Role of the colorless polypeptides in phycobilisome reconstitution from separated phycobiliproteins

A phycoerythrin (PE) and phycocyanin (PC) mixture was separated from allophycocyanin on calcium phosphate chromatography from completely dissociated phycobilisomes of the blue-green alga, Nostoc sp. After dialysis of the PE-PC mixture in 0.75 m potassium phosphate, pH 7, which allows reassociation of the dissociated pigment-proteins, complexes of PE and PC in a 2:1 m ratio (PE/PC complex) as well as complexes predominantly of PC (PC/PE complex) were then separated by sedimentation on linear sucrose gradients. These complexes resemble the rods of intact phycobilisomes and transfer energy efficiently from PE to PC. They contain the Group II colorless polypeptides described by Tandeau de Marsac and Cohen-Bazire (1977 Proc Natl Acad Sci USA 74: 1635 61639). Phycobilisomes can be reconstituted by combining the allophycocyanin pool with (a) the PE-PC mixture, (b) the PE/PC complex, or (c) the PC/PE complex. Successful reconstitution is measured by absorption, fluorescence, circular dichroism, and electron microscopy. The major requirement for reconstitution is the 29-kilodalton colorless polypeptide. In its absence, no phycobilisomes are formed. It is the only colorless polypeptide common to both the PE/PC complex and the PC/PE complex, and appears to be the polypeptide responsible for rod attachment to the allophycocyanin. In addition, high phosphate concentrations and 20 degrees C temperatures are needed for reconstitution.     

Isolation and Characterization of the Central Component of the Phycobilisome Core of Nostoc sp

Freshly isolated allophycocyanin is recovered from linear sucrose gradients made in 0.75 molar potassium phosphate buffer (pH 7.0) in three sizes: 19s, 10.3s, and 5.5s. The largest aggregate is a complex of a 680 nm fluorescing allophycocyanin I in the form (αβ)₃γ, where γ is the 95 kilodalton (kD) polypeptide, and two 660 nanometer fluorescing allophycocyanin II (αβ)₃ molecules; the complex, stabilized in high phosphate concentrations, fluoresces maximally at 675 nanometers. The 10.3s fraction is a hexamer of allophycocyanin of the 660 nanometer fluorescing type, perhaps attached through two polypeptides of 46 kD and 44 kD. The 5.5s component of the allophycocyanin pool is the usual trimeric form of allophycocyanin (αβ)₃. A similar 19s fraction is the major component of allophycocyanin I isolated under optimum conditions in the presence of the protease inhibitor, phenylmethylsulfonylfluoride. This 19s fraction is apparently a central component of the core of the phycobilisome with its 95 kD polypeptide the attachment point of the phycobilisome and membrane. The 95 kD polypeptide has both long wavelength absorption and fluorescence bands which seem to account for the long wavelength fluorescence properties of allophycocyanin I.     

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp

We have identified the function of the `extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.     

Spectral analysis of allophycocyanin I, II, III and B from Nostoc sp. phycobilisomes

Low temperature (-196C) and room temperature (25C) absorption spectra of a family of allophycocyanin spectral forms isolated from Nostoc sp. phycobilisomes as well as of the phycobilisomes themselves have been analyzed by Gaussian curve-fitting. Allophycocyanin I and B share long wavelength components at 668 and 679 nm, bands that are absent from allophycocyanin II and III. These long wavelength absorption components are apparently responsible for the 20 nm difference between the 680 nm fluorescence emission maximum of allophycocyanin I and B and the 660 nm maximum of II and III. This indicates that allophycocyanin I and B are the final acceptors of excitation energy in the phycobilisome and the excitation energy transfer bridge linking the phycobilisome with the chlorophyll-containing thylakoid membranes. These Gaussian components are also found in resolved spectra of phycobilisomes, are arguing against this family of allophycocyanin molecules being artifactual products of protein purification procedures.     

Cyanobacterial phycobilisomes

Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.     

