The primary light-induced events in the reversible Pr right harpoon over left harpoon 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.

The light-induced photoprocesses of phycocyanobilin were studied by femtosecond spectroscopy. After excitation with 100 fs pulses at 610 nm the transient absorption spectra were measured throughout the visible region as a function of time. Following excitation of phycocyanobilin bleaching of the initial ground-state absorption and broad excited-state absorptions located at the shortwave and longwave sides of the bleaching region were observed. The decay of both bleaching and the excited-state absorptions was accompanied by the build-up of a comparatively long-living transient absorption between 600 and 750 nm, which is ascribed to the formation of a photoproduct. On the basis of a global analysis the observed transient absorption changes are well described by three exponential components with lifetimes in the range of a few picoseconds (τ 1 ∼ 3 ps), some tens of picoseconds (τ 2 ∼ 30-50 ps) and some hundreds of picoseconds (τ 3 ∼ 350-600 ps) with the exact values depending on the solvent used for the dissolution of phycocyanobilin. The two fast components (τ 1 and τ 2 ) can be attributed to the relaxation of two different excited states populated with excitation, while the longer-lived component (τ 3 ) is associated with the decay of the photoproduct. A possible kinetic model that explains the mechanism of the light-induced relaxation processes in phycocyanobilin is presented. † Dedicated to Professor Wolfhart Ru ¨diger on the occasion of his 66th birthday.

Based on previous time-resolved absorption studies, phycocyanobilin undergoes a photoreaction from an A- into a B- and C-form, with the latter two photoproducts showing absorption spectra red-shifted from A. To identify the molecular mechanism involved in the excited-state reactions, the structural origin of the red shift in the absorption spectra is investigated. Using semiempirical AM1 calculations that include configuration interaction by pair doubles excitation configuration interaction, the absorption spectra of different conformers as well as different protonation states were calculated. The results clearly indicate a pronounced red shift in the spectra of structures either protonated or deprotonated at the basic/acidic centres of the tetrapyrrole chromophore whereas, in contrast, conformational changes alone result in a blue shift. Furthermore, it is shown by quantum chemical calculations that the basicity of phycocyanobilin is much higher in the excited than in the ground state, with a decrease in the excited-state pK(B)* of approximately 9.5 units. The acidity is only slightly enhanced with a drop in pK(A)* of only approximately 1.6 units. From these findings, a reaction model for the excited-state processes in phycocyanobilin is proposed. According to this model, photoexcitation of phycocyanobilin triggers an excited-state proton transfer giving rise to the formation of a protonated species. In parallel, the local increase in the medium pH associated with protonation then forwards a deprotonation at an acidic NH-group so that in effect both protonated and deprotonated phycocyanobilin would arise from the initial photoreaction and account for the observed red shift in the spectra of the B- and C-forms.

Abstract— The excited state behavior of the red light‐absorbing form of phytochrome (Pr) was studied on the femtosecond time scale. After excitation of Pr with 75 fs laser pulses at 616 nm the kinetics of the transient absorption changes was recorded at selected wavelengths probing mainly the bleaching of the Pr ground‐state absorption and the stimulated emission. The kinetic data obtained indicate the population of an excited state with a 3 ps lifetime immediately after excitation. This state precedes the formation of another excited state with a 32 ps lifetime. The decay of the latter state is followed by the appearance of a first product state that is assumed to represent lunii‐R. In addition, 2,3‐dihydrobiliverdin, which is considered to be an adequate model of the Pr chro‐mophore, was included in the femtosecond studies. The absorption difference spectra recorded at various delay times show an immediate bleaching of the ground‐state absorption. Simultaneously with bleaching a broad transient absorption appears between 410 and 525 nm. The data analysis yields similar kinetic components as they were observed in the decay of Pr. It is suggested from this finding that within the first tens of picoseconds after excitation the excited‐state properties of Pr are mainly determined by the properties of the chromophore itself.

The biologically active far-red-absorbing form of the plant photoreceptor phytochrome, Pfr, was studied by femtosecond spectroscopy. The transient absorption changes that occurred upon excitation of Pfr at 730 nm with laser pulses of 150 fs were measured as a function of time at selected probe wavelengths. Immediately after excitation, bleaching of the ground-state absorption bands in the red and blue spectral region was observed. Bleaching was accompanied by an absorption increase at wavelengths between 775 and 825 nm as well as at around 400 nm. The kinetic analysis of the absorption changes ΔOD(t) at different probe wavelengths yielded two decay components, one with a lifetime of about 700 fs and another with about 4.5 ps. A model that explains the relaxation processes following the excitation of Pfr is suggested. It assumes that the initially excited state decays within about 700 fs toward a secondary excited one which is depopulated with a rate constant of (4.5 ps)-1.