Elisabeth Gantt

Structure and Function of Phycobilisomes: Light Harvesting Pigment Complexes in Red and Blue-Green Algae

Publisher Summary Phycobilisomes are specialized aggregated structures composed of phycobiliproteins, which are photosynthetic accessory pigments in red and blue-green algae. Phycobiliproteins, which can account for as much as 24% of the dry weight of blue-green algal cells and 40–60% of the total soluble protein, are the major light harvesters in these organisms. Chlorophyll a, which absorbs light primarily in the blue region and the red region of the visible spectrum, leaves a large absorption gap. This is filled in by the phycobiliproteins, which have an absorption range of 500–660 nm. These pigments work in conjunction with chlorophyll a to optimize light harvesting for photosynthesis, particularly under light limiting conditions. The only phycobiliprotein in higher plants is phytochrome, which occurs in very small amounts and serves as a photoregulatory receptor. In cryptophyte algae (flagellated unicells of indefinite taxonomic position), phycobiliproteins also serve as major photosynthetic accessory pigments, but phycobilisomes are absent. There are three major classes of phycobiliproteins—the red-colored phycoerythrins (PEs), the blue-colored phycocyanins (PCs), and allophycocyanins (APCs). This chapter focuses on the phycobilisome characteristics, their relationship to the photosystems in the thylakoid membrane, and major problems to be addressed in future investigations.

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

Phycobilisomes, isolated in 500 mM Sorensens phosphate buffer pH 6.8 from the red alga, Porphyridium cruetum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounding 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 aggreagated 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-phycoertythrin 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 phycobilsome structure occurs. Reaction wbilisomes, 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.

PHOTOPHYSICAL PROPERTIES OF PHYCOBILIPROTEINS FROM PHYCOBILISOMES: FLUORESCENCE LIFETIMES, QUANTUM YIELDS, AND POLARIZATION SPECTRA

Abstract— Absorption and fluorescence polarization spectra, as well as absolute fluorescence quantum yields, and lifetimes of phycobiliproteins separated from intact phycobilisomes of Porphyridium cruentum, Nostoc sp. and Fremyella diplosiphon were measured. Two different types of phycoerythrin, in addition to phycocyanin and allophycocyanin, were separated from both Porphyridium cruentum and Nostoc sp. phycobilisomes. They were distinguishable by the shape of their absorption spectra, values of fluorescence quantum yields and their limiting polarization. Phycobilisomes of Fremyella diplosiphon had a type of phycoerythrin that was different from the above kinds. By the use of fluorescence quantum yields and lifetime data, the values of natural lifetimes, the decadic molar extinction coefficients, as well as Försters critical distances R0 for excitation energy transfer, between phycobiliproteins in phycobilisomes, were estimated. The values obtained of Försters critical distances indicate that for most efficient energy transfer from phycoerythrin to allophycocyanin, the outer layers of Porphyridium cruentum and Nostoc sp. phycobilisomes should be composed of bangiophycean, phycoerythrin and cyanophytan phycoerythrin‐II respectively.

Energy transfer in phycobilisomes from phycoerythrin to allophycocyanin.

Abstract Allophycocyanin appears to be the pigment through which energy trapped by phycobiliproteins is funneled to the chloroplast lamellae. Isolated, intact phycobilisomes from Porphyridium cruentum have a maximum fluorescence emission peak at 675–680 nm when excited at 545 nm. Upon dissociation, when the energy transfer is interrupted the 675–680-nm peak declines. Excitation at 435 nm produced no significant fluorescence at this wavelength.

ULTRASTRUCTURE OF PORPHYRIDIUM AERUGINEUM A BLUE-GREEN COLORED RHODOPHYTAN(1,2,3) .

The ulstrastructural study on Porphyridium aerugineum showed vesicles in the peripheral cytoplasm which contain fibrous material similar in appearance to the cell sheath. In most respects the morphology of P. aerugineum, a freshwater form, is very similar to that of P. cruenturn, a marine species. However, there is a marked difference in the shape of the phycobilisomes which are attached to the chloroplast lamellae. In P. cruentum the phycobilisomes are always spherical or oblate, whereas in P. aerugineum they are disk shaped. The possibility is considered that the shape of the phycobilisomes may be determined by the phycoerythrin and phycocyanin content.

