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Light-harvesting mechanisms in purple photosynthetic bacteria
Light-harvesting mechanisms in purple photosynthetic bacteria Neil W Isaacs, Richard J Cogdell, Andrew A Freer and Stephen M Prince University of Glasgow, Glasgow, UK The processes by which photosynthetic bacteria capture light and transfer the energy to the reaction centre continue to be studied using an array of methodologies, both physical and biological. With the publication this year of the crystal structure of the LH2 complex from Rhodopseudomonasacidophila and the projection structure of the LH1 complex from Rhodospirillum rubrum, structural models now exist for all the components in the bacterial photosynthetic apparatus. Current Opinion in Structural Biology 1995, 5:794-797 Introduction
Photosynthesis begins with the absorption of light. In purple bacteria this process is performed by light-harvesting complexes which are intricate assemblies of low molecular weight proteins and piglnents (bacteriochlorophyll and carotenoid). Most purple bacteria have two types of light-harvesting complexes, designated LH1 and LH2. LH1 complexes are closely associated with the reaction centre and form a so-called 'core' complex. LH2 complexes are peripheral to this core. When light is absorbed by the peripheral antenna LH2 complexes the energy is transferred to LH1, which then passes it on to the reaction centre (P,,C), in which a redox reaction causes charge separation across the membrane. This system has been studied extensively using biochemistry, molecular biology and spectroscopy techniques. The energy transfer events which take place in this process have been excellently reviewed by van Grondelle et al. [1"]. Until recently, our understanding of the processes by which light energy is captured and transferred to the reaction centre has been limited by a lack of information about the structures of these antenna complexes. In the past year this situation has changed dramatically with the publication of the three-dimensional crystal structure of the LH2 complex from Rps. acidophila [2"] and the low-resolution projection structure of the LH1 complex from Rhodospirillum mbmm [3"].
Structures of light-harvesting complexes The 2.5A resolution crystal structure of the LH2 antenna complex from Rps. acidophila strain 10050 revealed in detail the assembly and operation of this complex (Fig. 1) [2"]. The complex is formed from two apoproteins (an Ot unit of 53 amino acids and a ~ unit of 41 amino acids), monomeric bacteriochlorophyll a (Bchl a)
pigment molecules which absorb maximally at 800mn (B800s), exciton-coupled Bchl a molecules which absorb at 850 nm (B850s) and carotenoids (rhodopin glucoside). The a and [~ proteins each form single transmembrane helices and are arranged with nearly exact ninefold symlnetry in two concentric rings. The a apoproteins form the inner circle and the 1~ proteins form the outer one. Each protein co-ordinates a Bchl a through a highly conserved histidine residue. Eighteen Bchl a molecules are therefore sandwiched between the rings of proteins, with the plane of the pigment molecules perpendicular to the plane of the membrane, and with close contact between adjacent molecules. These 850ran absorbing pigments (B850s) form a complete overlapping ring. A further nine nlonomeric B800s are located between the outer ~ protein helices a further 16A into the membrane. The planes of these pigment molecules are parallel to the membrane plane and the central magnesium ion is co-ordinated to the formyl group of the N-terminal f - M e t residues of the 0t proteins. There is close contact between the phytol chains of both sets of Bchl a molecules. Carotenoid molecules span the complete assembly across the membrane, making contacts with both B800 and B850 molecules. A similar ring-like assembly exists in the LH2 complex from Rhodowdum su!fidophilum [4]. On the basis of data from native gels, gel filtration and ultracentrifugation, it was thought that this assembly was octameric, although the intrinsic error of the methods does not rule out the possibility of a heptamer or nonamer. Spectroscopic studies on the LH2 complex from Rhodospirillum molischiwmm ]5] show that the direction of the Qy transition dipoles of the B800 and B850 pigments are nearly in the membrane plane and that the orientation of the lycoprene carotenoid is predolninantly perpendicular to the naembrane plane. This is a similar arrangement to that found in the crystal structure of the Rps. acidophila
Abbreviations LH--light-harvesting complex; RC--reaction centre. 794
© Current Biology Lid ISSN 0959-440X
Light-harvesting mechanisms in purple photosynthetic bacteria Isaacs et al. Kinetic studies
Fig. 1. A schematic representation (produced by MOLSCRIPT [22]) of the LH2 complex structure viewed onto the membrane surface. The proteins are represented by helical ribbons, the Bchl a chromophores are shown as ball-and-stick models and the carotenoids as stick models.
