mesobiliverdin and phycocyanobilin

The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αβ)(α'β) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the β-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position β-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position β-Cys-50 and β-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the β-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.

Topics: Biliverdine; Chromatography, High Pressure Liquid; Cryptophyta; Light-Harvesting Protein Complexes; Mass Spectrometry; Molecular Weight; Phycobilins; Phycobiliproteins; Phycocyanin; Protein Subunits; Sequence Analysis, Protein

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.

Topics: Biliverdine; Chromatography, Liquid; Eukaryota; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Structure; Phycobilins; Phycocyanin; Pyrroles; Spectrophotometry, Ultraviolet; Tetrapyrroles

In vitro reaction of phycocyanobilin (PCB) with apophycocyanin results in the specific addition of the bilin to two of the cysteinyl residues, alpha-Cys-84 and beta-Cys-82, which normally function in PCB attachment (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). These bilin binding sites are designated alpha-1 and beta-1, respectively. Tryptic digestion of the apophycocyanin-PCB adduct releases two major bilin peptides, alpha-1 mesobiliverdin (MBV) and beta-1 MBV, which encompass the two bilin-binding sites. These peptides were examined by 1H NMR and fast atom bombardment mass spectroscopies. The NMR spectra show that the bilin is attached to each peptide through a thioether linkage identical to the linkage observed in the corresponding tryptic peptides, alpha-1 PCB and beta-1 PCB, derived from the natural product, C-phycocyanin. However, the NMR spectra of the adduct peptides lack the resonances corresponding to protons at positions C2 and C3 of ring A seen in the spectra of the alpha-1 PCB and beta-1 PCB peptides. Fast atom bombardment mass spectroscopy shows the masses of the alpha-1 MBV and beta-1 MBV peptides to be 2 atomic mass units lower than those of the alpha-1 PCB and beta-1 PCB peptides, respectively. Comparison of the bilin portion of the NMR spectra of the alpha-1 MBV and beta-1 MBV peptides to the NMR spectra of PCB and mesobiliverdin confirms that the bilin of the two adduct peptides resembles mesobiliverdin in having an extra double bond in the C2-C3 position of ring A. These results show that the major bilin products arising from the reaction of PCB with apophycocyanin differ from the bilins present in C-phycocyanin. The relevance of these results to the biosynthetic pathway for the attachment of tetrapyrroles to phycobiliproteins is discussed.

Topics: Apoproteins; Biliverdine; Magnetic Resonance Spectroscopy; Mass Spectrometry; Peptide Fragments; Phycobilins; Phycocyanin; Pigments, Biological; Pyrroles; Tetrapyrroles; Trypsin

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.

Topics: Aminolevulinic Acid; Bilirubin; Biliverdine; Chemical Phenomena; Chemistry; Chromatography, Thin Layer; Darkness; Heme Oxygenase (Decyclizing); Light; Mesoporphyrins; Mixed Function Oxygenases; Phycobilins; Phycocyanin; Pigments, Biological; Porphyrins; Pyrroles; Rhodophyta; Tetrapyrroles