Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment

Mutants of the cyanobacterium Synechococcus sp. PCC 7002 constructed by the insertional inactivation of either the cpcE or cpcF gene produce low levels of spectroscopically detectable phycocyanin. The majority of the phycocyanin produced in these strains appears to lack the α subunit phycocyanobilin (PCB) chromophore (Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A. (1992) J. Biol. Chem. 267, 16138-16145). Purification of the phycocyanin produced in the mutants revealed two fractions each with an aberrant absorption spectrum. Tryptic peptide maps of the major fraction showed that the α-84 PCB peptide was absent. The two PCB peptides derived from the β subunit were normal. Tryptic digests of the less abundant phycocyanin fraction contained a family of bilin peptides derived from the α subunit. Several distinct bilin adducts were present. A major component was a mesobiliverdin adduct, a previously described product of the in vitro reaction of PCB and apophycocyanin. The same results were obtained with both the cpcE mutant and the cpcF mutant. In vitro reactions with PCB and the fractions containing apoα subunit showed that the α-84 bilin attachment site was unmodified and competent for adduct formation. Pseudo-revertants of both strains were observed to arise at high frequency. Analysis of the phycocyanin from a cpcE pseudo-revertant, which produced a near wild-type level of phycocyanin with α subunit carrying PCB, revealed a single amino acid substitution, α-Tyr¹²⁹ → Cys. This residue, which is conserved in all phycocyanins sequenced to date, forms part of the α-84 bilin binding site and lies within 5 Å of α-Cys⁸⁴. A mutated cpcA gene containing this substitution was constructed by site-directed mutagenesis and transformed, along with cpcB, into a cpcBAC deletion strain containing an insertionally inactivated cpcE. This strain produces high levels of phycocyanin and the majority of the α subunit carries PCB at α-Cys⁸⁴.