Probing Bilin-Protein Interaction in the Protochromic Photocycle of Cyanobacteriochrome RcaE by Site-Directed Mutagenesis.

Cyanobacteriochromes (CBCRs) are members of the phytochrome superfamily of photosensor proteins that bind a bilin chromophore. CBCRs exhibit substantial diversity in their absorption wavelengths through a variety of bilin-protein interactions. RcaE is the first discovered CBCR as a regulator of chromatic acclimation, where cyanobacteria optimize the absorption wavelength of their photosynthetic antenna. RcaE undergoes a reversible photoconversion between green-absorbing (Pg) and red-absorbing (Pr) states, where the bilin chromophore adopts a deprotonated C15-Z,anti and a protonated C15-E,syn structures, respectively. This photocycle is designated as the 'protochromic photocycle' as the change in the bilin protonation state is responsible for the large absorption shift. With the guidance of recently determined Pg and Pr structures of RcaE, in this study, we investigated bilin-protein interaction by site-directed mutagenesis on three key residues referred to as a protochromic triad and also other conserved residues interacting with the bilin. Among the protochromic triad residues, Glu217 and Lys261 are critical for the formation of the Pr state, while Leu249 is critical for the formation of both Pg and Pr states. Substitution in other conserved residues, including Val218, Phe219 and Pro220 in the wind-up helix and Phe252, Phe214 and Leu209 in a part of the bilin-binding pocket, had less substantial effects on the spectral sensitivity in RcaE. These data provide insights into our understanding of the bilin-protein interaction in the protochromic photocycle and also its evolution in the CBCRs.