Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore
Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore
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DOI：
10.1074/jbc.m505493200
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发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Hildebrandt, P
中科院分区：
文献类型：
--
作者：
Borucki, B;von Stetten, D;Hildebrandt, P
用共振拉曼光谱和闪光光解研究了根癌农杆菌细菌光敏色素Agp 1的P-r-->P-fr光转化过程，并对胆绿素发色团的母体态和低温捕获的中间体间位-R-C的结构进行了研究。与植物光敏色素A的共振拉曼光谱的强烈相似性表明，在Agp 1中，发色团的次甲基桥异构化状态在P-r中为ZZZasa，在P-fr中为ZZEssa，所有吡咯氮被质子化。P-R的光激发之后是（至少）三个热弛豫组件在形成PFR的时间常数为230 μ s和3.1和260 ms。H2O/D2 O交换揭示动力学同位素效应为1.9，2.6，和1.3的相应的转变，伴随着振幅的变化。第二和第三弛豫分别对应于Meta-R-C的形成和衰变。共振拉曼光谱研究表明，该发色团为去质子化的ZZE构型。用pH指示剂染料的测量显示，Meta-R-C的形成和衰变分别与质子释放和吸收相关。质子释放的化学计量对应于每个光转换分子一个质子。在光敏色素P-r->P-fr光转化机制中，瞬时发色团去质子化和质子释放的耦合可能是一个重要的因素，它可能对Pfr形成的最后一步中感光体在活性和非活性状态之间转换的结构变化起着至关重要的作用。
The P-r-->P-fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R-C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P-r and ZZEssa in P-fr, with all pyrrole nitrogens being protonated. Photoexcitation of P-r is followed by ( at least) three thermal relaxation components in the formation of Pfr with time constants of 230 mu s and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R-C, respectively. Resonance Raman measurements of Meta-R-C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R-C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P-r-->P-fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.