Abstract
Cyanobacteriochromes are distinctive relatives of the red/far-red photoreversible phytochromes and are diversified in sensing light. Among them, GAF domain of AnPixJ (AnPixJ-GAF) shows reversible photoconversion between green- (Pg) and red-absorbing (Pr) forms. Using holoproteins prepared from cyanobacteria or PCB-producing Escherichia coli, Pr binds ZZZ-phycocyanobilin (PCB) and Z-to-E isomerization between C and D rings occurs during the photoconversion like Pr of phytochromes. However, final product, Pg, is blue-shifted in contrast with the red-shifted Pfr of phytochromes. In this study, we reconstituted in vitro the holoprotein by incubating apo-AnPixJ-GAF with PCB to understand the chromophorylation process. PCB was readily ligated to apo-AnPixJ-GAF and the reaction completed within 1 min. The dark reconstituted holoprotein was in the Pr form and showed reversible photoconversion between Pg and Pr. Absorption spectrum just after mixing showed broader peak and a shoulder around 680 nm, when compared with the native Pr, suggestive of an intermediate state during the chromophorylation process. The reaction scheme will be discussed with this unique intermediate state.
