PhotosynthesisResearch 48: 55-63, 1996. © 1996KluwerAcademicPublishers. Printedin theNetherlands. Minireview
The purple bacterial photosynthetic unit Richard J. Cogdell 1, Paul K. Fyfe 1, Smart J. Barrett 1, Stephen M. Prince2, Andrew A. Freer2, Neff W. Isaacs 2, Peter McGlynn 3 & C. Neil Hunter~ 1Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences; 2Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK; 3Department of Molecular Biology and Biotechnology, University of SheffteM, Western Bank, Sheffield $10 2TN, UK Received 13 February1996;acceptedin revisedform29 February1996
Key words: bacteriochlorophyll, energy transfer, light-harvesting complexes, membrane proteins, photosynthetic bacteria, reaction centres
Abstract
Now is a very exciting time for researchers in the area of the primary reactions of purple bacterial photosynthesis. Detailed slructural information is now available for not only the reaction center (Lancaster et al. 1995, in: Blankenship RE et al. (eds) Anoxygenic Photosynthetic Bacteria, pp 503-526), but also LH2 from Rhodopseudomonas acidophila (McDermott et al. 1995, Nature 374: 517-521) and LH1 from Rhodospirillum rubrum (Karrasch et al. 1995, EMBO J 14: 631-638). These structures can now be integrated to produce models of the complete photosynthetic unit (PSU) (Papiz et al., 1996, Trends Plant Sci, in press), which opens the door to a much more detailed understanding of the energy transfer events occurring within the PSU.
Abbreviations: Bchl-bacteriochlorophyll; LH-light-harvesting; PSU-photosynthetic unit Introduction
It is especially appropriate that a Minireview on the purple bacterial photosynthetic unit (PSU) should be included in this special issue of Photosynthesis Research honoring Bill Arnold, since he was involved in the original studies which led to the concept of the PSU (Emerson and Arnold 1932). The major light absorbing pigments in purple bacteria are bacteriochlorophylls a, b and the carotenoids. These pigments are non-covalently bound to two types of integral membrane proteins forming either the reaction centres or the antenna complexes (see Zuber and Cogdell 1995 for a recent review). Most of the pigments function as light harvesters, rapidly and efficiently transferring the absorbed solar radiation to a specialized few which form the reaction centers. This combination of antenna complexes with a reaction center constitutes the PSU. There is evidence suggesting that PSU units, rather than being isolated features, may
interact to form extended assemblies for energy transfer and trapping (van Grondelle 1987). In a seminal study on Rhodospirillum rubrum and Rhodobacter (then called Rhodopseudomonas) sphaeroides, Aagaard and Sistrom (1972) showed that the size of the PSU is variable in many species. Depending on the light intensity at which the cells were grown, the size of the PSU in Rb. sphaeroides varied from about 30 Bchl a molecules per reaction center (high-light) to 200-250 Bchl a molecules per reaction center (low ligh0. Therefore, some of the photosynthetic bacteria are able to adjust their capacity for photon capture in response to the levels of incident light, both in terms of the wavelengths of light absorbed, and in the area of the cell membrane occupied by the antenna. These strategies ensure that each reaction center is kept well supplied with photons with a minimal expenditure of metabolic energy on protein and pigment biosynthesis.
56 Implicit in the work of Aargard and Sistrom (1972) is the idea that there are two types of antenna complex, now referred to as LH1 (or B875) and LH2 (or B80(O 850). LH1 together with a reaction center forms the 'core' of the PSU the size of which appears to be fixed in most species of purple bacteria. Therefore, this structure represents the minimal size of the PSU, at least in wild-type bacteria (Zuber and Brunisholz 1991), and also the maximum size in species such as RhodospirriUum rubrum and Rhodopseudomonas viridis, which can only synthesize LH1 and reaction centers. When present, LH2 is arranged around the periphery of the LHl-reaction center 'core' complex and the ratio of this complex per reaction center is more variable. In general the lower the light-intensity at which the cells are grown the more LH2 per reaction center is synthesised. Species such as Rb. sphaeroides are thus able to regulate the size of their PSU. Still other species such as Rps. acidophila and Rps. cryptolactis (Hawthornthwaite and Cogdell 1991) are able to vary the absorbing range of their LH2 complexes. During growth at very low light-intensities these two species are able to produce B800-820 LH2 complexes instead of the B800-850. All of these antenna complexes are constructed on a common modular principle (Zuber and Cogdell 1995). The pigments are non-covalently attached to two types of low molecular weight, very hydrophobic apoproteins. These are called the a - and/~-apoproteins and are present in a 1:1 ratio. The intact, pigmented complexes are oligomers of these aft pairs. Many of these apoproteins, from both LH 1 and LH2 complexes, have been sequenced (Zuber and Cogdell 1995; Zuber and Brunisholz 1991) and appear to form a rather homologous family. For example, they all contain a conserved histidine residue at the equivalent point in their hydrophobic transmembrane domain, which is thought to be liganded to the Mg 2+ at the center of the Bchl a macrocycles which give rise to the absorption bands at 875 nm in LH1 and 850 nm in LH2. Over the past five to ten years, in the absence of information on the 3D-structure of these antenna complexes, a great deal of work was carried out using a combination of sequence comparisons, site-directed mutagenesis and various biophysical methods in order to construct models of these structures (e.g. see Olsen and Hunter 1994; Zuber and Cogdell 1995). However in the past year this situation has changed dramatically. The 3D-structure of the B800-850 complex from R. acidophila has been solved to a resolution of 2.5/k (McDermott et al. 1995) and an 8 ,~ projec-
tion map of LH1 from R. rubrum has been presented (Karrasch et al. 1995). Since the reaction center structure is also known (Lancaster et al. 1995), there is now structural information available for all the components of the purple bacterial PSU. This places the purple bacteria in a position unique in membrane biology. It is possible to combine the structural knowledge gained through crystallographic approaches with the modelling of spectroscopic techniques. The outcome should be the first detailed description of the architecture of an energy transducing membrane.
