1.3 The blue print of the photosynthetic apparatus

Transcription

1 by Frenkel (1954). Vishniac and Ochoa (1951), Arnon (1951) and Tolmach (1951) independently discovered the photochemical reduction of NADP + by chloroplasts. A number of useful chloroplast-driven reduction reactions other than CO 2 fixation are thermodynamically possible. By far the most interest has centred on the photobiological production of hydrogen from water. Gaffron and Rubin (1942) were the first to observe that certain algae, containing the enzyme hydrogenase, adapted in certain conditions to evolve hydrogen. Arnon et al. (1961) observed hydrogen evolution from isolated chloroplasts coupled to hydrogenase in the presence of artificial electron donors. Benemann et al. (1973) demonstrated hydrogen evolution from isolated chloroplasts plus hydrogenase in a system where water was clearly the electron donor. Enormous advances in our knowledge of the mechanisms of photosynthesis have been made since these pioneering studies by workers such as Witt, Duysens, Kok, Jagendorf, Clayton, Feher, Sauer, Joliot, Babcock and many others (see Ke, 2001). However, prior to the 1980s, the structures of reaction centres and other important photosynthetic components could only be inferred indirectly from spectroscopic and kinetic studies. Then in 1982, Hartmut Michel succeeded in crystallising the reaction centre protein of the purple photosynthetic bacterium Rhodopseudomonas viridis (Michel, 1982). X-ray crystallographic determination of the structure followed (Deisenhofer et al., 1984, 1985). This seminal achievement, for which Johann Deisenhofer, Robert Huber and Hartmut Michel shared the 1988 Nobel Prize in Chemistry, revealed for the first time the exact locations of the redox active cofactors involved in the earliest steps of photosynthesis and the arrangement of the bacteriochlorophyll special pair. Since then, several reaction centre proteins and lightharvesting complexes have been crystallised and their structures determined. 1.3 The blue print of the photosynthetic apparatus Our present-day understanding of the light-driven processes of photosynthesis has reached a high level, as detailed by Leibl and Mathis in Chapter 3, although there remain some unanswered questions. The dark, carbon fixation processes are also very well understood, as described by Edwards and Walker in Chapter 4. In this section, we shall summarise the main components of photosynthetic reaction centres and their structures and functions, and then briefly sketch in the dark chemistry that results in the fixation of atmospheric CO 2 as carbohydrates. Ch01 Archer and Barber ed MA July 2003 Page 18 of 41

2 1.3.1 Reaction centres All types of photosynthetic systems are constructed around an exquisitely designed basic blueprint. All contain a reaction centre (RC) protein complex in which the conversion of light energy to electrochemical potential occurs. This energy conversion process involves the movement of electrical charge in the form of an electron across a membrane, generating an electrical gradient as well as a chemical potential gradient in the form of redox energy, as indicated in Fig As stated earlier, the primary electron donor the pigment P is always a chlorophyll or bacteriochlorophyll, while the primary electron acceptor A can be either (bacterio)chlorophyll (as in Type 1 RCs) or (bacterio)pheophytin (as in Type II RCs). Typically the generation of the primary radical pair 10 P + A occurs within a few picoseconds at very high quantum efficiencies. The subsequent reduction of P + and oxidation of A occur on a slower time scale. In both Type I and Type II RCs, A is initially re-oxidised by a quinone molecule (Q). In Type I reaction centres, this quinone is phylloquinone, which typically has a low midpoint redox potential 11 of about 0.6 V and is strongly bound to the RC proteins. In Type II RCs, the quinone electron acceptor (called Q A ) is also tightly bound but has a higher midpoint potential of about 0.1 V. The identity of this Q A quinone depends on the organism: it is plastoquinone in plants and algae, and ubiquinone or menaquinone in purple photosynthetic bacteria. The transfer of electrons from A to Q occurs on a timescale of ~200 ps, resulting in the charge transfer state P + AQ. It is the next step in the reductive electron flow that clearly distinguishes Type I and Type II reaction centres. In Type I centres, the electron is passed to an iron sulphur centre (given the symbol F X ) which is contained within the reaction centre protein, as shown in Fig From F X, the electron proceeds to two further iron sulphur centres (F A and F B ) and ultimately to ferredoxin, which as a watersoluble protein, transfers the reducing equivalent away from the membrane. In contrast, Type II reaction centres transfer the electron on Q A to a second quinone, Q B (as shown in Fig. 3.1). When Q B receives a second electron from the next photochemical turnover, it is protonated to form a quinol, which diffuses away from the reaction-centre protein into the lipid matrix of the membrane. In plants and algae, Q B is a plastoquinone, while in purple photosynthetic bacteria it is a ubiquinone. These secondary electron transfer events leading to the ejection of reducing equivalents from the reaction centre occur on a timescale stretching from 10 The radical pair P + A is sometimes written P + A to emphasise the presence of unpaired electrons. 11 See Section for an explanation of midpoint potentials. Ch01 Archer and Barber ed MA July 2003 Page 19 of 41

