First Steps in the Phytochrome Phototransformation:  A Comparative Femtosecond Study on the Forward (Pr → Pfr) and Back Reaction (Pfr → Pr)

The primary light-induced events in the reversible Pr ⇌ Pfr phototransformation are investigated by femtosecond absorption spectroscopy using a pump−probe technique. After the selective electronic excitation of Pr and Pfr with pulses at 610 and 730 nm, respectively, the transient absorption spectra were measured as a function of the delay time and subjected to a global fit analysis. As a result of this analysis, the decay-associated spectra of the kinetic components involved in the formation of the first photoproducts in the forward and back reaction are obtained. These spectra provide a more detailed understanding of the primary stages in the light-induced transformations. In addition, the influence of the solvent viscosity on the initial reaction steps was studied. In each direction of reaction, a short-lifetime component is found to be strongly viscosity-dependent, indicating that the primary photochemistry encompasses intramolecular motions of the chromophore or its proximal amino acid side chains. H−D exchange has no significant effect on the kinetics of the initial photoprocesses. This suggests that the isomerization reaction in both directions is not accompanied by a rate-limiting proton transfer.