Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100.
While chromophore attachment to alpha-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in beta-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins beta84 and beta155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the beta-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-beta84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-beta155 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.
Zhao
Kai-Hong
KH
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, PR China. khzhao@163.com
Zhu
Jing-Ping
JP
Song
Bo
B
Zhou
Ming
M
Storf
Max
M
Böhm
Stephan
S
Bubenzer
Claudia
C
Scheer
Hugo
H
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol / Q000737:chemistry*; D052980:Phycobilins; D010798:Phycocyanin / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
D017931:DNA Primers; D052980:Phycobilins; C038397:phycoerythrocyanin; D010798:Phycocyanin; D017830:Octoxynol