SPECTRAL PROPERTIES OF PHYCOBILISOMES AND PHYCOBILIPROTEINS FROM THE BLUE‐GREEN ALGA‐NOSTOC SP.*

Abstract— An improved method for phycobilisome isolation from a blue‐green alga Nostoc sp. was developed using 1% Triton X‐100. The phycobilisome preparations showed little fragmentation and had structures similar in size to those observed in thin sections of the organism. Phycobiliproteins isolated from phycobilisomes and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, had subunits with the following molecular weights: phycoerythrin (PE), 20,000 and 16,900; phycocyanin (PC), 14,700 and 16,300; and allophycocyanin (APC), 14,000. Isoelectric focusing of each phycobiliprotein resulted in major bands isoelectric at the following pH values: PE, 4.43, 4.45; PC 4.32; APC, 4.38. Absorption spectra at ‐196°c showed maxima at 551 and 566 nm for PE; 598 and 631 nm for PC; and 590, 600, 629 and 650 nm for APC. Concentrated vs dilute difference spectra of phycobiliproteins showed increased absorption at 574 nm (PE), 630 nm (PC) and 651 nm (APC) suggesting that spectral changes resulted from aggregation. Fluorescence analysis of each phycobiliprotein and of intact phycobilisome preparations showed that energy absorbed by phycoerythrin is transferred to allophycocyanin, possibly by a resonance transfer mechanism. These observations support a model where allophycocyanin forms the base of the phycobilisome which is attached to the photosynthetic membrane. The next layer is assumed to be phycocyanin, which in turn is followed by a phycoerythrin layer that is the outermost layer (on the stroma side) of the phycobilisome.

MICROMORPHOLOGY OF THE PERIPLAST OF CHROOMONAS SP. (CRYPTOPHYCEAE)1,2

An ultrastructural examination of the periplast of Chroomonas sp. revealed a surface pattern composed of rows of plate areas. The plate areas are delineated by a series of ridges, which emanate from a common line at the posterior cell end, and lateral grooves which intersect the anterior‐posterior ridges. Small ejectosomes (trichocysts) are generally located at the intersection of the lateral grooves and the ridges. Size of the plate areas varies, being smallest at the posterior and anterior ends and largest in the midregion of the cell. The average plate area is 1 μ in length and 0.7 μ in width. In section the periplast is seen to consist of 3 intimately attached layers of which the middle (plasma membrane) layer is continuous with the gullet region, flagella, and ejectosome chambers. Trypsin digestion resulted in the disappearance of the inner and outer layers, and in the loss of periplast stiffness.

Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis.

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectively) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 mumol O2/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (-196 degrees C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O2/einstein (605 nm), with a lesser change in the Vmax values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.

EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue‐gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlsteins theory of energy migration devised for a three‐dimensional photosynthetic unit. It was also shown that Paillotins theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.

EFFECT OF LIGHT INTENSITY AND GLYCEROL ON THE GROWTH, PIGMENT COMPOSITION, AND ULTRASTRUCTURE OF CHROOMONAS SP.1,2,3

Growth of Chroomonas sp. increased with light intensity (100, 1800, and 2700 μW/cm2) with a fivefold increase from the lowest to the highest intensity. Chlorophyll and phycocyanin content per cell were greater in cells grown at low light intensity, but the ratio of chlorophyll a and c did not vary appreciably. Cells grown at low light intensity had 30% more phycocyanin than cells grown at high intensities of light. The chloroplast of cells with the higher phycocyanin content had average intrathyla‐koidal widths of 300 Å, whereas those cells with the lower phycocyanin content had average intrathylakoidal widths of 200 Å. This result is compatible with the hypothesis that phycocyanin is located in the intrathylakoidal space in the cryptophyte algae. Of the various energy sources tested, only glycerol was able to support limited growth tinder nonphotosynthetic conditions. Under no condition was the chloroplast reduced to an elioplast or proplastid state. Starch accumulation was greatest in cells grown in continuous while light in glycerol. Eye‐spots were commonest in cells grown in darkness and interrupted every 24 hr by a few seconds of white light. It was concluded that this organism is an obligate phototroph.