Purple bacterial antenna complexes

467

Purple bacterial antenna complexes Paul K Fyfe and Richard J Cogdell The purple bacterial antenna complexes continue to provide an area of very active and fertile research. During the past year, exciting advances have been made both on their structure and function, and on how their synthesis is regulated by various environmental factors.

Addresses Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK; e-mail: [email protected] Current Opinion in Structural Biology 1996, 6:46?-4?2 © Current Biology Ltd ISSN 0959-440X

Abbreviations BChl bacteriochlorophyll CD circular dichroism FT Fourier transform LDAO lauryldimethylamineoxide LH light-harvesting RC reactioncentre

Introduction

Photosynthesis begins with the absorption of light by pigment-protein complexes, known as light-harvesting ( L H ) complexes. Most purple bacteria have two types of light-harvesting complexes, designated LH1 and LH2. T h e LH1 complexes are found in close association with the reaction centre (Re), forming the so-called 'core' complex, whereas L H 2 complexes are arranged around the periphery, of the L H 1 - R C 'core' complex in amounts that vary depending on the light intensity. T h e s e two L H complexes collect and then rapidly transfer the absorbed light energy to the RCs, where the primary redox reaction produces charge separation across the membrane. Therefore these reactions are responsible for the initial conversion of light energy into a useful chemical form. T h e atomic structure of the photosynthetic RC has been known for over a decade [1]. During the past year, the first high-resolution crystal structure of a purple bacterial L H complex (LH2 from Rhodopseudomonas acidophi/a strain 10050) has been reported [2°°]. An 8.5 ~ resolution projection map of the LH1 complex from RhodospiHllum rubrum [3"] has also recently been described. T h e s e two structures have had a dramatic effect on our understanding of the structure and function of the purple bacterial photosynthetic unit. Light-harvesting

complexes

In the past, the lack of structural information on the purple bacterial L H complexes has limited our understanding of the processes by which light energy is captured and transferred to the RCs. This situation has now completely

changed. T h e three-dimensional crystal structure of the LH2(B800-850) complex from Rps. addophila 10050 [2"'] has revealed an elegant nonameric ring structure. Nine (xl3-apoprotein dimers pack together, forming a double ring of protein around a hollow cylinder with an internal radius o f - 1 8 ~ and an external radius o f - 3 4 A . Held within the protein rings are the pigments, three bacteriochlorophyll (BChl) molecules and two carotenoid molecules per (x[3 pair. T h e BChl molecules are organised into two sets. Eighteen BChl molecules, sandwiched between the (x- and [3-rings, absorb at 850 nm. T h e bacteriochlorin rings of these BChl molecules lie perpendicular to the plane of the membrane, parallel to the transmembrane (x-helices, and form a closely interacting molecular aggregate. A further nine monomeric BChls, which absorb at 800 nm, lie between the outer [3-polypeptides. One surprising finding was that the central magnesium atom of these B800 pigment molecules is co-ordinated to the formyl group of the N-terminal f-methionine residue of the (x-apoprotein. T h e carotenoid pigments span the entire complex, and make close contact with the rings of both sets of BChls. Several years ago, Michel [4] described crystals of the LH2 from Rhodospiri/lum molischianum that diffracted X-rays to a resolution of 2.4~. T h e structure of this antenna complex has also very recently been determined [5"]. Previously Hu eta]. [6"] had tried to model its structure. T h e results of this modelling procedure were then compared to the structure of the L H 2 complex from Rps. acidophi/a. T h e favoured model for the Rs. mo/ischianum was an octameric ring of eight (x[3 subunits. T h e results from this modelling procedure have now been confirmed by the crystal structure. Even thot, gh the LH2 from Rs. mo/isrhianum is an octamer, its structure is very similar to that of the Rps. acidophila complex. Spectroscopic studies by Visschers eta]. [7] have confirmed that the B800 and B850 pigments in the Rs. molischianum and the Rps. acidophi/a complexes are arranged similarly. T h e L H 2 complex from Rhodovulum su/fidophilum has been investigated by two-dimensional crystallography and electron microscopy [8,9"°]. Savage et al. [9"1 calculated a 7 ~ resolution projection map of this complex and were able to show that it was also a nonameric ring structure. Interestingl'> when this projection map was compared with the Rps. acidophila data, extra density was seen at the periphe~- of the ring. It was suggested that this represented the second carotenoid molecule per pair of (x[3 apoproteins, which is only partially visualised in the Rps. acidophila c~'stal structure. T h e primary structure of the (x-polypeptidc of the Rhv. sulfidophihem complex has some unique properties [10]. This sequence contains four methionine residues, a cysteine and a carboxyl group in the C-terminal domain. No counterparts of these residues

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have been found in any other known purple bacterial LH2 complex. The LH2 from t~v. sulfidophilum shows a range of spectral forms when dialysed into low salt or upon the addition of detergents. It will be interesting to see how these structural changes reflect the unusual primary structure of the cz-apoprotein. Two-dimensional crystallography has also been employed to study the LH1 complex, with an 8.5/k projection map produced for the complex from Rs. 17thrum [3"]. The map shows a 16-subunit ring, with an outer radius of 58~ and an internal radius of 34/~.. It was calculated that the size of hole in the centre of the ring would be sufficient to contain an RC complex. Karrasch et al. [3--] also suggested from the structure that the LH1 BChl molecules are organised into a ring sandwiched between the c~- and 13-apoproteins. It is very clear that the basic structural principle of the purple bacterial LH complexes has now been revealed. They are all rings! The wealth of detailed information that now exists for certain LH complexes, has increased the interest in the investigation of the more unusual complex forms. The ability of a small group of purple bacteria, such as Rps. acidophila and Rhodospirillum palustris, to synthesise two different forms of LH2 complex is one such topic of interest. Rhodopseudomonas cryptolactis is another newly described example of this class of bacteria. Hallorcn et al. [11] have purified and been successful in obtaining crystals of both complexes, B800-820 and B800-850, from this bacterium. One of the most unusual forms of LH2 complex would appear to be that from Chromatium purpuratum [12,13]. Chromatium purpuratum has many characteristics not shared by other purple bacteria. The complex can be resolved into six subunits by SDS-PAGE, and N-terminal sequence analysis of these subunits has suggested the presence of an extra BChl-binding site located outside the transmembrane domain. The proposed existence of an extra BChl pigment is supported by pigment ratio analysis. In addition, CD and amino acid sequencing indicate the presence of 13-type secondary structure as well as the more expected or-helix. For these reasons, a three-dimensional structure for this complex will be especially interesting. It is also interesting to note that the successful three-dimensional crystallization of another LH complex from the alga Mantonie//a squamata [14"] has also been described. So far, little structural similarity has been seen between the antenna complexes in purple bacterium and those from plants and algae. If the structure of the M. squamata complex can be successfully determined, it will be intriguing to see whether these differences still remain. Spectroscopic techniques such as Fourier transform Raman have the capability to probe the details of structure within proteins. Sturgis et al. [15] have F T Raman to study a range of purple bacterial

(FT) local used LH2

complexes. T h e y identified three amino acid residues that they believe are involved in hydrogen bonding to the 9-keto group of one of the B850 BChl molecules. The investigation of the B800-850 and B800-820 complexes revealed that the two acetyl groups of the 820nm-absorbing BChl are free from hydrogen bonding. This discovery, along with previous studies of mutant bacteria which yielded the B800-820 form, suggests that hydrogen bonding in these molecules is a major factor in the tuning of the functional properties. F T resonance Raman analysis of the B800-850 complex of Rhodobacter sulphidophilus has also been carried out by Sturgis et al. [16"]. This complex shifts to a B800-830 spectral form in the presence of LDAO. Previous CD and fluorescence experiments had failed to uncover any significant changes. However, F T resonance Raman identified an alteration in the hydrogen bonding of the 2-acetyl group of at least one of the B850 BChls. Sturgis eta/. propose that the detergent causes a conformational change in the C-terminus of the ~-polypeptide, which contains two tyrosine residues believed to act as hydrogen donors to this group. The findings of the F T Raman studies on the B800-850(LH2) complex from Rps. acidophila agree satisfyingly with the three-dimensional structure.