3 acceptor side (stroma) A Q hν A Q A Q P P + P D D donor side (lumen) D + REACTION PRIMARY CHARGE SECONDARY CENTRE SEPARATION ELECTRON FLOW Figure 1.6 Diagrammatic representation of a photosynthetic reaction centre embedded in a bilayer liquid membrane. The absorption of a quantum of light (h<) brings about charge separation across the membrane from a chlorophyll pigment P to a primary acceptor A, followed by secondary electron flows to a quinone Q and from a donor D. microseconds to milliseconds. Meanwhile, the reduction of P + by the electron donor D occurs on the nanosecond to millisecond time scale, depending on conditions. The nature of the electron donor also depends on the particular system. Cytochromes are usually the donors in both Type I and Type II RCs of photosynthetic bacteria, while in plants and algae, water is the electron donor to PSII and plastocyanin or cytochrome c 6 is the electron donor to PSI. In those photosynthetic organisms that evolve O 2 (plants, algae and cyanobacteria), the Type I (PSI) and Type II (PSII) reaction centres are coupled as shown schematically in Fig. 1.5b and in more detail in Fig. 1.7, so as to use two photons to drive each electron through the system, providing sufficient energy to oxidise water and reduce CO 2. In all cases, the fundamental principle is that energy storage is accomplished by rapidly separating the initial oxidants and reductants of the primary charge separation so as to avoid wasteful recombination reactions Light-harvesting systems As Alfred Holzwarth explains in detail in the next chapter, photosynthetic organisms have evolved light-harvesting (LH) antenna systems that service photosynthetic reaction centres so that they can operate efficiently under relatively low light intensities. The nature of these LH systems varies considerably according to the type of organism, but all function to intercept light and transfer the excitation energy rapidly to the reaction centre. The process is efficient, so the overall transfer rate must Ch01 Archer and Barber ed MA July 2003 Page 20 of 41

4 1.5 P700* midpoint redox potential at ph H 2 O Y Z P680* hν P680 Pheo Q A Q B Cyt b 6f A 0 [Chl] A 1 [Q] F X hν PC P700 PHOTOSYSTEM I F A F B F D FNR NADP + PHOTOSYSTEM II Figure 1.7 The Z-scheme for electron transfer in oxygenic photosynthesis. Y Z = tyrosine; P680 = primary electron donor of PSII composed of chlorophyll (Chl); Pheo = pheophytin; Q A and Q B = plastoquinone; Cyt b 6 f = cytochrome b 6 f complex, consisting of an Fe S Rieske centre, cytochrome f (Cyt f), cytochrome b low- and high-potential forms (Cyt b LP and Cyt b HP), plastoquinone binding sites, Q 1 and Q 0; PC = plastocyanin; P700 = primary electron Chl donor of PSI; A 0 = Chl; A 1[Q] = phylloquinone; F x, F A and F B = Fe S centres, F D = ferredoxin; FNR = ferredoxin NADP reductase; NADP + = oxidised nicotinamide adenine dinucleotide phosphate. be faster than the singlet lifetimes of the pigments, which are typically in the nanosecond time domain. In fact, overall transfer times of energy migration from the LH system to the RC are in the sub-nanosecond time domain, and in most cases transfer seems to occur by the Förster resonance mechanism. This requires good overlap between the absorption and emission spectra of the pigments, location of each pair of energy donor and acceptor pigment molecules to be close (typically within Å centre-to-centre) and with appropriate orientations. To achieve these properties, the pigment molecules are bound to a protein scaffold and these pigmentproteins associate with the RC. The number of light-harvesting pigment molecules servicing an RC varies according to the type of organism and the growth conditions, from 50 (in some purple photosynthetic bacteria) to many thousands (as in the case of the chlorosome of green sulphur bacteria). In plants and algae, the number is around 250 pigment molecules per reaction centre. The LH system and RC together comprise the photosynthetic unit. In the case of higher plants and green algae, the pigments bound to LH proteins are chlorophyll a, chlorophyll b and carotenoids. In addition to chlorophyll a and carotenoids, red algae Ch01 Archer and Barber ed MA July 2003 Page 21 of 41