The structure of LH2
In Rps. acidophila the B800-850 complex is a n a 9 • 9 oligomer as shown in Figure 1. The overall structure is like an elongated ring doughnut. The inner wall of radius 18/k, is formed by a ring a-apoproteins and the outer one of radius 34 ,~, by a ring of fl-apoproteins. The transmembrane portions of the apoproteins are folded into a-helices, and the structure is closed, top and bottom by the N- and C-termini folding back over and interacting with each other. The a-apoprotein a helices are almost perpendicular to the presumed plane of the membrane, while the/~-apoprotein a-helices are slightly tilted. All the pigments are arranged within the space between the two rings of helices. Interestingly at no point do the a - and ~-apoprotein transmembrane a-helices interact, a structural feature that none of the previous structural models predicted (Olsen and Hunter 1994). The Bchl a molecules are organized into two groups. Towards the cytoplasmic side of the complex there is a ring of monomeric 800 nm-absorbing Bchls a (21.2/k apart, center to center) lying flat in the plane of the membrane (perpendicular to the a helices) between the/~-apoprotein a-helices. A second ring of 18, tightly coupled, 850 nm-absorbing Bchl a molecules is located towards the periplasmic side of the complex. These Bchls a are approximately parallel to the transmembrane a-helices and are liganded to conserved histidine residues present on both the a- and /~-apoprotein. Two carotenoid molecules are present per a/~ pair. These span the thickness of the membrane and contact the two rings of Bchl a molecules. So far it appears that in a given species the size of the LH2 ring is constant, a9~ 9 in Rps. acidophila and Rhodovulum sulfidophilum (I. Sinning, personal communication). However, it may vary from species to species. In R. molischianum, for example, it is an as/38 oligomer (H. Michel, personal communication).
57
a
b Figure 1. Schematic representation of the LH2 nonamer complex (McDermott et al. 1995) (a) viewed from the periplasmic side of the membrane, and (b) viewed from within the membrane. The arrangement of the two rings of Bchl a molecules within the walls formed by the protein subunits are shown: the ring of 9 B800 Bchl a molecules parallel to the membrane; and the second ring of 18 B850 Bchl a molecules perpendicular to the membrane. The carotenoid molecule spans the membrane coming into close contact with the two Bchl a rings. Only the photoactive portions of the chromophores are shown. Figures produced using the package 'Molscript' (Kraulis 1991).
58 What controls the oligomeric size is not yet clear but is presumably a function of the exact primary sequence at the N- and C-termini where the a- and fl-apoproteins interact.
The structure of LH1 LH1-RC 'cores' were first clearly visualized in EM studies carried out on membranes from Rps. viridis (Stark et al. 1984, Miller 1982). This species has intracytoplasmic membranes in the form of lamellae that contain quasi-crystalline 2D arrays of LH1-RC 'cores'. These 'core' complexes are visualized as circular structures with a diameter of ,-~120 ~k. When these images were Fourier processed and averaged they showed 6fold symmetry. The ratio of Bchl a per reaction center was estimated to be 24:1 (Jay et al. 1982) and a structure was proposed where the LH1 ring surrounding the central reaction center was an al2fll2 oligomer (LH1 from Rps. viridis also contains a non-pigment binding 3' subunit, so in this case the oligomer was proposed to be c~12fl12"/12). These images of the 'core' complex have, until very recently, dominated thinking about the structure of 'core' of the PSU. Many studies showed that LH1-RC 'core' complexes were present in all species of purple bacteria (for example see Dawkins et al. 1988). There is a clear relationship between the size of an LH1 ring, and its ability to encircle a reaction center. Despite the lack of structural information, the stoichiometry of LH1 Bchls a per reaction center ought to provide a method by which to gain an insight into this problem. Thus, there have been numerous attempts to determine this stoichiometry, with most attempts giving values close to 24:1. However, these measurements are not straightforward, because LH1-RC complexes tend to be unstable when they are isolated. When a wider range of species was tested the values obtained varied from 21:1 to 40:1 (Dawkins et al. 1988). Two more recent attempts to determine the stoichiometry of Bchla : RC in LH1-RC 'core' complexes have been made (Gall 1995, Francke and Amesz 1995). Gall (1995) measured this ratio in 'core' complexes isolated from seven different species and, although there was some variability in the data, an average value of 334-4:1 was obtained. These are consistent with the model of Karrasch et al. (1995) described below, which implies a ratio of 32 Bchls a per reaction center. However, Francke and Amesz (1995) determined the Bchl a:RC ratio in 'core' complexes from 6 different
species and found values ranging from 24 + 2:1 to 28 4- 4:1; this would be more consistent with an c~12/~12 ring structure (which would predict a ratio of 24:1). Clearly more work is needed in this area but if the LH2 structure is enlarged (see Figure 2 below) to give either an O~12/~12or 0~16/~16 ring, only the Otl6~l 6 ring is large enough to accommodate a reaction center in its center. The observation leads us to favour the Oq6/~16 structure. More recently, Karrasch et al. (1995) produced 2D crystals of LH1 complexes from R. rubrum. When these were subjected to electron diffraction a projection map of LH1 was produced at 8.5 ,~ resolution which showed a ring structure comprising an 0~16/~16 oligomer. When their data was processed at lower resolution it appeared to show 6-fold symmetry, but at higher resolution this pseudo-6-fold symmetry broke down into 8-fold symmetry (R. Ghosh, personal communication). The diameter of the 'hole' left in their structure (68/k) is just, and only just, big enough to accommodate the reaction center. Interestingly the diameter of the LH1 ring seen by Karrasch et al. (1995) is very close to that seen in Rps. viridis membranes (e.g. Stark et al. 1984) and in 2D crystals of the equivalent 'core' complexes from Rhodopseudomonas marina (Meckenstock et al. 1994). It is possible that in these last two cases the 6-fold symmetry was also a result of low resolution. One way to reconcile a LH1 :reaction center Bchl a stoichiometry of less than 32:1, with the apparent need to encircle the reaction center, would be to include a non-pigment-binding protein. Indeed, the non-pigmented protein Puf X, has been proposed to position itself within the LHl-reaction center 'cores'. The possible roles of Puf X are discussed later in this review. However, for the remainder of this short review we will assume that a structure of a size equivalent to O~16~16 is the correct one.
