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 reversible arrow 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 abso rption 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-associate d 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-in duced 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, indi cating that the primary photochemistry encompasses intramolecular motions o f the chromophore or its proximal amino acid side chains. H-D exchange has no significant effect on the kinetics of the initial photoprocesses. This s uggests that the isomerization reaction in both directions is not accompani ed by a rate-limiting proton transfer.