Molecular architecture of a light-harvesting antenna. Structure of the 18 S core-rod subassembly of the Synechococcus 6301 phycobilisome

The 18 S subassembly particles obtained by partial dissociation of phycobilisomes from Synechococcus 6301 (Anacystis nidulans) strain AN 112 contain approximately one-half of the mass of the phycobilisome and include core-rod junctions (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086). The polypeptide composition of 18 S complexes, determined by analysis of uniformly 14C-labeled phycobilisomes, gave the following stoichiometry: 75K:27K:18.3K:alpha beta allophycocyanin monomer: alpha beta phycocyanin monomer of 1:2:1:5:6; where 75K, 27K, etc. represent polypeptides of 75, 27 kilodaltons, etc. The 18.3K polypeptide is a hitherto underscribed biliprotein bearing a single phycocyanobilin. The NH2-terminal sequence of this subunit was determined to be homologous to that of the beta subunit of allophycocyanin. Chromatography of products resulting from limited trypsin treatment of the 18 S complex led to the isolation of three subcomplexes: a mixture of (alpha beta)3 . 22K and (alpha beta)3 . 24K phycocyanin complexes, an (alpha beta)3 allophycocyanin trimer, and an (alpha beta)2 . 18.3K.40K.11K allophycocyanin-containing complex. The 22K and 24K components were products of the degradation of the 27K polypeptides, whereas the 40K and 11K components were derived from the 75K polypeptide. The subcomplexes accounted for the composition of the 18 S complex. Determination of the composition, stoichiometry, and spectroscopic properties of the subcomplexes has led to a model of the polypeptide arrangement within the 18 S complex and of the pathway of energy transfer among these polypeptides.     

Molecular architecture of a light-harvesting antenna. Quaternary interactions in the Synechococcus 6301 phycobilisome core as revealed by partial tryptic digestion and circular dichroism studies

The core of the phycobilisomes of Synechococcus 6301 (Anacystis nidulans) strain AN112 consists of two cylindrical elements each made up of the same four distinct subcomplexes: A (alpha AP beta AP)3; B (alpha AP beta AP)2 . 18.3K . 75K; C (alpha 1APB alpha 2AP beta 3AP) . 10.5K; and D (alpha AP beta AP)3 . 10.5K, where alpha AP and beta AP are the subunits of allophycocyanin, alpha APB is the subunit of allophycocyanin B, and 18.3K, 75K, and 10.5K are polypeptides of 18,300, 75,000, and 10,500 Da, respectively. An 18 S subassembly containing subcomplexes A and B has previously been characterized (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086; Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901, 902-908). A ternary core subassembly, containing complexes A, B, and C, was isolated from a limited tryptic digest of AN112 phycobilisomes and characterized with respect to composition and spectroscopic properties. Isolation of this ternary subassembly also establishes that subcomplex D must occupy a terminal position in each of the two core cylinders. Spectroscopic studies of the individual complexes, A-D, of the subassemblies AB and ABC, and of intact AN112 phycobilisomes showed core assembly-dependent changes in the circular dichroism spectra indicative of changes in the environment and/or conformation of the bilin chromophores within the individual subcomplexes. Two terminal energy acceptors are present in the phycobilisome core, alpha APB and 75K. No indication of interaction between the chromophores on these polypeptides was detected by circular dichroism spectroscopy. This result indicates that the bilins on alpha APB and 75K act as independent energy acceptors rather than as exciton pairs.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes

Two new allophycocyanin-containing complexes were found among the products of partial dissociation of the phycobilisomes of Synechococcus 6301 strain AN112. These complexes were purified to homogeneity and characterized with respect to composition, stability, and spectroscopic properties. The structures of the complexes were established to be (alpha AP beta AP)3 . 10.5K and (alpha 1APB alpha 2AP beta 3AP) . 10.5 K, where alpha AP and beta AP are subunits of allophycocyanin, and alpha APB is the subunit of allophycocyanin B (see Lundell, D. J., and Glazer, A. N. (1981) J. Biol. Chem. 256, 12600-12606), and 10.5K is an uncolored polypeptide of 10.5-kilodaltons. These complexes are derived from the core substructure of the phycobilisome. Electron microscopic studies of the morphology of the core of strain AN112 phycobilisomes (Yamanaka, G., Glazer, A. N., and Williams, R. C. (1980) J. Biol. Chem. 255, 11004-11010) as well as structural studies of an 18 S subassembly derived from the phycobilisomes by partial dissociation (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086) indicated that the core assembly consisted of two cylindrical elements each made up of the same four distinct "trimeric" biliprotein-containing complexes. Two such core components, (alpha AP beta AP)3 and alpha 2AP beta 2AP. 18.3K . 75K (where 18.3K and 75K are polypeptides of 18.3- and 75-kilodaltons), were shown to be contained within the 18 S subassembly (Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901). The isolation of the two allophycocyanin-containing complexes described here completes the characterization of the four types of components in the Synechococcus 6301 phycobilisome core. Two lines of evidence indicate that each of the four complexes is present twice in the core: comparison of the compositions (and yields) of the complexes with that of the intact AN112 phycobilisome, and near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes taken in appropriate molar proportions.     

Isolation and characterization of pigment-protein particles from the light-harvesting complex of Phaeodactylum tricornutum

The light-harvesting complex of the marine diatom Phaeodactylum tricornutum was fractionated into two large pigment-protein particles. One pigment-protein particle, which was contained in a yellow fraction, has a molecular weight, determined by gel filtration, of approx. 230 000 and can be dissociated in sodium dodecyl sulfate/mercaptoethanol solution to apopolypeptides of approx. 15 000. Characterization of particles with regard to molecular weights, subunits, protein and pigments suggests approx. 12 subunits per particle. The other pigment-protein particle, which was found in a green fraction, of approx. 95 000 molecular weight also reduces to apopolypeptide subunits of approx. 15 kDa. The relative molar proportions of chlorophyll a, chlorophyll c, fucoxanthin and total other accessory pigments in the former fraction are 3:1.3:6:2, whereas the proportions in the latter fraction are 5:1:3:1.     

The phycobilisome, a light-harvesting complex responsive to environmental conditions.

Photosynthetic organisms can acclimate to their environment by changing many cellular processes, including the biosynthesis of the photosynthetic apparatus. In this article we discuss the phycobilisome, the light-harvesting apparatus of cyanobacteria and red algae. Unlike most light-harvesting antenna complexes, the phycobilisome is not an integral membrane complex but is attached to the surface of the photosynthetic membranes. It is composed of both the pigmented phycobiliproteins and the nonpigmented linker polypeptides; the former are important for absorbing light energy, while the latter are important for stability and assembly of the complex. The composition of the phycobilisome is very sensitive to a number of different environmental factors. Some of the filamentous cyanobacteria can alter the composition of the phycobilisome in response to the prevalent wavelengths of light in the environment. This process, called complementary chromatic adaptation, allows these organisms to efficiently utilize available light energy to drive photosynthetic electron transport and CO2 fixation. Under conditions of macronutrient limitation, many cyanobacteria degrade their phycobilisomes in a rapid and orderly fashion. Since the phycobilisome is an abundant component of the cell, its degradation may provide a substantial amount of nitrogen to nitrogen-limited cells. Furthermore, degradation of the phycobilisome during nutrient-limited growth may prevent photodamage that would occur if the cells were to absorb light under conditions of metabolic arrest. The interplay of various environmental parameters in determining the number of phycobilisomes and their structural characteristics and the ways in which these parameters control phycobilisome biosynthesis are fertile areas for investigation.     

Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection

The photosystem II (PSII) light-harvesting system carries out two essential functions, the efficient collection of light energy for photosynthesis, and the regulated dissipation of excitation energy in excess of that which can be used. This dual function requires structural and functional flexibility, in which light-harvesting proteins respond to an external signal, the thylakoid DeltapH, to induce feedback control. This process, referred to as non-photochemical quenching (NPQ) depends upon the xanthophyll cycle and the PsbS protein. In nature, NPQ is heterogeneous in terms of kinetics and capacity, and this adapts photosynthetic systems to the specific dynamic features of the light environment. The molecular features of the thylakoid membrane which may enable this flexibility and plasticity are discussed.     

