Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins.

Marine Synechococcus strains WH8103, WH8020, and WH7803 each possess two different phycoerythrins, PE(II) and PE(I), in a weight ratio of 2-4:1. PE(II) and PE(I) differ in amino acid sequence and in bilin composition and content. Studies with strain WH7803 indicated that both PE(II) and PE(I) were present in the same phycobilisome rod substructures and that energy absorbed by PE(II) was transferred to PE(I). Strain WH8103 and WH8020 PE(I)s carried five bilin chromophores thioether-linked to cysteine residues in sequences homologous to those previously characterized in C-, B-, and R-PEs. In contrast, six bilins were attached to strain WH8103 and WH8020 PE(II)s. Five of these were at positions homologous to bilin attachment sites in other phycoerythrins. The additional bilin attachment site was on the alpha subunit. The locations and bilin types in these PE(s) and in the marine Synechocystis strain WH8501 PE(I) (Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9528-9534) are: (table; see text) Since phycourobilin (PUB) (lambda max approximately 495 nm) transfers energy to phycoerythrobilin (PEB) (lambda max approximately 550 nm), inspection of these data shows that the invariant PEB group at beta-82 is the terminal energy acceptor in phycoerythrins. The adaptations to blue-green light, high PUB content and the presence of an additional bilin on the alpha subunit, increase the efficiency of light absorption by PE(II)s at approximately 500 nm.

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content.

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.

R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins.

A new member of the phycocyanin family of phycobiliproteins, R-phycocyanin II (R-PC II) has been discovered in several strains of marine Synechococcus sp. R-PC II has absorption maxima at 533 and 554 nm, a subsidiary maximum at 615 nm, and a fluorescence emission maximum at 646 nm. It is the first phycoerythrobilin (PEB)-containing phycocyanin of cyanobacterial origin. The purified protein is made up of alpha and beta subunits in equal amounts and is in an (alpha beta)2 aggregation state. The alpha and beta subunits of this protein are homologous to the corresponding subunits of previously described C- and R-phycocyanins as assessed by amino-terminal sequence determination and analyses of sequences about sites of bilin attachment. R-PC II carries phycocyanobilin (PCB) at beta-84 and PEB at alpha-84 and beta-155 (residue numbering is that for C-phycocyanin), whereas in C-phycocyanin PCB is present at all three positions. In R-phycocyanin, the bilin distribution is alpha-84 (PCB), beta-84 (PCB), beta-155 (PEB). In both R-phycocyanin and R-phycocyanin II excitation at 550 nm, absorbed primarily by PEB groups, leads to emission at 625 nm from PCB. These comparative data support the conclusion that the invariant beta-84 PCB serves as the terminal energy acceptor in phycocyanins.

Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer.

Data on the wavelength and temperature dependence of both time-resolved and steady state fluorescence emission are presented for allophycocyanin (AP) and for a crosslinked allophycocyanin trimer (XL-AP) (Ong LJ and Glazer AN: Physiol Veg 23:777-787, 1985). AP dissociates at high dilution and is not stable above 40 degrees C even at moderate protein concentration. In contrast, XL-AP does not dissociate even at very low protein concentrations and is completely stable up to 60 degrees C in the presence of 0.75 M NaK-phosphate, pH 7.0. The results show that XL-AP is superior to AP for use in conjugates that absorb and emit in the red region of the spectrum. The high stability of XL-AP at elevated temperatures at high phosphate concentrations suggests that this derivative may be useful in conjunction with nucleic acid probes.

An unusual phycoerythrin from a marine cyanobacterium.

Phycoerythrin conjugates are reagents for cell sorting and analyses in which the argon-ion laser line at 488 nanometers is used for excitation. Many marine Synechococcus strains contain phycoerythrins with absorption maxima at approximately 490 and 550 nanometers; these maxima indicate the presence of phycourobilin and phycoerythrobilin prosthetic groups in the protein. Phycoerythrins of red algae contain both groups, but those of freshwater and soil cyanobacteria contain only phycoerythrobilin. Phycoerythrin purified from Synechococcus WH8103 has molecular properties typical of red algal phycoerythrins, but its phycourobilin content is higher than that of other phycoerythrins. The protein has absorption maxima at 492 and 543 nanometers and corresponding molar extinction coefficients of 2.78 and 1.14 x 10(6); it fluoresces maximally at 565 nanometers with a quantum yield of 0.5. Conjugates of Synechococcus WH8103 phycoerythrin could increase the sensitivity of cell analysis techniques to almost twice that possible with other phycoerythrin conjugates.