LH2 complex [2"°]. Equilibrium sedimentation and spectroscopic data have been interpreted as indicating an octameric state for the Rs. molischianum LH2 complex also. Although it is now apparent that LH2 complexes have a ring-like structure, the factors deternfining the ring size are still unknown. The low-resolution structure of the LH1 complex from RhMospirillim rubrunl [3°] reveals a similar ring-like structure but in this case it consists of 16 0t and proteins with associated pigments. The dimensions of this ring (116 fii outer diameter with a 68 fii central hole) are sufficient to incorporate a reaction centre within the ring. The structural elements of light capture and transfer can now be modelled as a close packing of ring-like structures, with LH2 complexes surrounding the LH1/1KC 'core complex'. With this arrangement, a 'special pair' of Bchl a molecules in the reaction centre is in the same plane of the membrane as are the rings o f B c h l a molecules in the LH1 and surrounding LH2 complexes, providing an optimal geometrical arrangement for energy transfer from LH2 to LH1 and on to RC. A review o f light-harvesting complexes [6] shows that good models had been devised for the folding of the individual protomer units (ct and [3 proteins, Bchl a molecules and carotenoids). That a circular arrangement o f proteins in the complex was not originally proposed can probably be attributed to both the inaccurate estimates o f the size o f the oligomeric state and a failure to realize the greater importance o f pigment-pigment interactions compared with protein-protein ones.
When light is absorbed by a Bchl a molecule, the lifetime of the excited electronic singlet state is of the order of 1 ns. The transfer of this energy to the reaction centre has to take place within this tmlefran~e. The direction of the energy transfer is driven by an energy gradient. Light is absorbed at high energy by LH2 B800 pigments. The energy is then transferred to LH2 B850, then to LH1 B875 pigments and finally to the reaction centre 'special pair', absorbing at 865-870nm. The transfer from B800 to B850 takes 0.7ps [1°°]. The energy is then rapidly distributed among the B850 molecules taking 200-300fs. Energy transfer from LH2 to LH1 then occurs within 1-Sps and from LH1 to 1KC in an estimated 20--40ps. There have been a number of papers (described below) investigating the composition and the kinetics of energy transfer between the pigment molecules. Davis and colleagues [7] have shown that LH1 complexes can be reconstituted from isolated 0t and proteins, Bchl a and carotenoid. This makes possible filrther functional analysis of the properties of the individual components. Pump-probe laser spectroscopy experiments using 40 g light pulses on both LH1 and LH2 complexes from Rhodobacter sphaeroides show that spectral equilibrium at 850nm (LH2) and at 875nm (LH1) occurs within 140fs [8]. This equilibrium is seen as a time-dependent migration of the excited-state bleaching towards the red end of the absorption band. Also, during the lifetime of these excited states, some ultrafast oscillations occur that may reflect more subtle relaxation dynanfics of both the pigments and their environments [9]. Introducing a nmtation at M210 in the reaction centre reduces the rate of primary electron transfer. This does not, however, significantly affect the overall rate of trapping when the light energy is absorbed by the antenna system, identifying the transfer from LH1 to IKC as the rate-limiting step m the light-harvesting process [10]. The absorption spectra of the B850s in LH2 and the Bchl a molecules absorbing at 873nm (1020nm in the Bchl b-containing organisms [B1020s]) in LH1 is relatively broad. There has been considerable discussion of the size o f these bandwidths. Monshouwer et al. [11] have presented evidence from low-temperature absorption and site-selected fluorescence data that indicates that in Rps. viridis the B1020 LH1 absorbing band is inhomogeneously broadened. In other words, there is heterogeneity in the site energies of the individual Bchl molecules which contribute to these absorptions. More systematic 'hole burning' experiments, where these absorption bands are subjected to illumination with high intensity, very narrow band laser light, are needed to resolve this issue. The migration of excitation energy to reaction centres, and the trapping o f this energy therein, has been measured on many occasions. The interpretation of these measurements depends on the assumptions that are made regarding the spatial organization of the pigments. Temperature-dependency studies show
