Deprotonation at Ring B Is an Intrinsic Property of the Bilin Chromophore in Cyanobacteriochrome RcaE

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors that utilize a linear tetrapyrrole (bilin) as a chromophore. Cyanobacteriochrome RcaE belongs to the green/red-type subfamily that photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. This subfamily shows a protochromic photocycle, leveraging a protonation state change at the B ring pyrrole nitrogen (N_B) to induce a large absorption shift. However, it is unclear why the deprotonation occurs at N_B among the four possible deprotonation sites (N_A to N_D), and its generality in other bilin-binding proteins remains unknown. In this study, we measured the Raman spectra of the Pg state of RcaE with isotopically labeled bilin chromophores. Vibrational analysis using quantum mechanics/molecular mechanics calculations led to a refinement of the structure of the N_B deprotonated bilin in the C5-Z,syn/C10-Z,syn/C15-Z,anti (ZZZssa) configuration. Density functional theory calculations of a series of chromophore models further revealed that N_B deprotonation most effectively minimizes the repulsion of the pyrrole NH moieties in the chromophore. Our data suggest that N_B deprotonation is a common property for the other CBCRs and phytochromes that harbor a bilin chromophore in the ZZZssa configuration and lack anionic groups interacting with the pyrroles. These findings provide new insights into the absorption tuning mechanism in the phytochrome superfamily of photosensors.