  Cyanobacteriochromes are bi-stable photoreceptor proteins with desirable
photochemical properties for biotechnological applications such as optogenetics
or fluorescence microscopy. Here, we investigated Slr1393-g3, a
cyanobacteriochrome that reversibly photo-switches between a red-absorbing (Pr)
and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to
track the sequence of intermediates during the photocycle over many orders in
magnitude in time. In the conversion from Pg to Pr, we have revealed a new
intermediate which precedes the Pr formation by using transient IR
spectroscopy. In addition, stationary and transient 2D~IR experiments measured
the vibrational couplings between different groups of the chromophore and the
protein during these intermediate states. Anharmonic QM/MM calculations predict
spectra in close-to-quantitative agreement with experimental 2D~IR spectra of
the initial and the final state of the photocycle. They facilitate the
assignment of the IR spectra and provide an atomistic insight into the coupling
mechanism. This serves as a basis for the interpretation of the spectroscopic
results and suggests structural changes of the intermediates along the
photocycle.