5 contain the phycobilin pigments that covalently bind to protein to form the phycobilisomes, large macromolecular structures that attach to the outer (stromal) surface of the photosynthetic membrane. Cryptomonads also contain phycobilins but they do not associate to form phycobilisomes and are located on the other side of the membrane. Like red algae, brown algae, dinoflagellates and diatoms do not contain chlorophyll b, but differ again in that they contain chlorophyll c as an LH pigment as well as chlorophyll a and carotenoids. Cyanobacteria also do not contain chlorophyll b, but like red algae they contain phycobiliproteins that assemble into phycobilisomes. However, as mentioned in Section 1.2, there are related prokaryotic organisms (oxyphotobacteria) known as prochlorophytes that do not contain phycobilins but instead have an LH system composed of chlorophyll a and chlorophyll b. In contrast, the purple and green sulphur bacteria contain different forms of bacteriochlorophyll and carotenoids. The photosynthetic unit is a marvellously tuned sunlight-gathering apparatus. The different spectral properties of LH pigments, coupled with fine-tuning of the IR spectra by interactions with the proteins to which they bind, allow photosynthetic organisms to absorb at all the wavelengths available in the solar spectrum at the Earth s surface ( nm) Photosynthetic membranes The reaction centres of purple and green sulphur bacteria are localised in membranes, often called chromatophore membranes, which lie close to or include the outer cell membrane. In purple photosynthetic bacteria, the LH proteins are also intrinsic to the chromatophore membrane. However, in green bacteria the very large LH chlorosome, packed with many thousands of molecules of bacteriochlorophyll, is stacked into rodlike structures attached to the cytoplasmic side of the photosynthetic membrane, which does not invaginate as it does in purple bacteria (Fig.1.8). chlorosome proteins BChl c rod element baseplate Baseplate containing BChl a containing BCHl proteolipid monolayer galactolipid monolayer enclosing rods of BChl c cytoplasmic membrane reaction centre containing P685 core antenna consisting of BChl a Figure 1.8 Model of the chlorosome in Chloroflexus aurantiacus (modified from Ke (2001)). Ch01 Archer and Barber ed MA July 2003 Page 22 of 41