Putting together the whole PSU Papiz et al. (1996) have produced a model for the whole of the PSU. The approach used was the following. Since the primary sequences of the LH1 and LH2 apoproteins are, in general terms very similar, the LH2 structure was used as the basis for producing a model of LH1. To model the O~16/~16 projection map of Karrasch et al. (1995), the LH2 Ct9/~9 ring was increased to produce the larger OZl6fll6 ring, with a diameter as predicted from the 8.5 ,~ LH1 projection map. The
59
Figure 2. A model of a section of the PSU illustrates how an LH2 complex may interact with an LH1-RC 'core' complex. (a) a view of a section taken in the plane of the membrane,and (b) a section viewed from within the membrane.The reaction center structure used is that of Rb. sphaeroides R-26 (Yeates et al. 1988). The basis for the modelis describedin the text and also in Papiz et al. (1996). The view from within the membranereveals the similarityin the depth at which the B850 Bchls a fromLH2, the B875 Bchls a from LHI and the Bchls a formingthe special pair in the reaction center, are positionedin the membrane.The positionsof the Bchls are muchclearer in the color versionsdeposited in the website given below. Figures produced using the package 'O' (Jones et al. 1991). Color versionsof the figures are lodged at the following website: http://www.chem.gla.ac.uk/protein/LH2/lh2.html
known structure of the reaction center was then placed in the LH1 ring. This is illustrated in Figure 2 where an LH2 ring is also shown next to the 'core' complex. Figure 2(b) shows a side-ways view from within the membrane. A striking feature of this model is the way in which the rings of 850 n m and 875 nm-absorbing Bchls a line up at the same depth in the membrane, and how this corresponds very closely to the position in the m e m b r a n e where the 'special pair' of reaction center Bchls a are also located. This minimizes the distances between Bchl a molecules, thus ensuring that the energy transfer from LH2 to LH1 and from LH1 to the RC is as fast as possible. Papiz et al. (1996) also showed that in this type of model i t is possible to pack eight LH2 nonameric rings closely around the
LH1. Of course if such a structure does exist it will only be seen in membranes from cells grown at very low light-intensities where the size of the PSU reaches its m a x i m u m of approximately 250 Bchls a per RC. More detailed EM studies on intact membranes are now required to test this model. There is another apparent problem with the model of the LH1-RC 'core' shown in Figure 2(a) apart from the ambiguity of the size of the LH 1 ring. In this picture the transmembrane c~-helices of antenna apoproteins appear to surround the reaction center rather like an impenetrable palisade. During its normal photochemical cycle the reaction center secondary quinone (QB) delivers reducing equivalents to the cytochrome blcl complex. However, if the reaction center is blockaded
60
A
B
C
Figure3. (A) Putative model for the location of Puf X as part of the LH 1 ring surrounding the reaction center. In this purely diagrammatic form, Puf X is represented as a dimer (A.R. Crofts, personal communication), which allows lateral ubiquinone transfer from the QB site of the reaction center to the cytochrornebcl complex (Barz et al. 1995b). (B) In a strain in which the aggregation state of the LH1 complex is limited (McGlynn et al. 1994), PufX is not required to expose the QB site. Therefore, strains carrying such mutations are photosynthetically competent, as is a RC-only Puf X-minus strain (McGlynn et al. 1994). (C) A strain which is not impaired in LH1 assembly and in which PufX is absent will be non-photosynthetic,as a consequence of blocking the Qn site. In this case, a small increase in the ratio of LH1 Bchls a:RC is observed in comparison with the model in (A) (McGlynn et al. 1994, 1996; Barz et al. 1995a).
by LH1 how can this happen? In some species such as Rb. sphaeroides there is a gene encoding a protein called Puf X which m a y function to alleviate this problem.
Puf X: An essential component of the PSU Several studies have identified Puf X as a component of the PSU. Its importance can be gauged by showing that strains o f Rb. sphaeroides lacking Puf X are incapable of photosynthetic growth. Following early observations of Farchaus and co-workers (Farchaus et al. 1990, 1992) using reaction center only and reaction center-LH1 strains, M c G l y n n et al. (1994) were able to show that the loss o f Puf X was deleterious only when the LH1 was present. In a further development of this approach, strains were constructed in which the L H I : reaction center 'core' size was genetically modified ( M c G l y n n et al. 1996). This manipulation resulted in a progressive truncation o f the C-terminal region of the LH1 c~ apoprotein. It was shown that if the 'core' size was reduced below 21 Bchls a : reaction center, the absence o f P u f X did not impair photosynthetic growth. In addition, for any given 'core' size, the loss of P u f X resulted in an increase in 'core' size of 1-2 Bchls a per reaction center. Finally the 'core' size was determined to be 25.7 4- 3.2 Bchls a in the presence of P u f X . The consequences o f these investigations are represented in
Figure 3, which depicts a reaction center encircled by LH1 apoproteins. The data indicate that Puf X could be part of the LH1 ring, and that it occupies perhaps 10-20% of the circumference of this antenna. In doing so, it allows the cytochrome bCl complex access to the QB site of the reaction center, as suggested by Lilburn et al. (1992). This becomes unnecessary when LH1 is either absent, or reduced in size. Recent work (Barz et al. 1995a, b) supports this, by demonstrating an essential role for Puf X in allowing access of ubiquinone to the QB site.
Energy transfer within the PSU When light is absorbed by a Bchl a molecule the lifetime of the first excited singlet state is of the order of a few nanoseconds. Therefore, energy transfer from the antenna system to the reaction center must take place well within this time or the absorbed energy will be lost in non-productive processes. The direction of energy transfer within the PSU is not random, but is guided by the energy gradient going from B800 to B850 to B875 and then into the RC. These energy transfer steps have now been resolved in the picosecond time domain using ultrafast laser spectroscopy (see Sunds t r r m and van Grondelle 1995 for a recent review). Taking Rb. sphaeroides as an example since it has been the most extensively studied system, energy transfer from B800 to B850 takes place in 0.7 ps. The energy is then extremely rapidly delocalized among the B850 molecules on the 5 0 - 1 5 0 fs timescale. Subsequent transfer between complexes involves the energy hopping from B850 in LH2, to B875 i n L H I , which takes 2 - 4 ps (Hess et al. 1995). Again, once the energy reaches the circular array of B875 molecules, it is very rapidly delocalized. Finally the energy is transferred from B875 to the RC in 20--40 ps. This last step is the slowest in the energy transfer pathway. The reason for this is suggested in the model shown in Figure 2b where the longest distance over which the energy transfer occurs (and hence the slowest transfer step) is from the B875 ring of Bchl a molecules to the RC special pair. Energy transfer within the PSU used to be modelled in terms o f 'random hopping' o f excitons between quasi-regular arrays o f chlorophylls (e.g. Robinson 1967). Clearly this is not a good physical description of the way in which the purple bacterial PSU functions. This system is one of interacting rings. The rings of B850 or B875 Bchls a act rather like 'storage rings', with the excited singlet state effective-
61 ly rapidly delocalized around the circumference. This makes the energy available for transfer from any part of a ring to any part of its neighbor, as long as they are close enough to each other. This appears to be one of the major reasons why the purple bacterial PSU is so efficient. The time resolved measurements on whole membrane systems, when compared to those carried out on the isolated complexes, show that there is only enough time for very few inter-ring 'hops'.
