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

Abstract

Photochemical interconversion between the red-absorbing (Pr) and the far-red-absorbing (P fr) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The Pr-to-Pfr transition commences with a rapid Z-to-E photoisomerization at the C 15AC16 methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C15-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted CAC stretch and NOH in-plane rocking modes within 500 fs, reveal that initial distortion of the C 15AC16 bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to Pr in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C 15-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C15AC16 Z-to-E isomerization occurs on the excited-state surface.photochemistry ͉ photoisomerization ͉ photosensory proteins ͉ plant signal transduction ͉ time-resolved vibrational spectroscopy L ight sensing and signaling responses mediated by photoreceptors are critical for the survival and growth of all life forms. Phytochromes are a class of biliprotein photoreceptors found in plants, bacteria, and fungi that are capable of sensing red/far-red light via interconversion between red-absorbing (P r ) and far-redabsorbing (P fr ) forms ( Fig. 1) (1). Light absorption by phytochrome triggers a rapid and reversible Z-to-E isomerization of the C 15 AC 16 methine bridge between the C and D rings of its bilin chromophore (2). This photochemistry subsequently drives changes in protein conformation that lead to changes in gene expression that influence growth and development (1, 3). Temporal resolution of the structure of the bilin chromophore during the photoisomerization process is important, not only to unravel common themes underlying ultrafast dynamics of biological reactions, but also for designing synthetic light-harvesting systems with rapid response times, efficient sensing, and energy storage.In the past, ultrafast pump-probe electronic spectroscopy has been used to probe the excited-state dynamics of plant and cyanobacterial (Cph1) phytochromes. 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 (4-10). 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. The two-state model for P r * decay has also received support from ultrafast transient absorption measurements on the plant phytochrome phyA, the cyanobacterial phytochrome Cph1, and the bacteriophytochrome Agp1, all of which exhibit two ...

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

“…In the Pr state, a substantial portion of the net positive charge is delocalized on the two inner rings B and C (43) (Fig. 4 A and B), whereas in the Pfr state, ring D carries the partial positive charge (16) (Fig. 4C).…”

“…Early NMR spectroscopic studies on proteolytic phytochrome fragments (12,13) indicated that this isomerization occurs at the C15═C16 double bond (for numbering, see Fig. 1A), a geometrical change in line with vibrational spectroscopic investigations (14)(15)(16) and results from recent 13 C solid-state NMR (17,18) in which the most significant changes during the light-triggered conversions are confined to rings C and D. Exact geometries of the chromophore in the Pr state have been resolved as periplanar ZZZssa configurations in bacteriophytochromes from Deinococcus radiodurans (19) and Rhodopseudomonas palustris (20) as well as in the more plant-phytochrome-like Cph1 from the cyanobacterium Synechocystis 6803 (21). On the other hand, the crystal structure of the unusual bacteriophytochrome PaBphP Pseudomonas aeruginosa (22) whose ground state is Pfr shows a ZZEssa conformation, consistent with the expected primary photochemistry at the C15═C16 double bond (Fig.…”

Elucidation of Primary Events in Bacteriophytochrome Photoreceptors

“…However, other studies suggest that the multiexponential relaxation rate reflects the influence of sequential intermediate states following photoexcitation (2,4). In this interpretation, heterogeneity in the equilibrium system is unrelated to the distribution of isomerization rates (i.e., the reaction dynamics are homogeneous within the ensemble).…”

“…Femtosecond laser spectroscopies conducted in recent years show that the isomerization dynamics of bilin cofactors in bacteriophytochromes span timescales of hundreds of femtoseconds to hundreds of picoseconds (2)(3)(4)(5)(6). The origin of this broad distribution in rate constants has been a matter of debate.…”

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

“…A complementary Raman spectroscopy study concluded that 85% of the molecules relax back from the excited P r * state to the P r ground state explaining the low quantum yield of phytochrome phototransformation. After isomerization, the residual 15% were supposed to reach a product-like electronically excited Lumi-R* state that decays to the Lumi-R ground state, which is formed within 30 ps (26). Recent multipulse pump-probe data indicate that isomerization proceeds, most likely via a conventional conical intersection, between the excited and ground state energy surfaces (27).…”