Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin.
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.
Heyne
Karsten
K
Institut für Experimentalphysik, Freie Universität Berlin, Germany.
Herbst
Johannes
J
Stehlik
Dietmar
D
Esteban
Berta
B
Lamparter
Tilman
T
Hughes
Jon
J
Diller
Rolf
R
D055592:Biophysical Phenomena; D001703:Biophysics; D002244:Carbon / Q000737:chemistry; D000458:Cyanobacteria / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D015233:Models, Statistical; D052980:Phycobilins; D010798:Phycocyanin / Q000737:chemistry*; D010799:Phycoerythrin / Q000737:chemistry*; D010834:Phytochrome / Q000737:chemistry*; D010944:Plants / Q000378:metabolism; D011487:Protein Conformation; D011758:Pyrroles / Q000737:chemistry*; D013053:Spectrophotometry; D045725:Tetrapyrroles; D013997:Time Factors
D052980:Phycobilins; D011758:Pyrroles; D045725:Tetrapyrroles; D010798:Phycocyanin; D010799:Phycoerythrin; D010834:Phytochrome; C011070:phycoerythrobilin; C011069:phycocyanobilin; D002244:Carbon; D003545:Cysteine