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

Abstract

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.

Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

“…The 26-ps decay of the stimulated emission (840-960 nm) is consistent with the 28-ps excited-state lifetime attributed to the P r * excited state from fluorescence quantum yield analysis (14). The 30-ps formation of Lumi-R is also in agreement with time-resolved transient absorption experiments on native and substituted chromophores in PhyA and Cph1 phytochromes (7,11). Therefore, the stimulated emission decay dynamics, together with the changes in intensity of the Raman bands, directly indicate that the 3-ps Z-to-E isomerization takes place on the long-lived excited state and that the ground-state Lumi-R photoproduct forms in 30 ps.…”

“…Such studies reveal that formation of the isomerized primary photoproduct Lumi-R, characterized by a red-shifted electronic absorption maximum at 700 nm, occurs 25-40 ps after excitation (4)(5)(6)(7)(8)(9)(10). The P r excited state exhibits multiexponential fluorescence decay dynamics with at least two lifetimes (10 ps and 45 ps) (5), thereby implicating the presence of at least two excited states.…”

Femtosecond Dynamics in the Lactim Tautomer of Phycocyanobilin: A Long‐Wavelength Absorbing Model Compound for the Phytochrome Chromophore

“…[18] The reaction pathway for photoconversion of Pr to Pfr is distinct from that for the back reaction of Pfr to Pr, involving different intermediates and time scales. The Pr-to-Pfr photoreaction was shown to occur along two distinct excited-state regions on the S 1 potential energy surface resulting in formation of the primary ground state product, lumi-R, which has already adopted the Pfr-like chromophore configuration, within % 30 ps (or even longer) [19][20][21][22][23][24][25][26][27] and % 15 % quantum yield. [19, 21-23, 25, 27, 28] On the other hand, the initial photoprocesses of the Pfr-to-Pr back reaction proceed on a much faster time scale.…”

Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes

“…As Pr, the bilin is cationic with all four of the pyrrole ring nitrogens protonated (12,13). Using plant Phys as the example, photoexcitation with red light converts Pr (absorption maximum of the Q band at 666 nm) within picoseconds to the partially bleached Lumi-R photoproduct (absorption maximum at 688 nm) (14,15). This conversion is predicted to involve a Z to E isomerization of the C15-C16 methine double bond between the C and D pyrrole rings of the chromophore (16 -18).…”