Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis

Phytochrome and cyanobacteriochrome utilize a linear methine-bridged tetrapyrrole (bilin) to control numerous biological processes. They show a reversible photoconversion between two spectrally distinct states. This photocycle is initiated by a CC double-bond photoisomerization of the bilin followed by its thermal relaxations with transient and/or stationary changes in the protonation state of the pyrrole moiety. However, it has never been identified which of the four pyrrole nitrogen atoms is deprotonated. Here, we report a resonance Raman spectroscopic study on cyanobacteriochrome RcaE, which has been proposed to contain a deprotonated bilin for its green-absorbing 15Z state. The observed Raman spectra were well reproduced by a simulated structure whose bilin B ring is deprotonated, with the aid of molecular dynamics and quantum mechanics/molecular mechanics calculations. The results revealed that the deprotonation of B and C rings has the distinct effect on the overall bilin structure, which will be relevant to the color tuning and photoconversion mechanisms of the phytochrome superfamily. Furthermore, this study documents the ability of vibrational spectroscopy combined with the advanced spectral analysis to visualize a proton of a cofactor molecule embedded in a protein moiety.