There and Back Again: Loss and Reacquisition of Two-Cys Photocycles in Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both families use linear tetrapyrrole (bilin) chromophores that are covalently attached to a conserved Cys residue. CBCRs are more spectrally diverse than phytochromes, with known examples detecting light from the near ultraviolet to the edge of the infrared (370-750 nm). Detection of ultraviolet to blue light by CBCRs is mediated by a second Cys residue, which forms a covalent linkage to the bilin C10 atom. Second linkage formation is best understood in a subfamily possessing a conserved Asp-Xaa-Cys-Phe (DXCF) motif. Some DXCF CBCRs can isomerize their phycocyanobilin (PCB) chromophores into phycoviolobilin (PVB), a property not reported for other lineages. Both the DXCF Cys and PVB formation have been lost during evolution of other CBCR subfamilies. Using phylogenetic analysis and characterization of recombinantly expressed CBCRs, we show that the DXCF Cys residue has also been reacquired during CBCR evolution. Guided by this knowledge, we successfully reintroduced a second cysteine into a red/green CBCR, restoring blue-light sensing and PVB formation with two additional substitutions. Our results validate the roles of these residues in CBCR spectral tuning and thus provide new insight into the molecular basis of their spectral diversity.