Molecular architecture of a light-harvesting antenna. Isolation and characterization of phycobilisome subassembly particles

Synechococcus 6301 mutant, strain AN112, produces phycobilisomes containing two major biliproteins, phycocyanin and allophycocyanin, and two major linker polypeptides of 27 and 75 kilodaltons (27K and 75K). These phycobilisomes have a molecular weight of approximately 2.5 X 10(6) and are the smallest of these particles known to date. Sucrose density gradient centrifugation of AN112 phycobilisomes partially dissociated in 50 mM N-[tris(hydroxymethyl)methyl]glycine, 5 mM CaCl2, 10% (w/v) glycerol, pH 7.8, separated three distinct fractions: (1) free trimeric biliproteins, (2) hexameric complexes of phycocyanin with 27K (11 S particles), and (3) phycobilisome subassemblies equivalent in mass to approximately 25% of the intact phycobilisome (18 S particles). The 18 S particles contained equimolar amounts of phycocyanin and allophycocyanin, which represented approximately 30 and 50%, respectively, of the content of these biliproteins in the AN112 phycobilisome. The 18 S particles also contained 75% and 100%, respectively, of 27K and 75K polypeptides; i.e. 75K was present in a 2-fold higher amount than in the intact phycobilisome. The absorption spectrum (lambda max 648 nm) of the 18 S particles was similar to that of allophycocyanin. Upon excitation at 580 nm, these particles exhibited a fluorescence emission spectrum consisting of 680 and 660 nm components, identical with that of intact phycobilisomes. The circular dichroism spectra of AN112 phycobilisomes and of the 18 S particles, in the region between 650 and 700 nm, were also very similar. Allophycocyanin B, which fluoresces at 680 nm, was found in fraction 1, and was totally absent from the 18 S particle. Thus, the long wavelength emission of the 18 S particle must have arisen from another terminal energy acceptor. The most probable candidate is the 75K polypeptide, which has been shown to carry a bilin chromophore and emit near 680 nm (Lundell, D. J., Yamanaka, G., and Glazer, A. N. (1980) J. Cell Biol. 91, 315-319). The 27K polypeptide, present in both fractions 2 and 3, was a component of different complexes in the two fractions. Fraction 2 displayed the physical and spectroscopic properties characteristic of the phycocyanin-linker complex, (alpha beta)6.27K. However, in the 18 S particle, 27K functioned in the assembly and attachment of phycocyanin trimers to a core domain. Based on the analysis of the components in fractions 1-3, a model is proposed which describes the structure of the AN112 phycobilisome, with emphasis on the roles of the linker polypeptides in the assembly of the core.     

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.     

Accessory polypeptides in phycobilisomes of red algae and cyanobacteria

Phycobilisomes of red algae and cyanobacteria contain small amounts of nonpigmented polypeptides in addition to the major constituent biliprotein pigments. The localization of these polypeptides is analyzed by gel electrophoresis of phycobilisome fragments obtained by selective dissociation and subsequent separation. Five groups of biliprotein aggregates are determined, belonging to the 6, 11, 16, 18 and 23 S categories. Accessory nonpigmented high molecular weight proteins (80,000 MW) are exclusively bound to phycobilisome core fractions and thylakoids, thus apparently serving as links between the phycobilisomes and the photosynthetic units of the thylakoids. In contrast, smaller nonpigmented accessory polypeptides of 20,000 to 60,000 MW are preferably found in the peripheral biliprotein stacks. They may either form a compatible link between the phycobilisome core and periphery or bind and co-polymerize with hexameric biliproteins in the peripheral stacks to enhance or effect binding of the aggregates. Furthermore, they may determine the arrangement and composition of the phycobilisomes during development and chromatic adaptation.Abbreviations PE phycoerythrin - PEC phycoerythrocyanin - PC phycocyanin - APC allophycocyanin     

Evidence for a Transient Association of New Proteins with the Spirulina maxima Phycobilisome in Relation to Light Intensity

Environmental parameters are known to affect phycobilisomes. Variations of their structure and relative composition in phycobiliproteins have been observed. We studied the effect of irradiance variations on the phycobilisome structure in the cyanobacterium Spirulina maxima and discovered the appearance of new polypeptides associated with the phycobilisomes under an increased light intensity. In high light, the six rods of phycocyanin associated with the central core of allophycocyanin contained only one to two phycocyanin hexamers instead of the two to three they contained in low light. The concomitant disappearance of a 33-kD linker polypeptide was observed. Moreover, in high light three polypeptides of 29, 30, and 47 kD, clearly unrelated to linkers, were found to be associated with the phycobilisome fraction: protein labeling showed that a specific association of these polypeptides was induced by high light. One polypeptide, at least, would play the role of a chaperone protein. Not only the synthesis of these proteins, which appeared slightly increased in high light, but also their association with phycobilisome structure are light intensity dependent.     