Energy transfer Energy transfer within the antenna systems of purple bacteria has recently been excellently reviewed by Sundstr6m and van Grondelle [17] (dealing mainly with bacteriochlorophyll-bacteriochlorophyll transfers) and by Frank and Christiansen [18] (dealing mainly with carotenoid to bacteriochlorophyll energy transfer). These reviews provide a comprehensive discussion of all the kinetic information up to the beginning of 1994. In an interesting paper published before the arrival of the high-resolution structure data on LH2 from Rps. acidophi/a [2"'], Novoderezhkin and Razjivin [19"] produced a theoretical treatment of exciton dynamics in circular arrays of BChls. Their theoretical analysis begins to provide a model to explain some of the interesting inhomogeneous effects seen in photosynthetic kinetic studies (for example, see [20]). Hess eta/. [21] have studied the energy-transfer kinetics on the femtosecond time scale in LH2 complexes from Rb. sphaeroides and Rps. palustris. They showed that energy transfer from B800 to B850 occurs with a time constant of 0.7+0.05ps at room temperature, and 1.8+0.2ps at 77K. Anisotropy measurements suggested limited B800 to B800 energy transfer with a time constant of 0.3ps. These results are well explained using the structure of LH2 determined by X-ray crystallography. Some species of purple bacteria (as described above) can produce two spectrally different types of LH2, B800-850 and B800-820. Kramer et al. [22] have investigated the

Purple bacterial antenna complexas Fyfe and Gogdell

469

energy transfer in membranes from Rps. co,ptolactis and showed that the presence of B800-820 (produced when the cell was grown at the lowest light intensity), prevented back transfer of the energy from the LH1-RC 'core'. This allows the more effective focusing of energy onto the RC under 'low-light' conditions. Interestingly, they concluded that the LH2 complexes were energetically coupled to very few LH1-RC 'cores', perhaps on]y one or two.

approach complements the earlier work of Zurdo et aL [30]. These workers showed that partial depletion of the carotenoid complement from the LH2 complex from R~odobacter capsu/atus led to a range of spectral effects in the BChl absorption. These observed changes were interpreted as evidence of a conformational change in the protein associated with a loss of stability attributable to the removal of the carotenoid.

Assembly of the photosynthetic apparatus

Carotenoids have two excited singlet states--S1 and $ 2 - - w h i c h might be involved in the function ofearotenoids as accessory LH pigments. In order to determine the molecular mechanisms involved in carotenoid to BChl singlet-singlet energy transfer, it is important to know the dynamics and energy levels of the carotenoid's two excited singlet states. Chynwat and Frank [31] have recently described a method for the deduction of the S1 state energies from the dynamics of the SI states, and they have calculated S1 energies for a number of biologically important carotenoids.

The visualization of the structure of the LH2 complex has highlighted our lack of understanding of how such a beautiful and complex structure is assembled. Loach and co-workers [23-25] have been engaged in an extensive programme of experiments aimed at tackling this problem. T h e y have carried out a series of reconstitution studies into the construction of LH1 and LH1-RC core complexes, using purified constituent molecules. Meadows eta/. [23] have described the minimal polypeptide size required for the correct assembly of the LH1 complex. The use of chemical and enzymatic cleavage of purified oc- and [3-polypeptides allowed the preparation of a range of modified polypeptides. When these were used in reconstitution experiments along with BChla molecules, truncation outside the conserved core region was found to have little or no effect on formation of the complex. Further shortening into this conserved region resulted in altered complex formation and stabilit'y: Reconstitution experiments with purified and unaltered (x- and lS-polypeptides, along with BChl and carotenoids enabled Davies eta/. [24] to produce complexes with apparently native characteristics. T h e success of this reconstitution system has allowed the Loach group to incorporate carotenoids not normally found in the complex being studied. Bustamante and Loach 125] also carried out reconstitution of the LH1-RC 'core' complex. Energy-transfer measurements revealed that the reconstituted 'corcs' had normal activity.

