Red, orange, green: Light- and Temperature-dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable UV-vis absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spec-troscopy, a further orange-absorbing state O600 was identified which is in thermal equilibrium with Pr. The different absorp-tion properties of the three states were attributed to different lengths of the conjugated -electron system of the phycocya-nobilin (PCB) chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations the most extended conjuga-tion holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by the inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicate a partially distorted ZZZssa geometry in which ring A is twisted, while ring D and the adjacent me-thine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ca. ~30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan (Trp) residue from the chromophore binding pocket to a solvent exposed position.