title: In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin.
abstract: Expression of cloned alpha and beta subunit genes of Synechococcus sp. PCC7002 C-phycocyanin in Escherichia coli led to the production of large amounts of apophycocyanin. The apophycocyanin was purified to homogeneity and shown to be an alpha beta monomer. The reactivity of the apoprotein toward a number of open chain and cyclic tetrapyrroles was examined. Phycocyanobilin (PCB), phycoerythrobilin, and biliverdin all formed covalent adducts with apophycocyanin in 50 mM sodium phosphate buffer at pH 7.0. Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with the apoprotein. None of these tetrapyrroles reacted with 2 mM 2-mercaptoethanol, cysteine, or reduced glutathione under the same conditions. The adduct with PCB was investigated in greater detail. Its visible absorption spectrum, with a maximum at 646 nm, is more similar to that of allophycocyanin than phycocyanin. Two PCBs are bound per alpha beta monomer when the reaction is performed with excess bilin. While tryptic digestion of the adduct generates numerous bilin peptides, amino acid analysis of these chromopeptides revealed that PCB reacted specifically at alpha-Cys-84 and beta-Cys-82, two of the three cysteinyl residues that serve as the attachment sites for PCB in native phycocyanin. The major bilin peptides arising from in vitro adduct formation at each of these sites differed both in chromatographic behavior and in spectroscopic properties from the corresponding PCB peptides isolated from tryptic digests of native C-phycocyanin.
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008970:Molecular Weight; D052980:Phycobilins; D010798:Phycocyanin / Q000031:analogs & derivatives* / Q000378:metabolism*; D010860:Pigments, Biological / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D013053:Spectrophotometry; D045725:Tetrapyrroles
chemical_list: D001059:Apoproteins; D052980:Phycobilins; D010860:Pigments, Biological; D011758:Pyrroles; D045725:Tetrapyrroles; C057786:apophycocyanin; D010798:Phycocyanin; C011069:phycocyanobilin; D003545:Cysteine