6 In oxygenic photosynthetic organisms, the photosynthetic apparatus involved in light reactions is embedded in the specialised thylakoid membrane (see Fig.1.3). In cyanobacteria, the thylakoid membranes tend to form concentric rings within the cytoplasm and are characterised by the presence of the large LH phycobilisomes attached to their surfaces, which induces a considerable spacing between them. In the green oxyphotobacteria (prochlorophytes), the same concentric rings are present but the membranes lie more closely together because of the absence of bulky phycobilisomes. The presence of phycobilisomes in the chloroplast of red algae leads to a thylakoid membrane organisation reminiscent of cyanobacteria. In striking contrast, the thylakoid membranes of higher plant chloroplasts, and to a lesser extent those of green algae, are arranged in stacked (grana) and unstacked regions (see Fig.1.3). The granal thylakoids are highly enriched in PSII, while PSI is found in the unstacked regions. However, this extreme lateral separation does not seem to occur in the thylakoid membranes of cyanobacteria and many forms of algae and therefore cannot be an absolute requirement for oxygenic photosynthesis to occur Energetics of electron-transfer processes in reaction centres Before discussing the structural and functional properties of the reaction centres of different types of photosynthetic organisms, it is necessary to appreciate their specific electron-transfer pathways in terms of redox potentials. Figure 3.2 compares the Type I and Type II reaction centres of anoxygenic and oxygenic organisms. In purple photosynthetic bacteria (specifically R. sphaeroides), the primary donor is called P870, because the long wavelength absorption peak of its special pair of bacteriochlorophylls is at 870 nm. Similar notation is used for other primary donors e.g. P840 (green sulphur bacteria), P870 (green non-sulphur bacteria), P700 (PSI) and P680 (PSII). However, as hinted in Section 1.1.4, P680 differs from the other primary electron donors in that it seems not to be a special pair (Barber and Archer, 2001). As we noted in Section 1.3.1, when excitation arrives at the RC from the LH system, primary charge separation occurs and this is followed by secondary electron flow to a terminal electron acceptor, ferredoxin (Fd) in the case of green sulphur bacteria and PSI (Type I RCs), or quinone (Q B ), in the case of purple bacteria and PSII (Type II RCs). In the case of anoxygenic bacteria, some of the reducing potential is used to convert NAD + to the NADH needed for CO 2 fixation and some is utilised in cyclic electron flow, whereby the reductant indirectly reduces the oxidised primary donor. This cyclic electron flow involves the cytochrome bc complex, which is also embedded in the chromatophore membrane and which couples the electron flow to Ch01 Archer and Barber ed MA July 2003 Page 23 of 41

7 the vectorial movement of protons across the membrane, as shown in Fig The resulting ph and electrical gradients are then used to drive the conversion of ADP to ATP in accordance with the chemiosmotic mechanism of Peter Mitchell (1966), a contribution for which he received the Nobel Prize for Chemistry in As already mentioned and shown diagrammatically in Figs. 1.4, 1.7 and 3.2, Photosystem I and Photosystem II work together in oxygenic photosynthetic organisms to oxidise water and reduce ferredoxin. PSII functions as the water plastoquinone oxidoreductase while PSI is a plastocyanin ferredoxin oxidoreductase. The redox coupling between the two reaction centres is accomplished by a cytochrome bc complex rather like that found in anaerobic photosynthetic bacteria but called, for historical reasons, the cytochrome b 6 f complex. An important feature of this scheme is that two photons are used to drive one electron from water to ferredoxin. The cytochrome b 6 f complex acts as a plastoquinol plastocyanin oxidoreductase and, like its counter part in photosynthetic bacteria, facilitates the maintenance of the electrochemical potential gradient of protons across the thylakoid membrane needed to convert ADP to ATP. In oxygenic photosynthesis, the reduced ferredoxin is used to convert NADP + to NADPH, which together with ATP is required to convert CO 2 to carbohydrate. Green sulphur bacteria also use reduced ferredoxin in the same way as PSI except that they, like purple bacteria, use non-phosphorylated nicotinamide adenine dinucleotide (NAD + ) rather than NADP +. The similarity in the redox properties and electron transport pathways of the Type I and Type II RCs is evident in Fig. 3.2 except for the important fact that P680 + is a much stronger oxidant (with a midpoint potential of ~1 V) than P700 +, P840 + and P870 + (~0.4 V). This is because P680 + must be sufficiently oxidising to remove electrons from water, which is a very stable molecule and difficult to oxidise compared with the substrates oxidised by other reaction centres. This oxidation reaction involves a cluster of 4 Mn atoms and the transfer of electrons and protons from the substrate water molecules is facilitated by a redox-active tyrosine, named Y Z, positioned between the (Mn) 4 -cluster and P680 (see Fig.1.7). As the production of dioxygen from water is a four-electron process 2H 2 O O 2 + 4e + 4 (1.4) and a dioxygen molecule is produced at a single PSII reaction centre, the Mn cluster must accumulate four oxidising equivalents. This is why the evolution of O 2 oscillates with a period of four when oxygenic organisms are subjected to single turnover flashes of light, as discovered by Pierre Joliot and colleagues in This discovery caused Kok et al. (1970) to propose the S-state cycle, whereby the absorption of four successive photons drives the series of reactions Ch01 Archer and Barber ed MA July 2003 Page 24 of 41