Regulation of the expression of the PSU In several species, e.g. Rb. capsulatus, Rb. sphaeroides and R. rubrum, all the genes encoding the reaction center polypeptides and the antenna apoproteins have been cloned and sequenced (reviewed by Williams and Taguchi 1995). The early studies of Cohen-Bazire et al. (1957) showed that light and oxygen exert a strong influence on the composition of the PSU. Nearly 40 years on, the molecular genetics of PSU formation continue to be studied in great detail. Our current knowledge of the molecular mechanism by which their expression is regulated has not progressed to a complete understanding of the mechanisms of environmental control. (This topic has recently been excellently reviewed by Bauer 1995.) However, this regulation appears to be similar to the classical two component sensory transduction cascade systems seen in other prokaryotes. One example of this is the regulation of light-harvesting and reaction center gene expression in response to oxygen tension by Reg A and Reg B. Reg B, which is believed to be a membrane spanning sensor kinase, appears to sense changes in the oxygen environment. In conditions of low oxygen tension, Reg B autophosphorylates itself before catalyzing the transfer of this phosphate onto Reg A, a cytosolic response regulator. This activated form of Reg A is then proposed to phosphorylate DNA binding proteins, which in turn control the induction of the light-harvesting and reaction center structural genes. The greatest progress has been made in Rb. capsulatus and Rb. sphaeroides, species in which the PSU consists of LH2, LH1 and the reaction center. In some species, however, the situation is much more complicated. In Rps. acidophila or Rps. palustris, for example, there is a multi-gene family encoding for the LH2 apoproteins, some of which in Rps. acidophila appear to be 'silent' (Tadros et al. 1993; Gardiner et al. 1992). There is at present very little detailed information on the significance of this or how they are regulated.
The genes encoding components of the PSU hold an extrinsic interest to those engaged in gene regulation studies. Aside from this, the availability of the genes for the reaction center and antenna apoproteins has opened the door to using site-directed mutagenesis to probe their structure/function relations (for example see Hunter 1995; Woodbury and Allen 1995). There is now extensive literature on site-directed mutants of the bacterial reaction center (reviewed in Woodbury and Allen 1995), which followed the elucidation of the three-dimensional structure of this complex (Deisenhofer et al. 1985). The mutagenesis of LH complexes has preceded the structural determinations (for example, Bylina and Youvan 1988; Babst et al. 1991; Fowler et al. 1992; Olsen et al. 1994) but it is now expected to receive fresh impetus from the availability of structural information on the LH1 and LH2 complexes.
Conclusions Now, with the discovery of the 'ring-like' structures for the purple bacterial antenna complexes, the underlying architecture is apparent for all of the components of the PSU. In most cases these structures are consistent with spectroscopic data on the mechanisms and kinetics of light capture and transfer. The way is now open for significant advances to be made in understanding not only the detailed molecular mechanisms of energy transfer, but also the role that the proteins play in determining the characteristics of light-harvesting and energy trapping. The model presented in Figure 2 focuses attention on the need to investigate the way the various components of the PSU actually interact within the intact photosynthetic membrane in vivo.
References Aagaard J and Sistrom WR (1972) Control of synthesis of reaction center bactcriochlorophylls in photosynthetic bacteria. Photochem Photobiol 15:209-225 Babst M, Albrecht H, Wegmann I, Brunisholz R and Zubcr H (1991) Single amino acid substitutions in the B870 alpha and beta lightharvesting polypcptides of Rhodobacter capsulatus. Structural and spectral effects. Eur J Biocbem 202:277-284 Barz WE Francia E Venturoli G, Melandri BA, Vermtglio A and Ocsterbelt D (1995a) Role of puf X protein in photosynthetic growth of Rhodobacter sphaeroides, l. Puf X is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions. Biochemistry 34:15235-15247 Barz WE Verm6glio A, Francia F, Venturoli G, Melandri BA and Ocsterhelt D (1995b) Role of the Puf X protein in photosynthetic growth of Rhodobacter sphaeroides. 2. Puf X is required for effi-
62 cient ubiquinone/ubiquinol exchange between the reaction center Q(B) site and the cytochrome bcl complex. Biochemistry 34: 15248-15258 Bauer CE (1995) Regulation of photosynthesis gene expression. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 1221-1234. Klnwer Academic Publishers, Dordrecht, The Netherlands Bylina EJ, Robles SJ and Youvan DC (1988) Directed mutations affecting the putative bacteriochlorophyll-binding sites in the light-harvesting-I antenna of Rhodobacter capsulatus. Israel J Chem 28:73-78 Cohen-Bazire G, Sistrom WR and Stanier RY (1957) Kinetic studies of pigment synthesis by non-sulphur purple bacteria. J Cell Comp Physiol 49:25-68 Dawkins DJ, Ferguson LA and Cogdell RJ (1988) The structure of the purple bacteria photosynthetic unit. In: Scheer H and Schneider S (eds) Photosynthetic Light-Harvesting Systems, pp 115127. Walter de Gruyter, Berlin Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1985) Structare of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viridis at 3 ,~ resolution. Nature, 318: 618-624 Emerson R and Arnold WA (1932) The photochemical reaction in photosynthesis. J Gen Physiol 16:191-205 Farchaus JW, Grnnberg H and Oesterhelt D (1990) Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype. J Bacteriol 172:977-985 Farchaus JW, Barz WP, Grunberg H and Oesterhelt D (1992) Studies on the expression of the puf X polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides. EMBO J 11:2779-2788 Fowler GJS, Crielaard W, Visschers RW, van Grondelle R and Hunter CN (1993) Site-directed mutagenesis of the LH2 light-harvesting complex of Rhodobacter sphaeroides - changing beta-lys23 to gin results in a shift in the 850 nm absorption peak. Photochem and Photobiol 5 7 : 2 - 6 Francke C and Amesz J (1995) The size of the photosynthetic unit in purple bacteria. Photosynth Res 46:347-352 Gall A (1995) Purification, characterisation and crystallisation from a range of Rhodospirillineae pigment protein complexes. PhD Thesis, Univ Glasgow, UK Gardiner AT, MacKensie RC, Barrett SJ, Kaiser K and Cogdell RJ (1992) The genes for the peripheral antenna complex from Rhodopseudomonas acidophila 7050 form a multigene family. In: Murata N (ed) Research in Photosynthesis, Vol 1, pp 77-80. Kluwer Academic Publishers, Dordrecht, The Netherlands Hawthornthwaite AM and Cogdell RJ (1991) Bacteriochlorophyll binding proteins. In: Scheer H (ed) Chlorophylls, pp 493-528. CRC Press, Boca Raton Hess S, Chachisvilis M, Jones MR, Hunter CN and Sundstr0m V (1995) Temporally and spectrally resolved subpicosecond energy transfer within the peripheral antenna complex (LH2) and from LH2 to the core antenna complex in photosynthetic purple bacteria. Proc Natl Acad Sci USA 92:12333-12337 Hunter CN (1995) Genetic manipulation of the antenna complexes of purple bacteria. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 473-501. Kluwer Academic Publishers, Dordrecht, The Netherlands Jay F, Lambillotte M and Muhlethaler K (1983) Localisation of Rhodopseudomonas viridis reaction center and light-harvesting protein using ferritin-antibody labelling. Eur J Cell Biol 30:1-8 Jones TA, Zou JY, Cowan SW and Kjeldgaard (1991) Improved methods for building protein models in electron density maps
