In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin.

D M Arciero,D A Bryant,A N Glazer

doi:10.1016/s0021-9258(19)81365-0

D M Arciero, D A Bryant + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)81365-0

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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.

Highlights

Expression of cloned a and @ subunit genes of Syne- linked to the protein through thioether bonds to cysteinyl chococcus sp
Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with theapoprotein
Two PCBs are bound bacteria are strict photoautotrophs, C. caldarium is a photoper a@monomer when the reaction is performed with heterotroph and can be grown in the dark on an exogenous excess bilin

Summary

In Vitro Attachment of Bilins to Apophycocyanin

The major bilin peptides arising from in vitroadduct formationat each of these sites differedboth in chromatographic behavior has been accumulated to show that exogenous [14C]5-aminolevulinic acid ( l l ) , [14C]heme( l l ) , and [14C]biliverdin[12, 13] can act as in vivo precursors to phycocyanin-bound PCB. C-phycocyanin is a light-harvestingcomponent of the photosynthetic apparatus of cyanobacteria and red algae This intensely blue-colored protein is made up of two dissimilar polypeptide chains, a and @o,f 162 and 172 residues, respectively. The nature of the immediate precursor to the protein-linked PCB remainsto be established unambiguously as do the features which designate specific cysteinyl residues as sites of bilin attachment. The results presented here and in the companion papers establish that PCB and phycoerythrobilin spontaneously form covalent adducts with apophycocyaninspecificallyat cysteinyl residues normally thioether-linked to bilin in native C-phycocyanin

RESULTS

PCB Adduct

WAVELENGTH lnml