Bilin organization in cryptomonad biliproteins

The bilin organization of three cryptomonad biliproteins (phycocyanins 612 and 645 and phycoerythrin 545) was examined in detail. Two others (phycocyanin 630 and phycoerythrin 566) were studied less extensively. Phycocyanin 645 and phycoerythrin 545 were suggested to have one bilin in each monomeric (alphabeta) unit of the dimer (alpha2beta2) isolated from the others, and the remaining six bilins may be in pairs. One pair was found across the monomer-monomer interface of the protein dimer, and two identical pairs were proposed to be within the monomer protein units. For phycocyanin 612, a major surprise was that a pair of bilins was apparently not found across the monomer-monomer interface, but the remaining bilins were distributed as in the other two cryptomonad proteins. The effect of temperature on the CD spectra of phycocyainin 612 demonstrated that two of the bands (one positive and one negative) behaved identically, which is required if they are coupled. The two lowest-energy CD bands of phycocyanin 612 originated from paired bilins, and the two higher-energy bands were from more isolated bilins. The paired bilins within the protein monomers contained the lowest-energy transition for these biliproteins. Using the bilins as naturally occurring reporter groups, phycocyanin 612 was shown to undergo a reversible change in tertiary structure at 40 degrees C. Protein monomers were shown to be functioning biliproteins. A hypothesis is that the coupled pair of bilins within the monomeric units offers important advantages for efficient energy migration, and other bilins transfer energy to this pair, extending the wavelength range or efficiency of light absorption.     

Effect of moderate UV-B irradiation on Synechocystis PCC 6803 biliproteins

In the present study, we investigated the mechanism of UV-B radiation induced damage to the light harvesting apparatus of the cyanobacterium Synechocystis 6803. Liquid chromatography analysis and spectroscopy investigations performed on phycobilisomes or isolated biliproteins irradiated with moderate UV-B intensity (1.3 W/m(2)) revealed rapid destruction of beta-phycocyanin and a slower damage of the other biliproteins, alpha-phycocyanin and both alpha and beta-allophycocyanin. EPR spin trapping measurements revealed that carbon centered adducts of the spin trap DMPO were formed. This evidence indicates that free radicals produced from bilins probably attack the polypeptide chain of protein inducing its degradation. Our results show that the bilin chromophore is the main target of UV-B irradiation, causing structural changes, which in turn induce reaction of the chromophore with atmospheric oxygen and lead to production of reactive radicals. Our results also demonstrate that beta-phycocyanin is the most affected biliprotein, probably due to the presence of two bilins as chromophore.     

Significant energy transfer from CpcG2-phycobilisomes to photosystem I in the cyanobacterium Synechococcus sp. PCC 7002 in the absence of ApcD-dependent state transitions

Hemidiscoidal phycobilisomes (PBS), the major light harvesting complexes of photosynthesis in most cyanobacteria, are composed of rods and cores, which are linked by the linker CpcG1 (L(RC)). Another type of PBS, CpcG2-PBS exits and their function in energy transfer has not been fully understood. We measured growth rates, absorption cross-sections and quantum efficiency of photosystem I in mutant strains of Synechococcus PCC sp. 7002 lacking the linker CpcG2. Our results showed that energy transfer from CpcG2-PBS to PSI in the absence of state transitions could be significant under PBS-absorbing light and energy transfer from two types of PBS is independent to each other. Evidence also suggested that CpcG2 anchors the CpcG2-PBS to thylakoid membranes.     

Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin 545.

Fluorescence (excitation) polarization spectroscopy in the wavelength region of the bilin chromophores was applied to phycoerythrocyanin (CV-phycocyanin), phycocyanins 645 and 612, and phycoerythrin 545. The cryptomonad biliproteins - phycoerythrin 545 and phycocyanins 612 and 645 - were studied as both protein dimers having an alpha(2)beta(2) polypeptide structure and as alphabeta monomers. The cyanobacterial phycoerythrocyanin (CV-phycocyanin) was a trimeric oligomer. The changes in polarization across the spectrum were attributed to transfers of energy between bilins. Cryptomonad biliproteins are isolated as dimers. The similarities between their steady-state fluorescence polarization spectra and those of the corresponding monomers suggested that the monomers' conformations were analogous to the dimers. This supports the use of monomers in the study of dimer bilin organization. The unusual polarization spectrum of phycoerythrin 545 was explained using a model for the topography of its bilins. Obtaining the emission spectra of phycoerythrin 545 at several temperatures and a deconvolution of the dimer circular dichroism spectrum also successfully tested the bilin model. Circular dichroism spectroscopy was used to determine which polarization changes are formed by F?rster resonance energy transfers and which may be produced by internal conversions between high- and low-energy states of pairs of exciton-coupled bilins. Attempts were made to assign energy transfer events to the corresponding changes in fluorescence polarization for each of the four biliproteins.     

Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: An overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Ultrafast light harvesting dynamics in the cryptophyte phycocyanin 645.

Steady-state and femtosecond time-resolved optical methods have been used to study spectroscopic features and energy transfer dynamics in the soluble antenna protein phycocyanin 645 (PC645), isolated from a unicellular cryptophyte Chroomonas CCMP270. Absorption, emission and polarization measurements as well as one-colour pump-probe traces are reported in combination with complementary quantum chemical calculations of electronic transitions of the bilins. Estimation of bilin spectral positions and energy transfer rates aids in the development of a model for light harvesting by PC645. At higher photon energies light is absorbed by the centrally located dimer (DBV, beta50/beta61) and the excitation is subsequently funneled through a complex interference of pathways to four peripheral pigments (MBV alpha19, PCB beta158). Those chromophores transfer the excitation energy to the red-most bilins (PCB beta82). We suggest that the final resonance energy transfer step occurs between the PCB 82 bilins on a timescale estimated to be approximately 15 ps. Such a rapid final energy transfer step cannot be rationalized by calculations that combine experimental parameters and quantum chemical calculations, which predict the energy transfer time to be 40 ps.     

Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane

In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome — a giant, multi-subunit pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 Å was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.     

CHARACTERIZATION OF PHYCOCYANIN-DEFICIENT PHYCOBILISOMES FROM A PIGMENT MUTANT OF PORPHYRIDIUM SP. (RHODOPHYTA)

The structure and function of phycobilisomes in the rhodophyte Porphyridium sp. were investigated by comparing the properties of the wild type with a pigment mutant called C12. When grown under low light, cells of C12 were bright orange, while wild-type cells were deep red. The results obtained from a characterization of purified phycobilisomes of the mutant C12 led us to propose the existence in Porphyridium sp. phycobilisomes of two types of rods, some containing only phycoerythrin and others containing phycoerythrin bound to phycocyanin, which is in turn linked to the core by the linker L_RC. By studying the partitioning of phycobiliproteins between phycobilisomes and pools of free phycobiliproteins, we found that phycocyanin in the C12 mutant was only present in the pool of free proteins and that its specific linker, L_RC, was totally absent. Phycoerythrin was present in the free pool and in the purified phycobilisomes as well. One of the three specific phycoerythrin linkers γ was missing. In light of the fact that in the C12 mutant, the linker L_RC is absent and that there is no phycocyanin bound to the phycobilisomes, we propose that the rods in the mutant contain only phycoerythrin. These phycobilisomes are nevertheless functional and exhibit an efficient excitation transfer from phycoerythrin directly to allophycocyanin. Electron microscopy showed the purified phycobilisomes of C12 to be less dense than those of the wild type. This change was attributed to the disappearance of the rods containing the combination phycocyanin/phycoerythrin. Light still regulates phycobiliprotein synthesis in the mutant, as shown by the change in the color of the culture, which turned green-yellow when cells were shifted from low light to high light growth conditions. Light also regulates the structure of the phycobilisomes, which have fewer rods under high light growth conditions.     

Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution

Cyanobacteria are the oldest life form making important contributions to global CO2 fixation on the Earth. Phycobilisomes (PBSs) are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs), and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state). We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.     

Light Harvesting and State Transitions in Cyanobacteria

Cyanobacteria are oxygenic phototrophic prokaryotes and are considered to be the ancestors of chloroplasts. Their photosynthetic machinery is functionally equivalent in terms of primary photochemistry and photosynthetic electron transport. Fluorescence measurements and other techniques indicate that cyanobacteria, like plants, are capable of redirecting pathways of excitation energy transfer from light harvesting antennae to both photosystems. Cyanobacterial cells can reach two energetically different states, which are defined as “State 1” (obtained after preferential excitation of photosystem I) and “State 2” (preferential excitation of photosystem II). These states can be distinguished by static and time resolved fluorescence techniques. One of the most important conclusions reached so far is that the presence of both photosystems, as well as certain antenna components, are necessary for state transitions to occur. Spectroscopic evidence suggests that changes in the coupling state of the light harvesting antenna complexes (the phycobilisomes) to both photosystems occur during state transitions. The finding that the phycobilisome complexes are highly mobile on the surface of the thylakoid membrane (the mode of interaction with the thylakoid membrane is essentially unknown), has led to the proposal that they are in dynamic equilibrium with both photosystems and regulation of energy transfer is mediated by changes in affinity for either photosystem.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.     

Excitation energy transfer from phycocyanin to chlorophyll in an apcA-defective mutant of Synechocystis sp. PCC 6803.