and the location of errors in these maps. Acta Cryst A 4 7 : 1 1 0 119 Karrasch S, Bullough PA and Ghosh R (1995) The 8.5 ~ projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14: 631-638 Kranlis PJ (I 991) Molscript - a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24:946-950 Lancaster CRD, Ermler U and Michel H (1995) The structures of photosynthetic reaction centers from purple bacteria as revealed by x-ray crystallography. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 503526. Kluwer Academic Publishers, Dordrecht, The Netherlands Lilbum TG, Halth CE, Prince RC and Beatty JT (1992) Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus. Biochim Biophys Acta 1100:160-170 McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374:517-521 McGlynn P, Hunter CN and Jones MR (1994) The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system. FEBS Lett 349:349-353 McGlynn P, Westerhuis WHJ, Jones MR and Hunter CN (1996) Consequences for the organization of reaction center-light harvesting antenna 1 (LHI) core complexes of Rhodobacter sphaeroides arising from deletion of amino acid residues from the C terminus of the LH1 cz polypeptides. J Biol Chem 271:3285-3292 Meckenstock R, Krusche K, Staehelin LA, Cyrklaff M, Brnnisholz RA and Zuber H (1994) The six fold symmetry of the B880 light-harvesting membranes of Rhodopseudomonas marina. Biol Chem Hoppe-Seyler 375:429--438 Miller KR (1982) Three dimensional structure of a photosynthetic membrane. Nature 300:53-55 Olsen JD and Hunter CN (1994) Protein structure modelling of the bacterial light-harvesting complex. Photochem Photobio160: 521-535 Olsen JD, Sockalingum GD, Robert B and Hunter CN (1994) Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides. Proc Natl Acad Sci USA 91:7124-7128 Papiz MZ, Prince SM, Hawthornthwaite-Lawless AM, McDermott G, Freer AA, Isaacs NW and Cogdell RJ (1996) A model for the photosynthetic apparatus of purple bacteria. Trends Plant Sci 1: 198-206 Robinson GW (1967) Excitation transfer and trapping in photosynthesis. Brookhaven Symp Biol 19:16-48 Stark W, Kuhlbrandt W, Wildhaber I, Wehrli E and Muhlethaler K(1984) The structure of the photoreceptor unit of Rhodopseudomonas viridis. EMBO J 3:777-783 Sundstr0m V and van Grondelle R (1995) Kinetics of excitation transfer and trapping in purple bacteria. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 349-372. Kluwer Academic Publishers, Dordrecht, The Netherlands Tadros MH, Katsiou E, Hoon MA, Yurkova N and Ramji DP (1991) Cloning of a new antenna gene cluster and expression analysis of the antenna gene family of Rhodopseudomonas palustris. Enr J Biochem 217:867-875 van Grondelle R, Hunter CN, Bakker JGC and Kramer HJM (1983) Size and structure of antenna complexes of photosynthetic bacte-
63 ria as studied by singlet-singlet quenching of the bacteriochlorophyll fluorescence yield. Biochim Biophys Acta 723:30-36 Williams JC and Taguchi AKW (1995) Genetic manipulation of purple photosynthetic bacteria. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 1029-1065. Kluwer Academic Publishers, Dordrecht, The Netherlands Woodbury NW and Allen JP (1995) The pathway, kinetics and thermodynamics of electron transport in wild-type and mutant reaction centers of purple non-sulphur bacteria. In: Blankenship RE, Madigan MT and Bauer CE(eds) Anoxygenic Photosynthetic Bacteria pp 527-557. Kluwer Academic Publishers, Dordrecht, The Netherlands
Yeates TO, Komiya H, Chirino A, Rees DC, Allen JP and Feher G (1988) Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: Protein-cofactor (bacteriochlorophyll, bacteriopheophytin and carotenoid) interactions. Proc Nail Acad Sci 85:7993-7997 Zuber H and Brunisholz RA (1991) Structure and function of antenna polypeptides and chlorophyll protein complexes: principles and variability. In: Scheer H (ed) Chlorophylls, pp 627-703. CRC Press, Boca Raton Zuber H and Cogdell RJ (1995) The structure and organisation of purple bacterial antenna complexes. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 315-348. Kluwer Academic Publishers, Dordrecht, The Netherlands
The purple bacterial photosynthetic unit Richard J. Cogdell 1, Paul K. Fyfe 1, Smart J. Barrett 1, Stephen M. Prince2, Andrew A. Freer2, Neff W. Isaacs 2, Peter McGlynn 3 & C. Neil Hunter~ 1Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences; 2Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK; 3Department of Molecular Biology and Biotechnology, University of SheffteM, Western Bank, Sheffield $10 2TN, UK Received 13 February1996;acceptedin revisedform29 February1996
Key words: bacteriochlorophyll, energy transfer, light-harvesting complexes, membrane proteins, photosynthetic bacteria, reaction centres
Abstract
Now is a very exciting time for researchers in the area of the primary reactions of purple bacterial photosynthesis. Detailed slructural information is now available for not only the reaction center (Lancaster et al. 1995, in: Blankenship RE et al. (eds) Anoxygenic Photosynthetic Bacteria, pp 503-526), but also LH2 from Rhodopseudomonas acidophila (McDermott et al. 1995, Nature 374: 517-521) and LH1 from Rhodospirillum rubrum (Karrasch et al. 1995, EMBO J 14: 631-638). These structures can now be integrated to produce models of the complete photosynthetic unit (PSU) (Papiz et al., 1996, Trends Plant Sci, in press), which opens the door to a much more detailed understanding of the energy transfer events occurring within the PSU.
Abbreviations: Bchl-bacteriochlorophyll; LH-light-harvesting; PSU-photosynthetic unit Introduction
It is especially appropriate that a Minireview on the purple bacterial photosynthetic unit (PSU) should be included in this special issue of Photosynthesis Research honoring Bill Arnold, since he was involved in the original studies which led to the concept of the PSU (Emerson and Arnold 1932). The major light absorbing pigments in purple bacteria are bacteriochlorophylls a, b and the carotenoids. These pigments are non-covalently bound to two types of integral membrane proteins forming either the reaction centres or the antenna complexes (see Zuber and Cogdell 1995 for a recent review). Most of the pigments function as light harvesters, rapidly and efficiently transferring the absorbed solar radiation to a specialized few which form the reaction centers. This combination of antenna complexes with a reaction center constitutes the PSU. There is evidence suggesting that PSU units, rather than being isolated features, may
interact to form extended assemblies for energy transfer and trapping (van Grondelle 1987). In a seminal study on Rhodospirillum rubrum and Rhodobacter (then called Rhodopseudomonas) sphaeroides, Aagaard and Sistrom (1972) showed that the size of the PSU is variable in many species. Depending on the light intensity at which the cells were grown, the size of the PSU in Rb. sphaeroides varied from about 30 Bchl a molecules per reaction center (high-light) to 200-250 Bchl a molecules per reaction center (low ligh0. Therefore, some of the photosynthetic bacteria are able to adjust their capacity for photon capture in response to the levels of incident light, both in terms of the wavelengths of light absorbed, and in the area of the cell membrane occupied by the antenna. These strategies ensure that each reaction center is kept well supplied with photons with a minimal expenditure of metabolic energy on protein and pigment biosynthesis.
