, termed xanthophylls, are also able to quench excitation energy by a phenomena known as ‘nonphotochemical’ quenching (Demmig-Adams, 1990; Owens, 1996). Under high-light conditions, violozanthin is rapidly converted to zeazanthin by enzymes associated with the thylakoid membrane lumen. Zeaxanthin is thought to have an energy level below chlorophyll, and so can act as an excited state quencher. Although the mechanism of quenching is not known in detail, the xanthophyll cycle, as it is now known, is thought to play a major role in regulating the dissipation of excitation energy in antenna complexes, before that energy can be used to initiate photochemical processes that could lead to photooxidative damage at high light. Light Absorption by Antennas Light energy is captured by pigment molecules in the antenna complexes and then migrates by excitation energy transfer to the specialized pigments, termed reaction centres, where electron transport is initiated. The vast majority of pigments in photosynthetic organisms function in light harvesting so that the light energy, which is relatively dilute, can be efficiently collected and used by the reaction centre. Photosynthesis is initiated when a photon of light is absorbed by a pigment molecule of the antenna. This causes the transition of a delocalized electron from the ground state to the lowest unoccupied molecular orbital, producing an excited state molecule. The excited state energy is then transferred to nearby pigments by a process termed resonance energy transfer. The rate of transfer is dependent upon the distance between the donor and acceptor molecules, the relative orientation of the transition dipoles, and the overlap between the donor emission spectra and the acceptor absorption spectrum (VanGrondelle and Amesz, 1986). The chlorophyll and other accessory pigments are thought to be organized by the protein so as to optimize transfer so that the excitation energy is rapidly funnelled to the reaction centre. The three-dimensional structure of several chlorophyll protein complexes, including the light-harvesting chlorophyll a/b complex associated with photosystem II (Kuhlbrandt et al., 1994), are beginning to reveal this precise organization in the case of chlorophyll a molecules. Photosystems I and II: Structures, Organization, Light Absorption, Mechanisms Photosystem II uses light energy to oxidize water and reduce plastoquinone. The photosystem II complex contains several different cofactors that are involved in mediating the transfer of electrons from water to plastoquinone (Diner and Babcock, 1996; Ruffle and Sayre, 1998). These include manganese (associated with the water-oxidizing complex described below), a tyrosine, the reaction centre chlorophyll a (P680), pheophytin a (Pheo; chlorophyll a lacking the central magnesium) and quinone (QA and QB). Additional cofactors in photosystem II are antenna chlorophyll a and cytochrome b559. Electron transfer is initiated when excitation energy reaches P680. The excited state reaction centre chlorophyll, P*680, transfers an electron to pheophytin to form the charge1 Phe 2 . Subsequently, the electron is separated state P680 rapidly transferred across the photosystem II complex (and across the thylakoid membrane) to the quinone acceptor QA. This further separation of the positive and negative charge across the membrane serves to minimize the rate of wasteful charge recombination, increasing the quantum yield of electron transfer to close to unity. The electron is then transferred from QA to a second quinone, QB, which serves as a two-electron acceptor, and is only loosely associated with photosystem II at the QB site. Each electron transfer to QB is also associated with a protonation of the quinone. The doubly reduced QB molecule dissociates from photosystem II, and migrates randomly through the thylakoid membrane until it reaches a cytochrome b6/f complex. Approximately nine unbound QB molecules (both reduced and oxidized) per photosystem II complex form the plastoquinone pool. The photosystem II complex contains numerous ( 4 26) individual polypeptide components that coordinate cofactors and/or serve a structural role (Ruffle and Sayre, 1998). Several key proteins in photosystem II make up what is known as the reaction centre core. In particular, two related proteins, D1 and D2, coordinate the cofactors involved in electron transfer, i.e. manganese, P680, pheophytin, QA and QB. The D1 and D2 polypeptides are also closely associated with the haem-containing cytochrome b559 complex. While cytochrome b559 is redox active, its role in photosystem II is unknown, although some evidence suggests a role in photoprotection of the reaction centre. Additional chlorophyll a molecules are coordinated by proteins named CP43 and CP47. CP43 and CP47 function as antenna pigment proteins, funnelling excitation energy to the reaction centre. The photosystem I complex catalyses the transfer of an electron from plastocyanin (PC) to ferredoxin (F). Photosystem I consists of approximately 13 individual proteins, and contains close to 100 chlorophyll a (Webber and ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3 Photophosphorylation Bingham, 1998). The structure of the photosystem I complex has been solved at 0.4 nm resolution, providing useful information on the organization of the different cofactors (Krauss et al., 1996). In photosystem I the antenna chlorophyll a, and chlorophyll a components of the electron transfer chain (six chlorophyll a), are associated with two proteins, PsaA and PsaB, that form the reaction centre heterodimer. Light energy is transferred from the antenna chlorophyll a to the reaction centre chlorophyll a dimer (P700). Excitation