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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 cyanobacteriochrome as a regulator of chromatic acclimation, where cyanobacteria optimize the absorption wavelength of their photosynthetic antenna. RcaE undergoes a reversible photoconversion between a green-absorbing (Pg) and a 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 "protochromic photocycle" as the change of 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-chromophore 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-chromophore interaction in the protochromic photocycle and also its evolution in the CBCRs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Cyanobacteriochromes (CBCRs) belong to the phytochrome superfamily of photoreceptors, the members of which utilize a linear tetrapyrrole (bilin) as a chromophore. RcaE is a representative member of a green/red-type CBCR subfamily that photoconverts between a green-absorbing dark state and red-absorbing photoproduct (Pr). Our recent crystallographic study showed that the phycocyanobilin (PCB) chromophore of RcaE adopts a unique C15- E , syn configuration in the Pr state, unlike the typical C15- E , anti configuration for the phytochromes and other CBCRs. Here, we measured Raman spectra of the Pr state of RcaE with 1064 nm excitation and explored the structure of PCB and its interacting residues under physiologically relevant aqueous conditions. We also performed measurements of RcaE in D 2 O as well as the sample reconstituted with the PCB labeled with 15 N or with both 13 C and 15 N. The observed Raman spectra were analyzed by quantum mechanics/molecular mechanics (QM/MM) calculations together with molecular dynamics simulations. The Raman spectra and their isotope effects were well-reproduced by the simulated spectra of fully protonated PCB with the C15- E , syn configuration and allowed us to assign most of the observed bands. The present vibrational analysis of the all syn bilin chromophore using the QM/MM method will advance future studies on CBCRs and the related proteins by vibrational spectroscopy.