56 Implicit in the work of Aargard and Sistrom (1972) is the idea that there are two types of antenna complex, now referred to as LH1 (or B875) and LH2 (or B80(O 850). LH1 together with a reaction center forms the 'core' of the PSU the size of which appears to be fixed in most species of purple bacteria. Therefore, this structure represents the minimal size of the PSU, at least in wild-type bacteria (Zuber and Brunisholz 1991), and also the maximum size in species such as RhodospirriUum rubrum and Rhodopseudomonas viridis, which can only synthesize LH1 and reaction centers. When present, LH2 is arranged around the periphery of the LHl-reaction center 'core' complex and the ratio of this complex per reaction center is more variable. In general the lower the light-intensity at which the cells are grown the more LH2 per reaction center is synthesised. Species such as Rb. sphaeroides are thus able to regulate the size of their PSU. Still other species such as Rps. acidophila and Rps. cryptolactis (Hawthornthwaite and Cogdell 1991) are able to vary the absorbing range of their LH2 complexes. During growth at very low light-intensities these two species are able to produce B800-820 LH2 complexes instead of the B800-850. All of these antenna complexes are constructed on a common modular principle (Zuber and Cogdell 1995). The pigments are non-covalently attached to two types of low molecular weight, very hydrophobic apoproteins. These are called the a - and/~-apoproteins and are present in a 1:1 ratio. The intact, pigmented complexes are oligomers of these aft pairs. Many of these apoproteins, from both LH 1 and LH2 complexes, have been sequenced (Zuber and Cogdell 1995; Zuber and Brunisholz 1991) and appear to form a rather homologous family. For example, they all contain a conserved histidine residue at the equivalent point in their hydrophobic transmembrane domain, which is thought to be liganded to the Mg 2+ at the center of the Bchl a macrocycles which give rise to the absorption bands at 875 nm in LH1 and 850 nm in LH2. Over the past five to ten years, in the absence of information on the 3D-structure of these antenna complexes, a great deal of work was carried out using a combination of sequence comparisons, site-directed mutagenesis and various biophysical methods in order to construct models of these structures (e.g. see Olsen and Hunter 1994; Zuber and Cogdell 1995). However in the past year this situation has changed dramatically. The 3D-structure of the B800-850 complex from R. acidophila has been solved to a resolution of 2.5/k (McDermott et al. 1995) and an 8 ,~ projec-
tion map of LH1 from R. rubrum has been presented (Karrasch et al. 1995). Since the reaction center structure is also known (Lancaster et al. 1995), there is now structural information available for all the components of the purple bacterial PSU. This places the purple bacteria in a position unique in membrane biology. It is possible to combine the structural knowledge gained through crystallographic approaches with the modelling of spectroscopic techniques. The outcome should be the first detailed description of the architecture of an energy transducing membrane.
The structure of LH2
In Rps. acidophila the B800-850 complex is a n a 9 • 9 oligomer as shown in Figure 1. The overall structure is like an elongated ring doughnut. The inner wall of radius 18/k, is formed by a ring a-apoproteins and the outer one of radius 34 ,~, by a ring of fl-apoproteins. The transmembrane portions of the apoproteins are folded into a-helices, and the structure is closed, top and bottom by the N- and C-termini folding back over and interacting with each other. The a-apoprotein a helices are almost perpendicular to the presumed plane of the membrane, while the/~-apoprotein a-helices are slightly tilted. All the pigments are arranged within the space between the two rings of helices. Interestingly at no point do the a - and ~-apoprotein transmembrane a-helices interact, a structural feature that none of the previous structural models predicted (Olsen and Hunter 1994). The Bchl a molecules are organized into two groups. Towards the cytoplasmic side of the complex there is a ring of monomeric 800 nm-absorbing Bchls a (21.2/k apart, center to center) lying flat in the plane of the membrane (perpendicular to the a helices) between the/~-apoprotein a-helices. A second ring of 18, tightly coupled, 850 nm-absorbing Bchl a molecules is located towards the periplasmic side of the complex. These Bchls a are approximately parallel to the transmembrane a-helices and are liganded to conserved histidine residues present on both the a- and /~-apoprotein. Two carotenoid molecules are present per a/~ pair. These span the thickness of the membrane and contact the two rings of Bchl a molecules. So far it appears that in a given species the size of the LH2 ring is constant, a9~ 9 in Rps. acidophila and Rhodovulum sulfidophilum (I. Sinning, personal communication). However, it may vary from species to species. In R. molischianum, for example, it is an as/38 oligomer (H. Michel, personal communication).
57
a
b Figure 1. Schematic representation of the LH2 nonamer complex (McDermott et al. 1995) (a) viewed from the periplasmic side of the membrane, and (b) viewed from within the membrane. The arrangement of the two rings of Bchl a molecules within the walls formed by the protein subunits are shown: the ring of 9 B800 Bchl a molecules parallel to the membrane; and the second ring of 18 B850 Bchl a molecules perpendicular to the membrane. The carotenoid molecule spans the membrane coming into close contact with the two Bchl a rings. Only the photoactive portions of the chromophores are shown. Figures produced using the package 'Molscript' (Kraulis 1991).
58 What controls the oligomeric size is not yet clear but is presumably a function of the exact primary sequence at the N- and C-termini where the a- and fl-apoproteins interact.
