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.     

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.     

Core substructure in cyanobacterial phycobilisomes

The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (alpha AP beta AP)3; B, (alpha AP beta AP)3 .10K; C, (alpha APB1 alpha AP2 beta AP3).10K; D, (alpha AP beta AP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.     

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.     

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.     

Properties and structure of the soluble ferredoxin from Synechococcus 6301 (Anacystis nidulans). Relationship to gene sequences.

Photoautotrophic cultures of the unicellular cyanobacterium Synechococcus 6301 (Anacystis nidulans) possessed a single [2Fe-2S] ferredoxin with a midpoint redox potential of -385 mV. Determination of the amino acid sequence of the ferredoxin showed that it consisted of 98 residues, with methionine and tryptophan both absent, and with only the four cysteine residues that are required to co-ordinate the iron-sulphur cluster. Comparisons with other ferredoxin sequences showed that most resemblance was to those from filamentous cyanobacteria, with up to 87% homology. There was less resemblance to the ferredoxins of unicellular cyanobacteria, with 25 differences when compared with that from another Synechococcus sp. However, the sequence of Synechococcus 6301 ferredoxin was identical with that derived for a gene sequence for a putative ferredoxin from the genotypically closely related Synechococcus 7942 (Anacystis nidulans R2). In contrast, the sequence showed substantial differences from that corresponding to a putative ferredoxin gene from Synechococcus 6301 reported by Cozens & Walker [(1988) Biochem. J. 252, 563-569].     

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.     

Asymmetrical core structure in phycobilisomes of the cyanobacterium Synechocystis 6701

The light-harvesting complex of cyanobacteria and red algae, the phycobilisome, has two structural domains, the core and the rods. Both contain biliproteins and linker peptides. The core contains the site of attachment to the thylakoid membrane and the energy transfer link between the phycobilisome and chlorophyll. There are also six rod-binding sites in the membrane-distal periphery of the core. The structure of phycobilisomes in the cyanobacterium Synechococcus 6301 was studied by Glazer, who proposed a model for the internal organization of the bicylindrical core. In the construction of that model, it was necessary to make arbitrary decisions between two possible locations for one of the trimeric protein complexes within a core cylinder and between two possible orientations of the basal core cylinders relative to one another. We isolated the tricylindrical cores from an ultraviolet-light-induced mutant of the cyanobacterium Synechocystis 6701 and obtained, by partial dissociation, a unique core substructure that maintained some contacts between the two basal cylinders. From its structure and spectral properties, we conclude that this particle is a central core substructure that resulted from dissociation of the two layers of peripheral trimers in the intact core. The compositions of this particle and the dissociated trimers were inconsistent with the proposed location of one of the trimers in the 6301 core model, but supported the placement of that trimer in the alternative position within the basal core cylinder. Rod-binding sites within the central core substructure were studied by partial dissociation of the short-rod phycobilisomes from another mutant of 6701. This dissociation generated particles that were interpreted as being central core substructures with the two basal rods attached. The appearance of these particles in the electron microscope suggested that both basal rods would be localized towards the same side of the intact core. Such an asymmetrical arrangement of basal rods is supported by previously published edge-views of intact cores with basal rods from strain 6701. These observations suggest a parallel arrangement of the basal cylinders with respect to each other, creating an asymmetrical core. A phycobilisome model was constructed that incorporated core asymmetry. This model predicts the energy transfer pathways from the basal and upper rods to specific trimers in the core.     

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.     

Isolation and localization of N4-methylasparagine in phycobiliproteins from the cyanobacterium Mastigocladus laminosus

The occurrence of post-translationally methylated asparagine residues in beta AP from Anabaena variabilis, Synechococcus PCC 6301 and Porphyridium cruentum has recently been reported (Klotz, A.V., Leary, J.A. & Glazer, A.N. (1986) J. Biol. Chem. 261, 15891-15894). We reinvestigated the amino-acid compositions of all phycobiliproteins from Mastigocladus laminosus. During total hydrolysis of beta AP, beta 16.2 and beta PC one mol methylamine per mol protein was released. These proteins were chemically and enzymatically fragmented and the sequences of the fragments containing the modified asparagine residue were determined by automated Edman degradation. Residues beta AP72, beta 16.2 72 and beta PC 72 were identified as N4-methylasparagine. This derivative of asparagine was also found at a homologous position in beta PE of Calothrix. In the x-ray structure model of C-phycocyanin (PC) the residue beta PC 72 points towards the chromophore beta 84, presumably having an effect on the spectroscopic characteristics of this light harvesting pigment protein complex.     

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisome to the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 microE m-2 sec-1), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 microE m-2 sec-1 (doubling time approximately 28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyll ratio and to a further decrease in light sensitivity. The resulting PSI-less/apcE- strain grew photoheterotrophically at normal light intensity (50 microE m-2 sec-1) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-less/apcE- strains had an approximately sixfold enrichment of PSII on a chlorophyll basis and were as active in oxygen evolution (on a per PSII basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSII studies and as background strains to analyze site- and region-directed PSII mutants in vivo.     

Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome

Anabaena sp. PCC 7120 mutants defective in phycobiliprotein biosynthesis or phycobilisome assembly were generated by transposon mutagenesis. Four mutants with grossly reduced content of the major phycobiliprotein, phycocyanin, were found to have insertions within the cpcBACDEFG1G2G3G4 operon coding for phycocyanin biosynthesis and assembly. The insertion in mutant B646 separated the promoter from the open reading frames and eliminated production of the phycocyanin (CpcA) and (CpcB) subunits. Insertion in cpcC in mutant B642 eliminated production of the L³⁶ _Rlinker polypeptide required for assembly of phycocyanin into the distal discs of the phycobilisome rod substructures. Mutants B64328 and B64407 had insertions, respectively, in cpcE and cpcF, genes coding for the subunits of the heterodimeric lyase which catalyzes the attachment of phycocyanobilin to the phycocyanin apo- subunit. Mutant SB12, often unable to survive under low light, was found to have an insertion in the apcE gene coding for the large core-membrane linker (L¹²⁸ _CM) that provides the scaffold for assembly of the phycobilisome core. DNA sequencing 3 of apcE revealed genes apcABC, coding for the and subunits of allophycocyanin and for the small core linker L^7.8 _C. Amino acid sequence comparisons showed that the ApcA and ApcB proteins are 37% identical and that each of these polypeptides is highly similar to corresponding polypeptides from the distantly related filamentous strains Calothrix sp. PCC7601 and Mastigocladus laminosus.     

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.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE(-) mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with--presumably--allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.     

A terminal energy acceptor of the phycobilisome: the 75,000-dalton polypeptide of Synechococcus 6301 phycobilisomes--a new biliprotein

A rapid procedure is described for the isolation of "linker" polypeptides (Lundell, D. J., R. C. Williams, and A. N. Glazer. 1981. J. Biol. Chem. 256:3580-3592) of cyanobacterial phycobilisomes. The 75,000-dalton component of the core of Synechococcus 6301 phycobilisomes isolated by this procedure has been shown to carry a bilin similar in spectroscopic properties to phycocyanobilin. "Renatured" 75,000-dalton polypeptide has absorption maxima at 610 and 665 nm and a fluorescence emission maximum at 676 nm, similar to that of intact phycobilisomes. A complex of allophycocyanin and a 40,000- dalton bilin-carrying fragment of the 75,000-dalton polypeptide, obtained by limited tryptic digestion, is described. This complex, which lacks allophycocyanin B, shows a fluorescence emission maximum at 676 nm. The above data indicate that the 75,000-dalton polypeptide functions as a terminal energy acceptor in the phycobilisome.