8 h< h< h< h< S 0 S 1 S 2 S 3 S 4 (1.5) When S 4 is formed, dioxygen is released and the cycle resets itself to the S 0 -state. Although the precise chemical mechanism of the S-state cycle is unknown, it is generally believed that the two water substrate molecules bind at the S 0 -state and that and electrons are extracted before arriving at the S 4 -state. The late Jerry Babcock and colleagues (Tommos and Babcock, 2000) have suggested an attractive hydrogenatom abstraction hypothesis for the water oxidation mechanism. Not surprisingly, the high redox potential of P680 + and the possibility of forming reactive oxygen species during the water-splitting reaction give rise to oxidative damage of the PSII RC. This manifests itself as rapid degradation and regular replacement of protein, as Godde and Bornman describe in Chapter 5. Plants and other oxygenic organisms have evolved a range of protective strategies that reduce the frequency of photoinduced PSII damage and allow the repair process to cope under normal conditions. The effect of this intrinsic and detrimental property of PSII is, however, observed when organisms are exposed to environmental stress, when the rate of repair does not match the rate of damage and photoinhibition occurs. When this happens, the efficiency of photosynthesis and biomass/crop productivity decline Reaction centre structures Figure 1.9 shows how PSI and PSII are functionally coupled with cyt b 6 f and ATP synthase in the thylakoid membrane. We now know for certain from x-ray crystallographic studies that all reaction centres are characterised by a pseudo-2 fold symmetry axis that relates the cofactors and the proteins that bind them. In Type II RCs, this symmetry gives rise to a redox-active branch and an inactive branch, as shown for PSII in Fig Despite intense studies on the purple bacterial RC it is still not clear how Type II centres are able to differentiate their active and inactive branches. However, this property has distinct advantages when the terminal acceptor (i.e. Q B ) requires two electrons to be fully reduced. In Type I reaction centres, where a single, centrally located iron sulphur centre F X is the electron acceptor (as for PSI in Fig. 1.9), it seems possible that primary charge separation occurs with similar probability up either branch; this must be so in green sulphur bacterial RCs, which are homodimeric while in the case of PSI the situation is less clear. The two protein subunits that constitute the RCs of PSI, PSII and purple bacteria are not identical, as in the case of green sulphur bacteria, but form a heterodimer. In purple bacteria, the two subunits are called L and M, while in PSII the closely related Ch01 Archer and Barber ed MA July 2003 Page 25 of 41

9 acceptor side (stroma) e á FD NADP + (CO2) e á ADP + Pi ATP + H2O FA FB Fe QA QB QH2 Q1 e á Fx Cyt bhp e á A1 A1 A-branch B-branch CF1 Pheo Chl hν P680 YZ YD Pheo Chl Cyt blp A0 e á QH2 Fe-S Chl Cyt f Q0 hν P700 A0 Chl CF0 (H2O) e á (Mn)4 PC donor side (lumen) O2 PHOTOSYSTEM II CYTOCHROME b6_f PHOTOSYSTEM I ATP SYNTHASE Figure 1.9 Schematic diagram of the electron proton transport chain of oxygenic photosynthesis in the thylakoid membrane, showing how Photosystem I (PSI) and Photosystem II (PSII) work together to use absorbed light to oxidise water and reduce NADP +, in an alternative representation to the Z scheme shown in Fig The diagram also shows how the proton gradient generated by the vectorial flow of electrons across the membrane is used to convert ADP to ATP at the ATP synthase complex (CF0CF1). In both PSI and PSII, the redox-active cofactors are arranged around a pseudo-twofold axis. In PSII, primary charge separation and subsequent electron flow occurs along one branch of the reaction centre. However, in the case of PSI, it is likely that electron flow occurs up both branches as shown. Electron flow through the cytochrome b6_f complex also involves a cyclic process known as the Q cycle. The symbols used for the various redox cofactors are defined in the legend of Fig. 1.7 except for YD = symmetrically related tyrosine to Yz but not directly involved in water oxidation, and QH2 = reduced plastoquinone (plastoquinol), which acts as a mobile electron/proton carrier from PSII to the cytochrome b6f complex. Ch01 Archer and Barber ed MA July 2003 Page 26 of 41