The structure of LH1 LH1-RC 'cores' were first clearly visualized in EM studies carried out on membranes from Rps. viridis (Stark et al. 1984, Miller 1982). This species has intracytoplasmic membranes in the form of lamellae that contain quasi-crystalline 2D arrays of LH1-RC 'cores'. These 'core' complexes are visualized as circular structures with a diameter of ,-~120 ~k. When these images were Fourier processed and averaged they showed 6fold symmetry. The ratio of Bchl a per reaction center was estimated to be 24:1 (Jay et al. 1982) and a structure was proposed where the LH1 ring surrounding the central reaction center was an al2fll2 oligomer (LH1 from Rps. viridis also contains a non-pigment binding 3' subunit, so in this case the oligomer was proposed to be c~12fl12"/12). These images of the 'core' complex have, until very recently, dominated thinking about the structure of 'core' of the PSU. Many studies showed that LH1-RC 'core' complexes were present in all species of purple bacteria (for example see Dawkins et al. 1988). There is a clear relationship between the size of an LH1 ring, and its ability to encircle a reaction center. Despite the lack of structural information, the stoichiometry of LH1 Bchls a per reaction center ought to provide a method by which to gain an insight into this problem. Thus, there have been numerous attempts to determine this stoichiometry, with most attempts giving values close to 24:1. However, these measurements are not straightforward, because LH1-RC complexes tend to be unstable when they are isolated. When a wider range of species was tested the values obtained varied from 21:1 to 40:1 (Dawkins et al. 1988). Two more recent attempts to determine the stoichiometry of Bchla : RC in LH1-RC 'core' complexes have been made (Gall 1995, Francke and Amesz 1995). Gall (1995) measured this ratio in 'core' complexes isolated from seven different species and, although there was some variability in the data, an average value of 334-4:1 was obtained. These are consistent with the model of Karrasch et al. (1995) described below, which implies a ratio of 32 Bchls a per reaction center. However, Francke and Amesz (1995) determined the Bchl a:RC ratio in 'core' complexes from 6 different
species and found values ranging from 24 + 2:1 to 28 4- 4:1; this would be more consistent with an c~12/~12 ring structure (which would predict a ratio of 24:1). Clearly more work is needed in this area but if the LH2 structure is enlarged (see Figure 2 below) to give either an O~12/~12or 0~16/~16 ring, only the Otl6~l 6 ring is large enough to accommodate a reaction center in its center. The observation leads us to favour the Oq6/~16 structure. More recently, Karrasch et al. (1995) produced 2D crystals of LH1 complexes from R. rubrum. When these were subjected to electron diffraction a projection map of LH1 was produced at 8.5 ,~ resolution which showed a ring structure comprising an 0~16/~16 oligomer. When their data was processed at lower resolution it appeared to show 6-fold symmetry, but at higher resolution this pseudo-6-fold symmetry broke down into 8-fold symmetry (R. Ghosh, personal communication). The diameter of the 'hole' left in their structure (68/k) is just, and only just, big enough to accommodate the reaction center. Interestingly the diameter of the LH1 ring seen by Karrasch et al. (1995) is very close to that seen in Rps. viridis membranes (e.g. Stark et al. 1984) and in 2D crystals of the equivalent 'core' complexes from Rhodopseudomonas marina (Meckenstock et al. 1994). It is possible that in these last two cases the 6-fold symmetry was also a result of low resolution. One way to reconcile a LH1 :reaction center Bchl a stoichiometry of less than 32:1, with the apparent need to encircle the reaction center, would be to include a non-pigment-binding protein. Indeed, the non-pigmented protein Puf X, has been proposed to position itself within the LHl-reaction center 'cores'. The possible roles of Puf X are discussed later in this review. However, for the remainder of this short review we will assume that a structure of a size equivalent to O~16~16 is the correct one.
Putting together the whole PSU Papiz et al. (1996) have produced a model for the whole of the PSU. The approach used was the following. Since the primary sequences of the LH1 and LH2 apoproteins are, in general terms very similar, the LH2 structure was used as the basis for producing a model of LH1. To model the O~16/~16 projection map of Karrasch et al. (1995), the LH2 Ct9/~9 ring was increased to produce the larger OZl6fll6 ring, with a diameter as predicted from the 8.5 ,~ LH1 projection map. The
59
Figure 2. A model of a section of the PSU illustrates how an LH2 complex may interact with an LH1-RC 'core' complex. (a) a view of a section taken in the plane of the membrane,and (b) a section viewed from within the membrane.The reaction center structure used is that of Rb. sphaeroides R-26 (Yeates et al. 1988). The basis for the modelis describedin the text and also in Papiz et al. (1996). The view from within the membranereveals the similarityin the depth at which the B850 Bchls a fromLH2, the B875 Bchls a from LHI and the Bchls a formingthe special pair in the reaction center, are positionedin the membrane.The positionsof the Bchls are muchclearer in the color versionsdeposited in the website given below. Figures produced using the package 'O' (Jones et al. 1991). Color versionsof the figures are lodged at the following website: http://www.chem.gla.ac.uk/protein/LH2/lh2.html
known structure of the reaction center was then placed in the LH1 ring. This is illustrated in Figure 2 where an LH2 ring is also shown next to the 'core' complex. Figure 2(b) shows a side-ways view from within the membrane. A striking feature of this model is the way in which the rings of 850 n m and 875 nm-absorbing Bchls a line up at the same depth in the membrane, and how this corresponds very closely to the position in the m e m b r a n e where the 'special pair' of reaction center Bchls a are also located. This minimizes the distances between Bchl a molecules, thus ensuring that the energy transfer from LH2 to LH1 and from LH1 to the RC is as fast as possible. Papiz et al. (1996) also showed that in this type of model i t is possible to pack eight LH2 nonameric rings closely around the
LH1. Of course if such a structure does exist it will only be seen in membranes from cells grown at very low light-intensities where the size of the PSU reaches its m a x i m u m of approximately 250 Bchls a per RC. More detailed EM studies on intact membranes are now required to test this model. There is another apparent problem with the model of the LH1-RC 'core' shown in Figure 2(a) apart from the ambiguity of the size of the LH 1 ring. In this picture the transmembrane c~-helices of antenna apoproteins appear to surround the reaction center rather like an impenetrable palisade. During its normal photochemical cycle the reaction center secondary quinone (QB) delivers reducing equivalents to the cytochrome blcl complex. However, if the reaction center is blockaded
60
A
B
C
Figure3. (A) Putative model for the location of Puf X as part of the LH 1 ring surrounding the reaction center. In this purely diagrammatic form, Puf X is represented as a dimer (A.R. Crofts, personal communication), which allows lateral ubiquinone transfer from the QB site of the reaction center to the cytochrornebcl complex (Barz et al. 1995b). (B) In a strain in which the aggregation state of the LH1 complex is limited (McGlynn et al. 1994), PufX is not required to expose the QB site. Therefore, strains carrying such mutations are photosynthetically competent, as is a RC-only Puf X-minus strain (McGlynn et al. 1994). (C) A strain which is not impaired in LH1 assembly and in which PufX is absent will be non-photosynthetic,as a consequence of blocking the Qn site. In this case, a small increase in the ratio of LH1 Bchls a:RC is observed in comparison with the model in (A) (McGlynn et al. 1994, 1996; Barz et al. 1995a).
by LH1 how can this happen? In some species such as Rb. sphaeroides there is a gene encoding a protein called Puf X which m a y function to alleviate this problem.