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Catalysis and regulation a spectral inhomogeneity at low temperature (4K) with a transition at 77K. Somsen et al. [12] explain these data assuming that all Bchl molecules in the LH1 complex do not have identical environments. A sinfilar temperature-dependent spectral inhomogeneity is invoked to explain the observed kinetics. In certain purple bacteria, a change in growth conditions can promote the synthesis of light-harvesting complexes that absorb at different wavelengths, for example 8 0 0 - 8 5 0 n m or 800--820nm in Rps. addophila strain 7750. This change in absorption is modulated by the apoprotein; a blue shift in the 850nm band can be induced by mutating highly conserved tyrosine residues at positions 44 and 45 in the 0t unit in Rb. sphaeroides. Evidence from fluorescence transfer resonance Raman spectroscopy suggests that a nmtation in the 0t subunit at Tyr44 to phenylalanine breaks a hydrogen bond to a 2-acetyl carbonyl group of a nearby B850 molecule [13]. This hydrogen bond is observed in the crystal structure of the Rps. acidophila LH2 complex. The data on the ct unit Tyr45--+Phe mutant is more equivocal, suggesting a disruption of the hydrogen bonding pattern for one of the 2-acetyl carbonyl groups, a weakening o f the other 2-acetyl carbonyl interaction and an enhancement of a 9-keto carbonyl interaction. These two nmtations have been used to study the rate of energy transfer as a function o f increasing spectral overlap o f the enfission of the donor-excited Bchl molecule with the absorption spectrum of the acceptor Bchl [14°]. As the spectral overlap between the wavelength absorbed by the B800s and the variable (the B850) bands increases, the rate of energy transfer also increases. A comparison o f the measured rates with those calculated by the F/Srster expression shows qualitative agreement. Sinlilar experiments that change the hydrogen bonding pattern of the Bchl molecules in the LH1 complex of Rb. sphaeroides have been carried out [15]. All of these papers were published before the crystal structure of the Rps. acidophila B800-850 LH2 complex was deternfined. Interpretation of the spectral data often depended on assumptions regarding the structure of the pigments and apoprotein. With the detailed structural information now available, these data might be further analyzed to provide more information. Crielaard et al. [16] and Visschers et al. [17] have described the result o f site-directed changes at [3 His21 and [~ Arg29 that affect the spectral properties of the B800s in LH2 from Rb. sphaeroides. A [~ His21-+Ser mutation prevents incorporation of Bchl 800 pigments into the complex. Changing [3 Arg29 to glutamine broadens the 800nm absorption band. Interestingly, both o f these mutant LH2s still bind carotenoid and still show the usual electrochromic carotenoid bandshift, even though its magnitude is greatly reduced. It will be interesting to re-analyze the structural consequences of these mutations with the recently elucidated structure of LH2 in mind. In the pufoperon, the pufX gene is next to the structural gene for the tLC 'M' subunit. McGlynn and co-workers
[18] have shown that photosynthetic cells that lack LHI do not require the presence of the correct pl!/X gene product. This interesting observation suggests that the pufX gene product may function by creating a gap in the LH1 ring to allow the passage of ubiquinone to and from the ILC as required in cyclic electron transport.
Carotenoids Carotenoid molecules have two important roles in lightharvesting complexes. Firstly, they capture light in the visible region o f the spectrum (where the Bchl molecules do not absorb) and transfer it to the Bchl molecules (a process known as accessory light-harvesting). Secondly, they protect the complex from damage induced by singlet oxygen species. Carotenoid molecules have two excited singlet states: S 1 and S2. It is not clear whether one or both of these states is the energy donor for light harvesting. The mechanism of transfer is also unclear. Nagae et a[. [19] have presented a theoretical model for this singlet-singlet energy transfer and suggest $2/S 1 mixing may well be important in this process. One powerful way of probing carotenoid function in the light-harvesting process is to change the carotenoid composition of the system while keeping everything else the same. Hunter et al. [20] have described a novel genetic method of achieving this, which involves expressing genes for carotenoid biosynthesis from Em,inia in Rb. sphaeroides. These new carotenoids are then functionally inserted into the antenna complexes in Rb. sphaeroides. This is a way of investigating the functional consequences of introducing a wider range of carotenoids into the antenna complexes than would normally be possible. The presence of carotenoids is also essential for the assembly of LH2 complexes. Lang and Hunter [21] showed that although the LH2 ot and [~ apoproteins are synthesized in the absence of bound carotenoids, they are unstable and are rapidly turned over under these conditions.