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.     

Light Intensity Adaptation and Phycobilisome Composition of Microcystis aeruginosa

Phycobilisomes isolated from Microcystis aeruginosa grown to midlog at high light (270 microeinsteins per square meter per second) or at low light intensities (40 microeinsteins per square meter per second) were found to be identical. Electron micrographs established that they have a triangular central core apparently consisting of three allophycocyanin trimers surrounded by six rods, each composed of two hexameric phycocyanin molecules. The apparent mass of a phycobilisome obtained by gel filtration is 2.96 × 10⁶ daltons. The molar ratio of the phycobiliproteins per phycobilisome is 12 phycocyanin hexamers:9 allophycocyanin trimers. The electron microscopic observations combined with the phycobilisome apparent mass and the phycobiliprotein stoichiometry data indicate that M. aeruginosa phycobilisomes are composed of a triangular central core of three stacks of three allophycocyanin trimers and six rods each containing two phycocyanin hexamers. Adaptation of M. aeruginosa to high light intensity results in a decrease in the number of phycobilisomes per cell with no alteration in phycobilisome composition or structure.     

Light-Harvesting System of the Red Alga Gracilaria tikvahiae: II. Phycobilisome Characteristics of Pigment Mutants

Phycobilisomes were isolated from wild type Gracilaria tikvahiae and a number of its genetically characterized Mendelian and non-Mendelian pigment mutants in which the principal lesions result in an increase or decrease in the accumulation of phycoerythrin. Both the size and phycoerythrin content of the phycobilisomes are proportional to the phycoerythrin content of the crude algal extracts. In most of the strains examined, the structure and function of the phycocyanin-allophycocyanin phycobilisome cores are the same as in wild type. The phycobilisome architecture is derived from wild type by the addition or removal of phycoerythrin. The same pattern is observed for the phycobilisome of mos² which contains a large excess of phycocyanin that is not bound to the phycobilisome. The single exception is a yellow, non-Mendelian mutant, NMY-1, which makes functional phycobilisomes composed of phycoerythrin and allophycocyanin with almost no phycocyanin. Characterization of the `linker' polypeptides of the phycobilisome indicates that a 29 kilodalton protein is required for the stable incorporation of phycocyanin into the phycobilisome. Evidence is provided for the requirement of nuclear and cytoplasmic genes in phycobilisome synthesis and assembly. The symmetry properties of the phycobilisome are considered and a structural model for the reaction center II-phycobilisome organization is presented.     

Phycobilisome Structure of Porphyridium cruentum: POLYPEPTIDE COMPOSITION

Purified phycobilisomes of Porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the α and β subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the γ subunit of phycoerythrin. Another colored polypeptide had a molecular weight of 95,000 and the characteristics of long wavelength-emitting allophycocyanin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin. In addition, the core fraction was enriched in a colored 95,000 dalton polypeptide. Inasmuch as a polypeptide with the same molecular weight is found in thylakoid membranes (free of phycobilisomes), it is suggested that this polypeptide is involved in anchoring phycobilisomes to thylakoid membranes.     

Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201.

The phycobiliproteins of the unicellular cyanobacterium Synechocystis sp. strain BO 8402 and its derivative strain BO 9201 are compared. The biliproteins of strain BO 8402 are organized in paracrystalline inclusion bodies showing an intense autofluorescence in vivo. These protein-pigment aggregates have been isolated. The highly purified complexes contain phycocyanin with traces of phycoerythrin, corresponding linker polypeptides LR35PC and LR33PE (the latter in a small amount), and a unique colored polypeptide with an M(r) of 55,000, designated L55. Allophycocyanin and the core linker polypeptides are absent. The substructure of the aggregates has been studied by electron microscopy. Repetitive subcomplexes of hexameric stacks of biliproteins form extraordinary long rods associated side by side in a highly condensed arrangement. Evidence that the linker polypeptides LR35PC and LR33PE stabilize the biliprotein hexamers is presented, while the location and function of the colored linker L55 remain uncertain. The derivative strain BO 9201 contains established hemidiscoidal phycobilisomes comprising phycoerythrin, phycocyanin, and allophycocyanin as well as the corresponding linker polypeptides. The core-membrane linker protein (LCM), and two polypeptides with M(r)s of 40,000 and 45,000 which are present in small amounts, exhibit strong cross-reactivity in Western blot (immunoblot) analysis using an antibody directed against the colored LCM of a Nostoc sp. In contrast, strain BO 8402 exhibits no polypeptide with a significant immunological cross-reactivity in Western blot analysis. Physiological and genetic implications of the unusual pigment compositions of both strains are discussed.     