Carotenoids The roles of carotenoids in light harvesting and photoprotection are well documented (for recent reviews, see [26,27]). A body of evidence, however, which dates back as far as the 1950s [28], suggests that these pigments are also essential for the correct assembly of LH2 complexes. Lang and Hunter [29] have recently shown in Rb. sphaeroides that although the LH2 apoproteins and BChl pigments are synthesised in the absence of carotenoids, any complex formed is unstable and turned over rapidly. Mutants capable of producing the LH2 polypeptides but with a carotenoid biosynthesis pathway blocked after the synthesis of phytoene, by removal of a functional crtI gene, did not synthesise intact LH2 complexes. Upon complementation with a functional crtI gene and the consequent return of 'coloured' carotenoid biosynthesis, however, stable LH2 complexes reappeared. This genetic

Genetic organization and regulation The carotenoids have also come under investigation in terms of the sequence and organization of the carotenoid biosynthetic genes. The arrangement of this cluster in Rb. sphaeroides has been described by Lang et al. [32"]. These genes lie in the same cluster as those responsible for the biosynthesis of BChl, the RC and the LH proteins, and is very, similar to that found in Rb. capsulatus. [33]. The determination of the structure of the LH2 complexes from Rps. acidop/ff/a and Rs. molischianum opened the door to the use of site-directed mutagenesis to probe structure-function relationships. It is unfortunate, therefore, that the elegant site-directed mutagenesis work on purple bacterial antenna complexes has so far only been possible in species for which no direct structural information is available [34,35]. No genetic manipulation system has yet been described for use in either Rps. acidophila or Rs. molischianurn. Therefore, great interest surrounds the possibility of using an heterologus expression system. Fowler eta/. [36 °°] have described the successful expression of LH complexes from Rps. acidophi/a and Rubrivivax gelatinosus in mutants of Rb. sphaeroides in which the host LH2 genes had been deleted. This development opens the door for the creation of site-directed mutants of LH2 complexes from strains that lack their own system for genetic manipulation. The synthesis of purple bacterial antenna complexes is regulated by a variety of external stimuli. The process by which this is controlled is now beginning to be unravelled (see [37"] for an excellent review of the regulation of gene expression in purple bacteria). The majority of this work has been carried out in Rb. capsulatus. A sensory transduction pathway that exerts control on the expression of RCs and LH complexes has been identified. Two protein components of this pathway have been identified

470

Membrane proteins

Figure 1 The overlapping regulatory circuits responsible for the regulation of photosynthetic structural genes in response to environmental stimuli. The regulation of photosynthetic gene cluster from puhA (the gone encoding the RC H subunit) to pufX, and the puc genes (encoding LH2, located elsewhere in the chromosome) is shown as a combination of the effects of light intensity (hvrA), oxygen tension (the Reg system) and ORF469. Adapted from [ 3 7 " ] .

Light hut

I(+) Aerobic ORF469

j

(-)

bchH

bchD

crtl

bchC

pucBACDE

Fl [ puhA

pufQBALMX

t ADP~(P,-RegB-P'-,~ ATP J'~-

]-4

pucBACDE

t

RegA ~ k ~ R e g X - P

RegB ~*""~ RegA-P J ~ -

RegX

Anaerobic c 1996 CurrentOpinion in Structural Biology

and termed RegA and RegB. Mosley eta/. [38] suggest that RegB functions as a membrane-spanning sensor kinase.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

Inoue et al. [39] have proposed a model for how this pathway functions. Under conditions of low oxygen tension, RegB autophosphorylates itself before catalyzing the transfer of this phosphate onto RegA, a cytosolic response regulator. This activated form of RcgA then phosphorylates DNA-binding proteins, which in turn control the induction of the LH and RC structural genes (Fig.l)

2. *•

An additional layer of control is provided by the product of the gene ORF469 [40], which acts as a repressor during aerobic growth. A light-responsive regulatory element from Rb. capsulatus has also been described [41]. This gene, hvrA, regulates the expression of the puh and puf genes, controlling the biosynthesis of the LH1 and Re. It appears to have no effect on the LH2 or the pigment biosynthesis genes.