of P700 results in electron transfer to the first electron acceptor, a chlorophyll a molecule named A0. An additional chlorophyll a molecule, termed A, is positioned between P700 and A0, but has not yet been detected as an electron transfer intermediate. Electrons are then transferred from A0 to ferredoxin through several electron carriers, called A1 (a phylloquinone), FX, FA and FB. FX, FA and FB are iron– sulfur clusters. FA and FB are 4Fe–4Fe clusters coordinated by a small polypeptide very similar to ferredoxin. The detailed structure of photosystem I indicates that there are two potential branches for electron transfer between P700 and FX. The structure shows that P700 is a dimer, and that between P700 and FX are four chlorophyll a molecules. These chlorophylls reside on either side of a symmetry axis running through P700 and FX. The two chlorophylls furthest from P700 represent A0. The chlorophyll a molecules between P700 and A0 are named A and serve an as yet unknown function. Although two quinone molecules have been identified in the photosystem I crystal structure, only a single quinone radical (per P700) is detected spectrophotometrically, raising the intriguing possibility that only one of the electron transfer branches is operative. This is similar to the case in reaction centres of purple sulfur bacteria, described below. Establishment of a Proton Electrochemical Potential and Use for ATP Synthesis Electron transfer from water to NADP 1 requires the operation of three complexes – photosystem II, cytochrome b6/f and photosystem I – acting in series. The doubly reduced QB dissociates from photosystem II and becomes a part of the plastoquinone pool. The PQH2 diffuses to the cytochrome b6/f complex where it is oxidized, concomitantly releasing protons that are deposited in the thylakoid lumen. The cytochrome b6/f complex contains cytochrome b6, cytochrome f, Rieske protein, and several additional polypeptides. Electrons are next transferred from the cytochrome b6/f complex to photosystem I by plastocyanin, a small soluble copper-containing protein located in the thylakoid lumen. 4 Photosynthetic electron transfer and water oxidation lead to the accumulation of protons on the lumenal side of the thylakoid membrane. In addition, primary charge separation generates a charge difference across the membrane. The combination of a proton concentration and charge difference across the thylakoid membrane is termed the electrochemical proton gradient, and drives the synthesis of ATP from ADP and phosphate. The proton electrochemical energy is used to make ATP by a single thylakoid protein complex called the ATP synthase. The ATP synthase is composed of two major components termed CF0 and CF1. CF0 is comprised of several different subunits, and spans the thylakoid membrane forming a proton channel through the membrane. The CF1 component is made up of five different proteins termed a, b, d, e and g. Each CF1 contains three ab dimers, which together form three distinct catalytic sites. Each interface of an ab dimer can bind ADP and phosphate, and form ATP. However, each of the three sites has a different affinity for the nucleotides. Passage of protons through the CF0 component is thought to change the conformation of one of the sites, causing ATP to be released. One model suggests that the passage of protons causes CF1 to rotate on top of CF0 so that there is rapid interconversion of the three different nucleotide binding sites. As the catalytic sites interconvert, ATP is released. Production of Oxygen Photosynthesis has produced all the oxygen in the Earth’s atmosphere. Despite the importance of this reaction, it is one of the most poorly understood areas of photosynthesis. Water oxidation is mediated by photosystem II, which is the only complex that can oxidize water. On the lumenal side of photosystem II are several extrinsic proteins that form the oxygen-evolving complex, and are required for water oxidation. Water oxidation requires the removal of four electrons from two molecules of water. The oxidation–reduction potential of water is 1 0.82 mV, which means that it is an extremely difficult compound to oxidize. In photosystem 1 II, the oxidation of water is driven by P680 , which has a midpoint potential estimated at 1 1.2 mV, making it a very 1 oxidizes a tyrosine amino acid strong oxidizing agent. P680 on the D1 protein. The tyrosine radical cation then is able to oxidize the manganese ions of the oxygen-evolving complex by an as yet unknown mechanism. There are four manganese atoms associated with the oxygen-evolving complex. Four electrons need to be removed from two molecules of water to produce oxygen. Each electron is removed sequentially from components of the oxygenevolving complex, with oxygen evolution occurring only after the fourth electron is removed. This was shown by subjecting photosystem II to a series of saturating light ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Photophosphorylation flashes, such that each flash initiates one photochemical turnover, and following the pattern of oxygen release (Kok et al., 1970). It was found that oxygen is released every fourth flash. It is now believed, based on X-ray absorption spectroscopy, that it is some of the manganese ions that undergo progressive oxidation following each photosystem II turnover, and oxidize water when sufficient charge has been accumulated. Anoxygenic Photosynthetic Organisms Anoxygenic photosynthetic bacteria are unable to oxidize water and instead use a range of electron donors with less positive reduction potentials, such as sulfide, thiosulfate or succinate. All anoxygenic photosynthetic bacteria contain only a single type of reaction