Phycobilisome: architecture of a light-harvesting supercomplex

The phycobilisome (PBS) is an extra-membrane supramolecular complex composed of many chromophore (bilin)-binding proteins (phycobiliproteins) and linker proteins, which generally are colorless. PBS collects light energy of a wide range of wavelengths, funnels it to the central core, and then transfers it to photosystems. Although phycobiliproteins are evolutionarily related to each other, the binding of different bilin pigments ensures the ability to collect energy over a wide range of wavelengths. Spatial arrangement and functional tuning of the different phycobiliproteins, which are mediated primarily by linker proteins, yield PBS that is efficient and versatile light-harvesting systems. In this review, we discuss the functional and spatial tuning of phycobiliproteins with a focus on linker proteins.     

Isolation and characterization of a subunit form of the light-harvesting complex of Rhodospirillum rubrum

A new method is described for the isolation of subunits of the light-harvesting complex from Rhodospirillum rubrum (wild type and the G-9 mutant) in yields that approach 100%. The procedure involved treating membrane vesicles with ethylenediaminetetraacetic acid-Triton X-100 to remove components other than the light-harvesting complex and reaction center. In the preparation from wild-type cells, a benzene extraction was then employed to remove carotenoid and ubiquinone. The next step involved a careful addition of the detergent n-octyl beta-D-glucopyranoside, which resulted in a quantitative shift of the long-wavelength absorbance maximum from 873 to 820 nm. This latter complex was then separated from reaction centers by gel filtration on Sephadex G-100. The pigment-protein complex, now absorbing at 820 nm, contained two polypeptides of about 6-kilodalton molecular mass (referred to as alpha and beta) in a 1:1 ratio and two molecules of bacteriochlorophyll (BChl) for each alpha beta pair. This complex is much smaller in size than the original complex absorbing at 873 nm but probably is an associated form such as alpha 2 beta 2 X 4BChl or alpha 3 beta 3 X 6BChl. The 820-nm form could be completely shifted back to a form once again having a longer wavelength lambda max near 873 nm by decreasing the octyl glucoside concentration. Thus, the complex absorbing at 820 nm appears to be a subunit form of the original 873-nm complex.     

Oligomerization of light-harvesting I antenna peptides of Rhodospirillum rubrum.

We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated alpha beta BChl(2) subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl-beta-D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperature-mediated transition at low detergent concentration, a set of spectral forms with maxima slightly blue-shifted from 873 nm were observed, possibly due to opened rings with one or only a few alpha beta BChl(2) units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of -216 kJ mol(-1) and an entropy change of -0.59 kJ mol(-1)K(-1), at a detergent concentration of 0.8% n-octyl-beta-D-glucopyranoside.     

The light-harvesting antenna of the diatom Phaeodactylum tricornutum. Evidence for a diadinoxanthin-binding subcomplex

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex ( approximately 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where the de-epoxidation of diadinoxanthin into diatoxanthin mostly occurs.     

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect

By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 degrees C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 degrees C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F(680)/F(655) fluorescence intensity ratio. In contrast, heat treatment at 60 degrees C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F(655)), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F(680) originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600-7500 micromol photons m(-2) s(-1)) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 degrees C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.     

Disordered exciton model for the core light-harvesting antenna of Rhodopseudomonas viridis.

In this work we explain the spectral heterogeneity of the absorption band (. Biochim. Biophys. Acta. 1229:373-380), as well as the spectral evolution of pump-probe spectra for membranes of Rhodopseudomonas (Rps.) viridis. We propose an exciton model for the LH1 antenna of Rps. viridis and assume that LH1 consists of 24-32 strongly coupled BChl b molecules that form a ring-like structure with a 12- or 16-fold symmetry. The orientations and pigment-pigment distances of the BChls were taken to be the same as for the LH2 complexes of BChl a-containing bacteria. The model gave an excellent fit to the experimental results. The amount of energetic disorder necessary to explain the results could be precisely estimated and gave a value of 440-545 cm(-1) (full width at half-maximum) at low temperature and 550-620 cm(-1) at room temperature. Within the context of the model we calculated the coherence length of the steady-state exciton wavepacket to correspond to a delocalization over 5-10 BChl molecules at low temperature and over 4-6 molecules at room temperature. Possible origins of the fast electronic dephasing and the observed long-lived vibrational coherence are discussed.