Karrasch S, Bullough PA, Ghosh R: The 8.5/~ projection map of the light-harvesting complex I from Rhodospiril/um rubrum reveals a ring composed of 16 subunits. EMBQ J 1995, 14:631-638. This paper demonstrates that the LH1 complex also has a circular structure, at the centre of which lies a hole large enough to accommodate an Re.

• •*

Deisenhofer J, Epp O, Miki K, Huber R, Michel H: Structure of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viritis at 3 A resolution. Nature 1985, 318:618-624. 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 reveals the underlying architecture of an LH complex from a purple bacterium. This structure provides the molecular basis for understanding the mechanism and kinetics of energy transfer. 3. •.

Michel H: General and practical aspects of membrane protein crystallization. In Crysta//ization of Membrane Proteins. Edited by Michel H. Boca Raton, FL: CRC Press; 1991:74-88.

Conclusion The determination of the crystal structure of the LHZ complex from Rps. acid•phi/a, together with the visualization of the size of the LH1 ring from Rs. rubrum, has transformed our understanding of the structure of the purple bacterial photosynthetic unit. This structural information is already opening the door to a deeper understanding of the molecular mechanisms involved in the LH process. The next few years promise a great deal as structure-function studies are combined with detailed kinetic analyses to reveal the LH process in all its glory.

Acknowledgements T h i s work has been supported by grants from the BBSRC, the EU and the H u m a n Frontiers of Science Program. P K F is a B B S R C postgraduate student.

of special interest of outstanding interest

Hu X, Schulten K, Koepke J, Michel H: Structure of the lightharvesting complex-II of Rhodospirillum molischianum. Biophys J 1996, 70:A130. This paper describes the structure of the LH2 complex from R. molischianum. It shows that the LH2 complex from this species, although very similar to that from R. acidophi/a is an octamer rather than a nonamer. 5. .-

Hu X, Xu D, Hamer K, Schulten K: Predicting the structure of the light-harvesting complex of Rhodospirillum molischianum. Protein Sci 1995, 4:1617-1682. This paper presents an interesting molecular modelling study and successfully predicts the structure of the LH2 complex from R. mohschianum. 6. •

Visschers RW, Germeroth L, Michel H, Monshouwer R, van Grondelle R: Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum, Biochim Biophys Acta 1995, 1230:147-154.

Purple bacterial antenna complexes Fyfe and Cogdell

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Montoya G, Cyrklaff M, Sinning h Two-dimensional crystallisation and preliminary structure analysis of light harvesting I1(B800-850) complex from the purple bacterium Rhodovulum sulfidophilum. J Mol Biol 1995, 250:1-10.

Savage H, Cyrklaff M, Montoya G, KLihlbrandt W, Sinning I: T w o dimensional structure of light-harvesting complex II ( L H I I ) from the purple bacterium Rhodovulum sulfidophilum and comparison with LHII from Rhodopseudomonas acidophila. Structure 1996, 4:243-252. Two-dimensional crystallography is used to determine the structure of the LH2 complex from R. su/fidophi/um and shows that, like the case of the LH2 complex of R. acidophi/a, it is a nonamer.

pigment protein complexes of purple bacteria. Biophys J 1995, 69:2211-2225. 22.

Kramer H, Deinum G, Gardiner AT, Cogdell RJ, Francke C, Aartsma TJ, Amesz J: Energy transfer in the photosynthetic anntanna system of the purple non-sulfur bacterium Rhodopseudomonas cryptolactis. Biochim Biophys Acta 1995, 1231:33-40.

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Meadows KA, lida K, Tsuda K, Recchia PA, Heller BA, Antonio B, Nango M, Loach PA: Enzymatic and chemical cleavage of the core light-harvesting polypeptides of photosynthetic bacteria: determination of the minimal polypeptide size and structure required for subunit and light-harvesting complex formation, Biochemistry 1995, 34:1559-1574.

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Davies CM, Bustamante PL, Loach PA: Reconstitution of the bacterial core light-harvesting complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with isolated c~- and 13-polypeptides, bacteriochlorophyll a, and carotenoid. J Biol Chem 1995, 270:5793-5804.

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