centre. In the case of the purple bacteria (either nonsulfur purple bacteria or sulfur purple bacteria) the reaction centre contains a quinone as the terminal electron acceptor in the complex. In purple bacteria electron transfer is cyclic. The reduced quinone produced by charge separation is reoxidized by the cytochrome b/c complex. The b/c complex then reduces cytochrome c2, which then transfers its electron to the 1 . Associated with the reaction oxidized primary donor, P870 centre are antenna complexes, termed light-harvesting complexes I and II, that are intrinsic membrane proteins binding bacteriochlorophyll and carotenoids. The structure of the reaction centre from purple bacteria has been solved at very high resolution (described below). The reaction centre of purple bacteria is very similar to photosystem II, and has served as an excellent model to Figure 2 Structure of the reaction centre from purple photosynthetic bacteria. The L, M and H subunits making up the reaction centre complex are shown in yellow, blue and green. Cofactors are shown in red. Reproduced courtesy of Jim Allen. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5 Photophosphorylation guide our understanding of the structure and function of photosystem II. Green sulfur bacteria contain reaction centres that have iron–sulfur centres as the terminal electron acceptor, and are therefore similar to photosystem I. The iron–sulfur centre can reduce ferredoxin, which then reduces NAD 1 . Green sulfur bacteria contain an antenna complex associated with the surface of the membrane. This complex contains bacteriochlorophyll and carotenoids and is termed a chlorosome. Green gliding bacteria also have chlorosomes, but their reaction centre is similar to that of purple bacteria. In the 1980s a group of Gram-positive photosynthetic bacteria, called Heliobacteria, were discovered. These bacteria also have reaction centre complexes similar to photosystem I, but contain bacteriochlorophyll g as the main photosynthetic pigment. Reaction Centre of Purple Bacteria The reaction centre complex from anoxygenic purple bacteria is the best understood of all reaction centres, and most of our information on the molecular basis of the early stages of photosynthesis is based on studies of this system. In particular, the X-ray structure of this class of reaction centre has been known for over a decade (Figure 2); this provides a very good understanding of cofactor organization and protein interaction. The reaction centre from purple bacteria contains two related integral membrane proteins called the L and M subunits. Many also contain an H subunit and a c-type cytochrome complex. The L and H subunits bind the cofactors involved in electron transport. These include four bacteriochlorophyll a, two bacteriopheophytin, two quinones (either ubiquinone or menaquinone), an iron atom and, in most cases, a carotenoid. The reaction centre proteins L and M are very hydrophobic and each have five membrane-spanning a helical regions. The H subunit also contains a single transmembrane a helix. The L and M subunits are organized such that they show a pseudo-C2 symmetry around an axis running perpendicular to the membrane plane. The electron transfer cofactors are similarly organized about the C2 symmetry axis. The primary 6 electron donor is a dimer of two bacteriochlorophyll a molecules. The subsequent cofactors, two bacteriochlorophyll a, two bacteriopheophytin and two quinones, are organized along either side of the symmetry axis running through the primary donor and the nonhaem iron located between QA and QB. Thus there are two potential branches of electron transfer, referred to as the A and B branches. However, for reasons that remain unclear, electron transfer occurs predominantly along the A (active) branch cofactors. References Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for carotenoid zeaxanthin. Biochimica et Biophysica Acta 1020: 1– 24. Diner BA and Babcock GT (1996) Structure, dynamics and energy conversion efficiency in photosystem II. In: Ort D and Yocum C (eds) Oxygenic Photosynthesis: the Light Reactions, pp. 41–58. Dordrecht: Kluwer Academic. Kok B, Forbush B and McGloin M (1970) Cooperation of charges in photosynthetic O2 evolution. I. A linear four step mechanism. Photochemistry and Photobiology 11: 457–475. Krauss N, Schubert W-D, Klukas O, Fromme P, Witt HT and Saenger W (1996) Photosystem I at 4 Å resolution: a joint reaction center and core antenna system. Nature Structural Biology 3: 965–973. Kuhlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light harvesting complex by electron crystallography. Nature 367: 614–621. Owens TG (1996) Processing of excitation energy by antenna pigments. In: Baker NR (ed.) Photosynthesis and the Environment, pp. 1–23. Dordrecht: Kluwer Academic. Ruffle SV and Sayre RT (1998) Functional analysis of photosystem II. In: Rochaix J-D, Goldschmidt-Clermont M and Merchant S (eds) Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 287–322. Dordrecht: Kluwer Academic. VanGrondelle R and Amesz J (1986) Excitation energy transfer in photosynthetic systems. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Photosynthetic Bacteria, pp. 191–223. Dordrecht: Kluwer Academic. Webber AN and Baker NR (1996) Control of thylakoid membrane development and assembly. In: Ort D and Yocum C (eds) Oxygenic Photosynthesis: the Light Reactions, pp. 41–58. Dordrecht: Kluwer Academic. Webber AN and Bingham SE (1998) Structure and function of photosystem I. In: Rochaix J-D, Goldschmidt-Clermont M and Merchant S (eds) Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 323–348. Dordrecht: Kluwer Academic. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net