Puf X: An essential component of the PSU Several studies have identified Puf X as a component of the PSU. Its importance can be gauged by showing that strains o f Rb. sphaeroides lacking Puf X are incapable of photosynthetic growth. Following early observations of Farchaus and co-workers (Farchaus et al. 1990, 1992) using reaction center only and reaction center-LH1 strains, M c G l y n n et al. (1994) were able to show that the loss o f Puf X was deleterious only when the LH1 was present. In a further development of this approach, strains were constructed in which the L H I : reaction center 'core' size was genetically modified ( M c G l y n n et al. 1996). This manipulation resulted in a progressive truncation o f the C-terminal region of the LH1 c~ apoprotein. It was shown that if the 'core' size was reduced below 21 Bchls a : reaction center, the absence o f P u f X did not impair photosynthetic growth. In addition, for any given 'core' size, the loss of P u f X resulted in an increase in 'core' size of 1-2 Bchls a per reaction center. Finally the 'core' size was determined to be 25.7 4- 3.2 Bchls a in the presence of P u f X . The consequences o f these investigations are represented in
Figure 3, which depicts a reaction center encircled by LH1 apoproteins. The data indicate that Puf X could be part of the LH1 ring, and that it occupies perhaps 10-20% of the circumference of this antenna. In doing so, it allows the cytochrome bCl complex access to the QB site of the reaction center, as suggested by Lilburn et al. (1992). This becomes unnecessary when LH1 is either absent, or reduced in size. Recent work (Barz et al. 1995a, b) supports this, by demonstrating an essential role for Puf X in allowing access of ubiquinone to the QB site.
Energy transfer within the PSU When light is absorbed by a Bchl a molecule the lifetime of the first excited singlet state is of the order of a few nanoseconds. Therefore, energy transfer from the antenna system to the reaction center must take place well within this time or the absorbed energy will be lost in non-productive processes. The direction of energy transfer within the PSU is not random, but is guided by the energy gradient going from B800 to B850 to B875 and then into the RC. These energy transfer steps have now been resolved in the picosecond time domain using ultrafast laser spectroscopy (see Sunds t r r m and van Grondelle 1995 for a recent review). Taking Rb. sphaeroides as an example since it has been the most extensively studied system, energy transfer from B800 to B850 takes place in 0.7 ps. The energy is then extremely rapidly delocalized among the B850 molecules on the 5 0 - 1 5 0 fs timescale. Subsequent transfer between complexes involves the energy hopping from B850 in LH2, to B875 i n L H I , which takes 2 - 4 ps (Hess et al. 1995). Again, once the energy reaches the circular array of B875 molecules, it is very rapidly delocalized. Finally the energy is transferred from B875 to the RC in 20--40 ps. This last step is the slowest in the energy transfer pathway. The reason for this is suggested in the model shown in Figure 2b where the longest distance over which the energy transfer occurs (and hence the slowest transfer step) is from the B875 ring of Bchl a molecules to the RC special pair. Energy transfer within the PSU used to be modelled in terms o f 'random hopping' o f excitons between quasi-regular arrays o f chlorophylls (e.g. Robinson 1967). Clearly this is not a good physical description of the way in which the purple bacterial PSU functions. This system is one of interacting rings. The rings of B850 or B875 Bchls a act rather like 'storage rings', with the excited singlet state effective-
61 ly rapidly delocalized around the circumference. This makes the energy available for transfer from any part of a ring to any part of its neighbor, as long as they are close enough to each other. This appears to be one of the major reasons why the purple bacterial PSU is so efficient. The time resolved measurements on whole membrane systems, when compared to those carried out on the isolated complexes, show that there is only enough time for very few inter-ring 'hops'.
Regulation of the expression of the PSU In several species, e.g. Rb. capsulatus, Rb. sphaeroides and R. rubrum, all the genes encoding the reaction center polypeptides and the antenna apoproteins have been cloned and sequenced (reviewed by Williams and Taguchi 1995). The early studies of Cohen-Bazire et al. (1957) showed that light and oxygen exert a strong influence on the composition of the PSU. Nearly 40 years on, the molecular genetics of PSU formation continue to be studied in great detail. Our current knowledge of the molecular mechanism by which their expression is regulated has not progressed to a complete understanding of the mechanisms of environmental control. (This topic has recently been excellently reviewed by Bauer 1995.) However, this regulation appears to be similar to the classical two component sensory transduction cascade systems seen in other prokaryotes. One example of this is the regulation of light-harvesting and reaction center gene expression in response to oxygen tension by Reg A and Reg B. Reg B, which is believed to be a membrane spanning sensor kinase, appears to sense changes in the oxygen environment. In conditions of low oxygen tension, Reg B autophosphorylates itself before catalyzing the transfer of this phosphate onto Reg A, a cytosolic response regulator. This activated form of Reg A is then proposed to phosphorylate DNA binding proteins, which in turn control the induction of the light-harvesting and reaction center structural genes. The greatest progress has been made in Rb. capsulatus and Rb. sphaeroides, species in which the PSU consists of LH2, LH1 and the reaction center. In some species, however, the situation is much more complicated. In Rps. acidophila or Rps. palustris, for example, there is a multi-gene family encoding for the LH2 apoproteins, some of which in Rps. acidophila appear to be 'silent' (Tadros et al. 1993; Gardiner et al. 1992). There is at present very little detailed information on the significance of this or how they are regulated.
The genes encoding components of the PSU hold an extrinsic interest to those engaged in gene regulation studies. Aside from this, the availability of the genes for the reaction center and antenna apoproteins has opened the door to using site-directed mutagenesis to probe their structure/function relations (for example see Hunter 1995; Woodbury and Allen 1995). There is now extensive literature on site-directed mutants of the bacterial reaction center (reviewed in Woodbury and Allen 1995), which followed the elucidation of the three-dimensional structure of this complex (Deisenhofer et al. 1985). The mutagenesis of LH complexes has preceded the structural determinations (for example, Bylina and Youvan 1988; Babst et al. 1991; Fowler et al. 1992; Olsen et al. 1994) but it is now expected to receive fresh impetus from the availability of structural information on the LH1 and LH2 complexes.
Conclusions Now, with the discovery of the 'ring-like' structures for the purple bacterial antenna complexes, the underlying architecture is apparent for all of the components of the PSU. In most cases these structures are consistent with spectroscopic data on the mechanisms and kinetics of light capture and transfer. The way is now open for significant advances to be made in understanding not only the detailed molecular mechanisms of energy transfer, but also the role that the proteins play in determining the characteristics of light-harvesting and energy trapping. The model presented in Figure 2 focuses attention on the need to investigate the way the various components of the PSU actually interact within the intact photosynthetic membrane in vivo.
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