Conclusions With the discovery of the ring-like structure for lightharvesting complexes in purple bacteria, the underlying architecture of light harvesting is now apparent. In most cases these structures support the spectroscopic data available on the mechanisms and kinetics of light capture and transfer. In other cases a re-analysis of the data will be valuable. Site-directed nmtagenesis studies have already identified some key residues in the apoproteins and now that with the structure is available this work will have a sharper focus. The way is now open for significant advances to be made in understanding not only the mechanics of energy transfer in more detail, but also the role that the proteins play in determining the characteristics of light harvesting.
L i g h t - h a r v e s t i n g mechanisms in p u r p l e p h o t o s y n t h e t i c b a c t e r i a Isaacs et al.
Acknowledgements This work has been supported by the Membrane Initiative of the Biotechnology and Biological Sciences Research Council.
site-directed fluorescence of the light-harvesting antenna of Rhodopseudomonas viridis. Evidence for heterogeneity. Biochim Biophys Acta 1995, 1229:373-380.
12.
Somsen OJG, Van Mourik F, Van Grondelle R, Valkunas L: Energy migration and trapping in a spectrally and spatially inhomogeneous light-harvesting antenna. 8iophys J 1994, 66:1580-1.596.
13.
Fowler GJS, Sockalingum GD, Robert B, Hunter CN: Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at ct-Tyr-44 and o:-Tyr-45. Biochem I 1994, 299:695-700.
References Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. "•
Van Grondelle R, Dekker JP, Gillbro T, Sundstrom V: Energy transfer and trapping in photosynthesis. Biochim Biophys Acta 1994, 1187:1-65. A clear and comprehensive review of the fundamental processes involved in bolh energy transfer and primary charge separation in photosynthesis.
14. •
Hess S, Visscher KJ, Pullerits 1-, Sundstrom V, Fowler GJS, Hunter CN: Enhanced rates of subpicosecond energy transfer in blue-shifted light harvesting LH2 mutants of Rhodobacter sphaeroides. Biochem 1994, 33:8300-8305. An elegant use of site-directed mutagenesis to show the effect of the extent of spectral overlap on the F6rster rate of energy transfer.
15.
Olsen JD, Sockalingum GD, Robert 8, Hunter CN: Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides. Proc Nag Acad Sci USA 1994, 91:7124-7128.
16.
Crielaard W, Visschers RW, Fowler GJS, Van Grondelle R, Hellingwerf KJ, Hunter CN: Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutanls. I. Mutagenesis, effects on binding, function and eleclrochromic behaviour of its carolenoids. Biochim Biophys Acta 1994, 1183:473-482.
17.
Montoya G, Cyrklaff M, Sinning I: Two-dimensional crystallization and preliminary structure analysis of light harvesting II (8800-850) complex from the purple bacterium Rhodovulum sulfidophilum. J Mol Biol 1995, 250:1-10.
Visschers RW, Crielaard W, Fowler GJS, Hunter CN, Van Grondelle R: Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. II. A low-temperature spectroscopy study of structural aspects of the pigment-protein conformation. Biochim Biophys Acta 1994, 1183:483-490.
18.
Visschers RW, Germeroth L, Michel H, Monshouwer R, Van Grondelle R: Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum. Biochim Biophys Acta 199.5, 1230:147-154.
McGlynn P, Hunter CN, Jones MR: The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system. FEBS Lett 1994, 349:349-353.
19.
Nagae H, Kakitani T, Katoh T, Mimuro M: Calculalion of the excitation transfer matrix elements between the S2 or S1 state of bacteriochlorophyll. J Chem Phys 1993, 98:8012-8023.
20.
Hunter CN, Hundle BS, Hearst JE, Lang HI:', Gardiner AT, Takaichi S, Cogdell RJ: Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides. J Bacteriol 1994, 176:3692-3697.