10 subunits are known as the D1 and D2 proteins (see Figs. 3.3 and 3.10). All four proteins show considerable homologies, and all have five transmembrane helices related to each other in their reaction centres by the same pseudo-2 fold axis that relates the cofactors. The two proteins that make up Type I reaction centres, PsaA and PsaB, are also arranged around the pseudo-2 fold axis that relates the cofactors (see Fig. 3.8), but in this case they have eleven transmembrane helices. Interestingly the five transmembrane helices at the C-terminal ends of these Type I RC proteins are arranged in a similar, but not identical, manner as in Type II RCs (Rhee et al., 1998; Schubert et al., 1998). The structural details briefly described above have emerged from X-ray crystallographic studies which began with the elucidation of the structure of a Type II RC isolated from the purple bacterium R. viridis in the 1980s by Deisenhofer, Huber, Michel and colleagues (Deisenhofer et al., 1984, 1985) and have recently advanced to the determination of the structure of PSI at 2.5 Å (Jordan et al., 2001) and PSII at resolutions ranging from 3.8 Å to 3.5 Å (Zouni et al., 2001; Kamiya and Shen, 2003; Ferreira et al., 2004). As discussed in detail in Chapter 3, these studies have given a structural basis for the interpretation of data obtained by a variety of spectroscopic techniques and for developing general theories of electron transfer in proteins. Moreover, the determination of the structures of the cytochrome bc (Iwata et al., 1998; Zhang et al., 1998) and ATP synthase (Abrahams et al., 1994) complexes of the respiratory membranes allows realistic structural extrapolations to the corresponding complexes of photosynthesis The dark reactions of photosynthesis The sequences of reactions by which CO 2 is reduced to carbohydrate are sometimes referred to as the dark reactions of photosynthesis because CO 2 can be fixed in the dark by a leaf or photosynthetic organism if the appropriate reagents are available. The dark reactions take place separately from the light-driven reactions in the stroma or cytoplasm, as indicated in Fig Electrons from the light-driven process in the thylakoid membrane reduce either nicotinamide adenine dinucleotide (NAD + ) or its phosphorylated form (NADP + ), and the reduced forms NADH or NADPH provide the reducing power for CO 2 fixation, with the help of some additional free energy in the form of adenosine triphosphate (ATP) generated by photosynthetic phosphorylation. There are several mechanisms of CO 2 reduction, characteristic of different photosynthetic species. The reductive pentose cycle or C 3 cycle (so called because the Ch01 Archer and Barber ed MA April 2003 Page 27 of 41

11 2H 2O CO 2 8 hν light-driven electron transport 2NADPH ATP carbon fixation cycle thylakoid membrane 2NADP + + 3P i + 3ADP stroma O 2 LIGHT-DRIVEN REACTION (oxidation half-reaction) 1/6 C 6H 12O 6 + H 2O DARK REACTION (reduction half-reaction) Figure 1.10 The light and dark reactions of oxygenic photosynthesis. The light-driven production of oxygen occurs in reaction centres embedded in the thylakoid membrane. The electrons from this process reduce NADP to NADPH, and also enable the production of ATP from ADP and inorganic phosphate (P i ). The dark carbon fixation cycle occurring in the stroma is driven by NADPH and ATP. first stable product of CO 2 reduction is a three-carbon compound) is the commonest mechanism, operating in algae and most plants. Some plants, especially those indigenous to hot climates, such as corn (maize) and sugar cane, operate the C 4 cycle. Edwards and Walker discuss these and other carbon fixation cycles such as the CAM cycle in Chapter 4; Blankenship (2002) provides a full account. 1.4 Energy-storage efficiency of photosynthesis Photosynthesis is the only natural process able to store a significant amount of solar energy as chemical energy in biomass: terrestrial plants, particularly trees, are the main repositories. However, nature has not entered photosynthesis for any energy efficiency awards the imperative for any photosynthetic organism is replication, not the accretion of biomass. Nonetheless, if the energy-storage process were not adequately efficient, it would not serve this primary purpose. In our context, the energy-storage efficiency of photosynthesis is of course of great interest. It determines the flux of energy into the biosphere, the land area required to produce a given number of food calories, and the biomass yield from a Ch01 Archer and Barber ed MA April 2003 Page 28 of 41