Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100

While chromophore attachment to α-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in β-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins β84 and β155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the β-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-β84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-β155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV.