- Authors: Walter A., Andresen M., Jakobs S., Schroeder J., Schwarzer D.
- Title: Primary Light-Induced Reaction Steps of Reversibly Photoswitchable Fluorescent Protein Padron0.9 Investigated by Femtosecond Spectroscopy
- Journal: The Journal of Physical Chemistry
- Volume: B119
- Volume: 5136–5144
- DOI: 10.1021/jp512610q
The reversible photoswitching of the photochromic fluorescent protein Padron0.9 involves a cis-trans isomerization of the chromophore. Both isomers are subject to a protonation equilibrium between a neutral and a deprotonated form. The observed pH dependent absorption spectra require at least two protonating groups in the chromophore environment modulating its proton affinity. Using femtosecond transient absorption spectroscopy we elucidate the primary reaction steps of selectively excited chromophore species. Employing kinetic and spectral modelling of the time dependent transients we identify intermediate states and their spectra. Excitation of the deprotonated trans species is followed by excited state relaxation and internal conversion to a hot ground state on a timescale of 1.1-6.5 ps. As the switching yield is very low (Φtrans→cis = 0.0003±0.0001) direct formation of the cis isomer in the times-resolved experiment is not observed. The reverse switching route involves excitation of the neutral cis chromophore. A strong H/D isotope effect reveals the initial reaction step to be an excited state proton transfer with a rate constant of kH = (1.7 ps)-1 (kD = (8.6 ps)-1) competing with internal conversion (kic = (4.5 ps)-1). The deprotonated excited cis intermediate relaxes to the well-known long lived fluorescent species (kr = (24 ps)-1). The switching quantum yield is determined to be low as well, Φcis→trans = 0.02±0.01. Excitation of both the neutral and deprotonated cis chromophore is followed by a ground state proton transfer reaction partially re-establishing the disturbed ground state equilibrium within 1.6 ps (deuterated species: 5.6 ps). The incomplete equilibration reveals an inhomogeneous population of deprotonated cis species which equilibrate on different timescales.