2, •"
McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM, Papiz MZ, Cogdell RJ, Isaacs NW: Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 1995, 374:517-521. The determination of this three-dimensional structure clearly reveals the underlying architecture of light harvesting in bacteria. Relationships among the pigments and between pigments and protein now provide a structural basis for understanding the mechanism and kinetics of energy transfer. 3.
Karrasch S, Bullough PA, Ghosh R: 8.5A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 1995, 14:631-638. This paper demonstrates that the LH1 complex is circular and contains a central hole large enough to accommodate the reaction centre, and gives the basic structures of all the components of bacterial photosynthetic units. •
4.
5.
6.
Olsen JD, Hunter CN: Protein structure modelling of the bacterial light-harvesting complex. Photochem Photobiol 1994, 60:521-535.
7.
Davis CM, Bustamante PL, Loach PA: Reconslitution of the bacterial core light-harvesting complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with isolated c~- and ]3-polypeptides, bacteriochlorophyll a, and carotenoid. J Biol Chem 1995, 270:5793-5804.
21.
Pullerits T, Chachisvilis M, Jones MR, Hunter CN, Sundstrom V: Exciton dynamics in the light-harvesting complexes of Rhodobacter sphaeroides. Chem Phys Lett 1994, 224:355-365.
Lang HP, Hunter CN: The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides. Biochem J 1994, 298:197-205.
22.
Kraulis PJ: MOLSCRIPT: A program to produce both detailed and schematic plots of prolein structure. J Appl Crystallogr
8.
9.
Chachisvilis M, Pullerits T, Jones MR, Hunter CN, Sundstrom V: Vibrational dynamics in the light-harvesting complexes of the photosynthetic bacterium Rhodobacter sphaeroides. Chem Phys tett 1994, 224:345-351.
10.
Beekman LMP, Van Mourik F, Jones MR, Visser HM, Hunter CN, Van Grondelle R: Trapping kinetics in mutants of the photosynthetic purple bacterium Rhodobacter sphaeroides: influence of the charge separation rate and consequences for the rate-limiting step in the light-harvesting process. Biochemistry 1994, 33:3143-3147.
11.
Monshouwer R, Visschers RW, Van Mourik F, Freiberg A, Van Grondelle R: Low-temperature absorption and
1991, 24:946-950. N W lsaacs, AA Freer and SM Prince, ])epartment of Chemistry, University of Glasgow, Glasgow G 12 8QQ, UK. N W lsaacs e-mail: [email protected] AA Freer e-mail: [email protected] SM Prince e-mail: [email protected] RJ Cogdell, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. E-mail: gbta 18@udc f.gla.ac.uk
797
Photosynthesis begins with the absorption of light. In purple bacteria this process is performed by light-harvesting complexes which are intricate assemblies of low molecular weight proteins and piglnents (bacteriochlorophyll and carotenoid). Most purple bacteria have two types of light-harvesting complexes, designated LH1 and LH2. LH1 complexes are closely associated with the reaction centre and form a so-called 'core' complex. LH2 complexes are peripheral to this core. When light is absorbed by the peripheral antenna LH2 complexes the energy is transferred to LH1, which then passes it on to the reaction centre (P,,C), in which a redox reaction causes charge separation across the membrane. This system has been studied extensively using biochemistry, molecular biology and spectroscopy techniques. The energy transfer events which take place in this process have been excellently reviewed by van Grondelle et al. [1"]. Until recently, our understanding of the processes by which light energy is captured and transferred to the reaction centre has been limited by a lack of information about the structures of these antenna complexes. In the past year this situation has changed dramatically with the publication of the three-dimensional crystal structure of the LH2 complex from Rps. acidophila [2"] and the low-resolution projection structure of the LH1 complex from Rhodospirillum mbmm [3"].
