Heyne Karsten, Herbst Johannes, Stehlik Dietmar, Esteban Berta, Lamparter Tilman, Hughes Jon, Diller Rolf
Institut für Experimentalphysik, Freie Universität Berlin, Germany.
Biophys J. 2002 Feb;82(2):1004-16. doi: 10.1016/S0006-3495(02)75460-X.
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.
飞秒时间分辨瞬态吸收光谱首次用于表征细菌光敏色素系统在光循环的两个热稳定状态下的初级光异构化动力学。在大肠杆菌中表达的来自集胞藻PCC 6803的85 kDa光敏色素Cph1用藻蓝胆素（Cph1-PCB）和藻红胆素（Cph1-PEB）进行了重组。在650 nm光激发后，Cph1-PCB的红光吸收形式Pr在S(1)态显示出约150 fs的弛豫。线性四吡咯发色团的C环和D环之间随后的Z-E异构化最好用速率常数分布来描述，其第一矩为(16 ps)(-1)。在615 nm激发导致分布略有变宽。从远红光吸收形式Pfr开始的反向E-Z异构化的特征是两个较短的时间常数，分别为0.54和3.2 ps。在Cph1-PEB的情况下，双键异构化不发生，激发态寿命延伸到纳秒范围。除了40到150 fs之间的受激发射上升时间外，未观察到快速弛豫过程。这表明沿着A、B和C环的发色团-蛋白质相互作用对在Cph1-PCB中观察到的皮秒动力学贡献不大，而是在异构化C(15)[双键]C(16)双键附近的D环周围区域。发现Cph1-PCB在环境温度下的初级反应动力学表现出与植物A型光敏色素所描述的非常相似的特征，即相对较慢的Pr和快速的Pfr光反应。这表明初始反应在植物光敏色素进化开始之前就已经确立。