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation.

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.     

Further evidence for a phycobilisome model from selective dissociation,fluorescence emission,immunoprecipitation, and electron microscopy

Phycobilisomes, isolated in 500 mM Sorensen's phosphate buffer pH 6.8 from the red alga, Porphyridium cruentum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounded by a hemispherical layer of R-phycocyanin, with phycoerythrin being on the periphery. Such a structure also allows maximum energy transfer.Intact phycobilisomes transfer excitation energy ultimately to a pigment with a fluorescence emission maximum at 675 nm. This pigment is presumed to be allophycocyanin in an aggregated state. Uncoupling of energy transfer among the pigments, and physical release of the phycobiliproteins from the phycobilisome follow a parallel time-course; phycoerythrin is released first, followed by R-phycocyanin, and then allophycocyanin. In 55 mM phosphate buffer, the times at which 50% of each phycobiliprotein has dissociated are: phycoerythrin 40 min, R-phycocyanin 75 min, and allophycocyanin 140 min.The proposed arrangement of phycobiliproteins within phycobilisomes is also consistent with the results from precipitation reactions with monospecific antisera on intact and dissociated phycobilisomes. Anti-phycoerythrin reacts almost immediately with intact phycobilisomes, but reactivity with anti-R-phycocyanin and anti-allophycocyanin is considerably delayed, suggesting that the antigens are not accessible until a loosening of the phycobilisome structure occurs. Reaction with anti-allophycocyanin is very slow in P. cruentum phycobilisomes, but is much more rapid in phycobilisomes of Nostoc sp. which contains 6–8 times more allophycocyanin. It is proposed that allophycocyanin is partially exposed on the base of isolated intact phycobilisomes of both algae, but that in P. cruentum there are too few accessible sites to permit a rapid formation of a precipitate with anti-allophyocyanin.Phycobilisome dissociation is inversely proportional to phosphate concentration (500 mM to 2 mM), and is essentially unaffected by protein concentration in the range used (30–200 μg/ml). Phycobiliprotein release occurs in the same order (phycoerythrin > R-phycocyanin > allophycocyanin) in the pH range 5.4–8.0.     

Characterization of the biliproteins of Gloeobacter violaceus chromophore content of a cyanobacterial phycoerythrin carrying phycourobilin chromophore

The biliproteins of the unicellular, thylakoid-less cyanobacterium Gleobacter violaceus were resolved by chromatography on hydroxylapatite and DEAE-cellulose into five components: phycoerythrin I and II, phycocyanin I and II, and allophycocyanin. Allophycocyanin B was not detected. Three of these components, phycoerythrin II, phycocyanin II, and allophycocyanin, were purified to homogeneity. Phycoerythrin II crystallized as hexagonal prisms. G. violaceus allophycocyanin crystallized as thin plates; unter similar conditions other cyanobacterial allophycocyanins crystallize as needles. The biliproteins in the phycoerythrin I and phycocyanin I components were present in polydisperse, high molecular weight aggregates, which may represent incompletely dissociated substructures of the phycobilisome.Both phycoerythrin components from G. violaceus carry phycoerythrobilin and phycourbilin groups in the ratio of 6:1. Separation of the and subunits of these biliproteins revealed that the phycoerythrobilins were equally distributed between the two subunits, and that the subunit alone carried the phycourobilin. These phycoerythrins are the first cyanobacterial phycobiliproteins found to carry a phycourobilin prosthetic group.Abbreviations used PE poycoerythrin - PC phycocyanin - AP allophycocyanin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - B Bangiophycean - R Rhodophytan - C Cyanobacterial     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR_L) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR_L accumulated. FNR_L has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR_L and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.