Ultrafast Dynamics of Phytochrome from the Cyanobacterium Synechocystis, Reconstituted with Phycocyanobilin and Phycoerythrobilin

Abstract

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

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

“…The intensity of all three FC bands decays within 150 fs concomitant with the rise of dispersive features. These decay dynamics are in agreement with the Ϸ100-fs rise time of stimulated emission in transient absorption studies on Cph1 and plant phytochromes (8). Spectra from 200 to 400 fs consist of dispersive Raman features at 1,668, 1,330, 1,245, and 816 cm Ϫ1 , each of which correlates with a prominent P r ground-state feature.…”

“…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 excited-state lifetimes characterized by a short (5-16 ps) and a long (25-40 ps) time constant (8,10,11). Despite the kinetic insight of these transient electronic spectroscopic studies, these techniques do not establish when the double bond isomerization occurs, nor do they resolve the molecular basis for the low P r -to-P fr photochemical quantum yields (7-16%) (12,13).…”

“…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.…”

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.…”