Structures of light-harvesting complexes The 2.5A resolution crystal structure of the LH2 antenna complex from Rps. acidophila strain 10050 revealed in detail the assembly and operation of this complex (Fig. 1) [2"]. The complex is formed from two apoproteins (an Ot unit of 53 amino acids and a ~ unit of 41 amino acids), monomeric bacteriochlorophyll a (Bchl a)
pigment molecules which absorb maximally at 800mn (B800s), exciton-coupled Bchl a molecules which absorb at 850 nm (B850s) and carotenoids (rhodopin glucoside). The a and [~ proteins each form single transmembrane helices and are arranged with nearly exact ninefold symlnetry in two concentric rings. The a apoproteins form the inner circle and the 1~ proteins form the outer one. Each protein co-ordinates a Bchl a through a highly conserved histidine residue. Eighteen Bchl a molecules are therefore sandwiched between the rings of proteins, with the plane of the pigment molecules perpendicular to the plane of the membrane, and with close contact between adjacent molecules. These 850ran absorbing pigments (B850s) form a complete overlapping ring. A further nine nlonomeric B800s are located between the outer ~ protein helices a further 16A into the membrane. The planes of these pigment molecules are parallel to the membrane plane and the central magnesium ion is co-ordinated to the formyl group of the N-terminal f - M e t residues of the 0t proteins. There is close contact between the phytol chains of both sets of Bchl a molecules. Carotenoid molecules span the complete assembly across the membrane, making contacts with both B800 and B850 molecules. A similar ring-like assembly exists in the LH2 complex from Rhodowdum su!fidophilum [4]. On the basis of data from native gels, gel filtration and ultracentrifugation, it was thought that this assembly was octameric, although the intrinsic error of the methods does not rule out the possibility of a heptamer or nonamer. Spectroscopic studies on the LH2 complex from Rhodospirillum molischiwmm ]5] show that the direction of the Qy transition dipoles of the B800 and B850 pigments are nearly in the membrane plane and that the orientation of the lycoprene carotenoid is predolninantly perpendicular to the naembrane plane. This is a similar arrangement to that found in the crystal structure of the Rps. acidophila
Abbreviations LH--light-harvesting complex; RC--reaction centre. 794
© Current Biology Lid ISSN 0959-440X
Light-harvesting mechanisms in purple photosynthetic bacteria Isaacs et al. Kinetic studies
Fig. 1. A schematic representation (produced by MOLSCRIPT [22]) of the LH2 complex structure viewed onto the membrane surface. The proteins are represented by helical ribbons, the Bchl a chromophores are shown as ball-and-stick models and the carotenoids as stick models.
LH2 complex [2"°]. Equilibrium sedimentation and spectroscopic data have been interpreted as indicating an octameric state for the Rs. molischianum LH2 complex also. Although it is now apparent that LH2 complexes have a ring-like structure, the factors deternfining the ring size are still unknown. The low-resolution structure of the LH1 complex from RhMospirillim rubrunl [3°] reveals a similar ring-like structure but in this case it consists of 16 0t and proteins with associated pigments. The dimensions of this ring (116 fii outer diameter with a 68 fii central hole) are sufficient to incorporate a reaction centre within the ring. The structural elements of light capture and transfer can now be modelled as a close packing of ring-like structures, with LH2 complexes surrounding the LH1/1KC 'core complex'. With this arrangement, a 'special pair' of Bchl a molecules in the reaction centre is in the same plane of the membrane as are the rings o f B c h l a molecules in the LH1 and surrounding LH2 complexes, providing an optimal geometrical arrangement for energy transfer from LH2 to LH1 and on to RC. A review o f light-harvesting complexes [6] shows that good models had been devised for the folding of the individual protomer units (ct and [3 proteins, Bchl a molecules and carotenoids). That a circular arrangement o f proteins in the complex was not originally proposed can probably be attributed to both the inaccurate estimates o f the size o f the oligomeric state and a failure to realize the greater importance o f pigment-pigment interactions compared with protein-protein ones.