A greenish mutant of the normally blue-green cyanobacterium Synechocystis sp. PCC 6803, designated UV6p, has been isolated and characterized. UV6p possesses functional photosystems I and II (PSI and PSII) but lacks normal light harvesting phycobilisomes because allophycocyanin is absent and core-specific linker proteins are almost entirely absent. The mutation responsible for the UV6p phenotype has been identified; it is a base substitution which results in the creation of a termination codon within the coding region of the apcA gene. Phycocyanin (PC) and phycobilisome rod linker proteins are present in UV6p and, despite the absence of core components, at least 35% of the PC is associated with rod linker proteins. At 77 K, light absorbed by PC of UV6p elicits PSI fluorescence comparable to that of wild type cells but produces greatly diminished PSII fluorescence. The results indicate that the assembly of rods is independent of cores and that light energy absorbed by rods can be transferred principally and directly to PSI. This energy transfer pathway, which may also be present in wild type, may have a regulatory role in maintaining the balance of input of excitation energy into PSI versus PSII during photosynthesis.     

Adaptation of the Photosynthetic Apparatus of Anacystis nidulans to Irradiance and CO2-Concentration

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m², and supplied with 0.03 and 3 % CO₂ enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO₂, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO₂ concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L³⁰_R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO₂ adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO₂ adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L⁶⁰_CM and L⁷⁵_CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.     

The immunologically conserved phycobilisome-thylakoid linker polypeptide

We have isolated phycobilisomes from two classes of red algae, several subdivisions of the cyanobacteria, and the cyanelles of Cyanophora paradoxa. In addition to the major light harvesting biliproteins, these phycobilisomes also contain several other polypeptides, the largest of which ranges from 75 to 120 kilodaltons in the different species surveyed. This protein, previously isolated and characterized from three species, was shown to be the final emitter of excitation energy in phycobilisomes and is also thought to be involved in the attachment of the phycobilisomes to the thylakoid membrane. We have obtained polyclonal antibodies to the 95 kilodalton polypeptide isolated from phycobilisomes of the cyanobacterium, Nostoc sp. This protein shares no common antigenic determinants with either the α or β subunits of allophycocyanin, or any of the other biliproteins, as determined by the sensitive Western immunoblotting technique. However, this antiserum cross-reacts with the highest molecular weight polypeptide of all the rhodophytan and cyanobacterial phycobilisomes tested. That these proteins are immunologically related, but are unrelated to other biliproteins, is reminiscent of previous immunological studies of biliproteins which showed that while the three major spectroscopically distinct classes of biliproteins (phycoerythrin, phycocyanin, and allophycocyanin) shared no common antigenic determinants, there was a strong antigenic determinant to specific biliprotein classes which crossed taxonomic divisions.     

Two novel phycoerythrin-associated linker proteins in the marine cyanobacterium Synechococcus sp. strain WH8102

The recent availability of the whole genome of Synechococcus sp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of alpha and beta subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marine Synechococcus spp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed.     

Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803.

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.     

Functional studies of the Synechocystis phycobilisomes organization by high performance liquid chromatography on line with a mass spectrometer.

This study was designed to yield data on the supramolecular organization of the phycobilisome apparatus from Synechocystis, and the possible effects of environmental stress on this arrangement. Phycobilisomes were dissociated in a low ionic strength solution and a quantitative estimation of the protein components present in each subcomplex was obtained using liquid chromatography coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). An advantage of this approach is that information can be collected on the initial events, which take place as this organism adapts to environmental changes. Ultracentrifugation of whole phycobilisomes revealed five subcomplexes; the lightest contained four linker proteins plus free phycocyanin, the second the core complex, while the last three bands contained the rod complexes. Four linkers were found in band 1 with higher molecular masses than those expected from the DNA sequence, indicating that they also contain linked chemical groups. UV-B irradiation specifically destroyed the beta-phycocyanin and one rod linker, which resulted in the disintegration of the rod complexes. The two bilins present in beta-phycocyanin give a greater contribution to the UV absorption than the single bilin of the other bilinproteins and probably react with atmospheric oxygen forming toxic radicals. The protein backbone is, in fact, protected from damage in anaerobic conditions and in the presence of radical scavengers. Cells grown in sulfur- and nitrogen-deficient medium contained significantly reduced levels of beta-phycocyanin and one rod linker.     

Protective dissipation of excess absorbed energy by photosynthetic apparatus of cyanobacteria: role of antenna terminal emitters

Two mechanisms of photoprotective dissipation of the excessively absorbed energy by photosynthetic apparatus of cyanobacteria are described that divert energy from reaction centers. Energy dissipation, monitored as nonphotochemical fluorescence quenching, occurs at different steps of energy transfer within the phycobilisomes or core antenna of photosystem I. Although these mechanisms differ significantly, in both cases, energy dissipates mainly from terminal emitters: allophycocyanin B or core membrane linker protein (L_CM) in phycobilisomes, or the longest-wavelength chlorophylls in photosystem I antenna. It is supposed that carotenoid-induced energy dissipation in phycobilisomes is triggered by light-induced transformation of the nonquenched state of antenna into quenched state due to conformation changes caused by orange carotinoid-binding protein (OCP)–phycobilisome interaction. Fluorescence of the longest-wavelength chlorophylls of photosystem I antenna is strongly quenched by P700 cation radical or by P700 triplet state, dependent on redox state of the acceptor side cofactors of photosystem I.