When light is absorbed by a Bchl a molecule, the lifetime of the excited electronic singlet state is of the order of 1 ns. The transfer of this energy to the reaction centre has to take place within this tmlefran~e. The direction of the energy transfer is driven by an energy gradient. Light is absorbed at high energy by LH2 B800 pigments. The energy is then transferred to LH2 B850, then to LH1 B875 pigments and finally to the reaction centre 'special pair', absorbing at 865-870nm. The transfer from B800 to B850 takes 0.7ps [1°°]. The energy is then rapidly distributed among the B850 molecules taking 200-300fs. Energy transfer from LH2 to LH1 then occurs within 1-Sps and from LH1 to 1KC in an estimated 20--40ps. There have been a number of papers (described below) investigating the composition and the kinetics of energy transfer between the pigment molecules. Davis and colleagues [7] have shown that LH1 complexes can be reconstituted from isolated 0t and proteins, Bchl a and carotenoid. This makes possible filrther functional analysis of the properties of the individual components. Pump-probe laser spectroscopy experiments using 40 g light pulses on both LH1 and LH2 complexes from Rhodobacter sphaeroides show that spectral equilibrium at 850nm (LH2) and at 875nm (LH1) occurs within 140fs [8]. This equilibrium is seen as a time-dependent migration of the excited-state bleaching towards the red end of the absorption band. Also, during the lifetime of these excited states, some ultrafast oscillations occur that may reflect more subtle relaxation dynanfics of both the pigments and their environments [9]. Introducing a nmtation at M210 in the reaction centre reduces the rate of primary electron transfer. This does not, however, significantly affect the overall rate of trapping when the light energy is absorbed by the antenna system, identifying the transfer from LH1 to IKC as the rate-limiting step m the light-harvesting process [10]. The absorption spectra of the B850s in LH2 and the Bchl a molecules absorbing at 873nm (1020nm in the Bchl b-containing organisms [B1020s]) in LH1 is relatively broad. There has been considerable discussion of the size o f these bandwidths. Monshouwer et al. [11] have presented evidence from low-temperature absorption and site-selected fluorescence data that indicates that in Rps. viridis the B1020 LH1 absorbing band is inhomogeneously broadened. In other words, there is heterogeneity in the site energies of the individual Bchl molecules which contribute to these absorptions. More systematic 'hole burning' experiments, where these absorption bands are subjected to illumination with high intensity, very narrow band laser light, are needed to resolve this issue. The migration of excitation energy to reaction centres, and the trapping o f this energy therein, has been measured on many occasions. The interpretation of these measurements depends on the assumptions that are made regarding the spatial organization of the pigments. Temperature-dependency studies show
795
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Catalysis and regulation a spectral inhomogeneity at low temperature (4K) with a transition at 77K. Somsen et al. [12] explain these data assuming that all Bchl molecules in the LH1 complex do not have identical environments. A sinfilar temperature-dependent spectral inhomogeneity is invoked to explain the observed kinetics. In certain purple bacteria, a change in growth conditions can promote the synthesis of light-harvesting complexes that absorb at different wavelengths, for example 8 0 0 - 8 5 0 n m or 800--820nm in Rps. addophila strain 7750. This change in absorption is modulated by the apoprotein; a blue shift in the 850nm band can be induced by mutating highly conserved tyrosine residues at positions 44 and 45 in the 0t unit in Rb. sphaeroides. Evidence from fluorescence transfer resonance Raman spectroscopy suggests that a nmtation in the 0t subunit at Tyr44 to phenylalanine breaks a hydrogen bond to a 2-acetyl carbonyl group of a nearby B850 molecule [13]. This hydrogen bond is observed in the crystal structure of the Rps. acidophila LH2 complex. The data on the ct unit Tyr45--+Phe mutant is more equivocal, suggesting a disruption of the hydrogen bonding pattern for one of the 2-acetyl carbonyl groups, a weakening o f the other 2-acetyl carbonyl interaction and an enhancement of a 9-keto carbonyl interaction. These two nmtations have been used to study the rate of energy transfer as a function o f increasing spectral overlap o f the enfission of the donor-excited Bchl molecule with the absorption spectrum of the acceptor Bchl [14°]. As the spectral overlap between the wavelength absorbed by the B800s and the variable (the B850) bands increases, the rate of energy transfer also increases. A comparison o f the measured rates with those calculated by the F/Srster expression shows qualitative agreement. Sinlilar experiments that change the hydrogen bonding pattern of the Bchl molecules in the LH1 complex of Rb. sphaeroides have been carried out [15]. All of these papers were published before the crystal structure of the Rps. acidophila B800-850 LH2 complex was deternfined. Interpretation of the spectral data often depended on assumptions regarding the structure of the pigments and apoprotein. With the detailed structural information now available, these data might be further analyzed to provide more information. Crielaard et al. [16] and Visschers et al. [17] have described the result o f site-directed changes at [3 His21 and [~ Arg29 that affect the spectral properties of the B800s in LH2 from Rb. sphaeroides. A [~ His21-+Ser mutation prevents incorporation of Bchl 800 pigments into the complex. Changing [3 Arg29 to glutamine broadens the 800nm absorption band. Interestingly, both o f these mutant LH2s still bind carotenoid and still show the usual electrochromic carotenoid bandshift, even though its magnitude is greatly reduced. It will be interesting to re-analyze the structural consequences of these mutations with the recently elucidated structure of LH2 in mind. In the pufoperon, the pufX gene is next to the structural gene for the tLC 'M' subunit. McGlynn and co-workers
[18] have shown that photosynthetic cells that lack LHI do not require the presence of the correct pl!/X gene product. This interesting observation suggests that the pufX gene product may function by creating a gap in the LH1 ring to allow the passage of ubiquinone to and from the ILC as required in cyclic electron transport.
Carotenoids Carotenoid molecules have two important roles in lightharvesting complexes. Firstly, they capture light in the visible region o f the spectrum (where the Bchl molecules do not absorb) and transfer it to the Bchl molecules (a process known as accessory light-harvesting). Secondly, they protect the complex from damage induced by singlet oxygen species. Carotenoid molecules have two excited singlet states: S 1 and S2. It is not clear whether one or both of these states is the energy donor for light harvesting. The mechanism of transfer is also unclear. Nagae et a[. [19] have presented a theoretical model for this singlet-singlet energy transfer and suggest $2/S 1 mixing may well be important in this process. One powerful way of probing carotenoid function in the light-harvesting process is to change the carotenoid composition of the system while keeping everything else the same. Hunter et al. [20] have described a novel genetic method of achieving this, which involves expressing genes for carotenoid biosynthesis from Em,inia in Rb. sphaeroides. These new carotenoids are then functionally inserted into the antenna complexes in Rb. sphaeroides. This is a way of investigating the functional consequences of introducing a wider range of carotenoids into the antenna complexes than would normally be possible. The presence of carotenoids is also essential for the assembly of LH2 complexes. Lang and Hunter [21] showed that although the LH2 ot and [~ apoproteins are synthesized in the absence of bound carotenoids, they are unstable and are rapidly turned over under these conditions.
Conclusions With the discovery of the ring-like structure for lightharvesting complexes in purple bacteria, the underlying architecture of light harvesting is now apparent. In most cases these structures support the spectroscopic data available on the mechanisms and kinetics of light capture and transfer. In other cases a re-analysis of the data will be valuable. Site-directed nmtagenesis studies have already identified some key residues in the apoproteins and now that with the structure is available this work will have a sharper focus. The way is now open for significant advances to be made in understanding not only the mechanics of energy transfer in more detail, but also the role that the proteins play in determining the characteristics of light harvesting.
L i g h t - h a r v e s t i n g mechanisms in p u r p l e p h o t o s y n t h e t i c b a c t e r i a Isaacs et al.
Acknowledgements This work has been supported by the Membrane Initiative of the Biotechnology and Biological Sciences Research Council.
site-directed fluorescence of the light-harvesting antenna of Rhodopseudomonas viridis. Evidence for heterogeneity. Biochim Biophys Acta 1995, 1229:373-380.
12.
Somsen OJG, Van Mourik F, Van Grondelle R, Valkunas L: Energy migration and trapping in a spectrally and spatially inhomogeneous light-harvesting antenna. 8iophys J 1994, 66:1580-1.596.
13.
Fowler GJS, Sockalingum GD, Robert B, Hunter CN: Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at ct-Tyr-44 and o:-Tyr-45. Biochem I 1994, 299:695-700.
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1991, 24:946-950. N W lsaacs, AA Freer and SM Prince, ])epartment of Chemistry, University of Glasgow, Glasgow G 12 8QQ, UK. N W lsaacs e-mail: [email protected] AA Freer e-mail: [email protected] SM Prince e-mail: [email protected] RJ Cogdell, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. E-mail: gbta 18@udc f.gla.ac.uk
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