Lamparter Tilman, Michael Norbert
Pflanzenphysiologie, Freie Universität Berlin, Königin Luise Strasse 12-16, D-14195 Berlin, Germany.
Biochemistry. 2005 Jun 14;44(23):8461-9. doi: 10.1021/bi047510g.
Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.
光敏色素从吸收红光的形式Pr向吸收远红光的形式Pfr的光转化是由环C与环D连接双键周围的Z到E异构化引发的；发色团经历ZZZ到ZZE异构化。在体内，光敏色素发色团与蛋白质共价结合，但已报道了一些非共价体外加合物的例子，它们也经历Pr到Pfr的光转化。我们表明，高度富集ZZE异构体的游离胆绿素或藻蓝胆素可以很容易地从以非共价方式与农杆菌光敏色素Agp1结合的发色团中获得，并用于光谱分析。甲醇/盐酸溶液中游离胆绿素从ZZE到ZZZ的光转化量子产率为1.8%，但在中性甲醇溶液中可忽略不计，表明该过程依赖于质子。分别测试了胆绿素和藻蓝胆素的ZZE形式与Agp1和蓝细菌光敏色素Cph1组装的能力。在这两种情况下，都形成了类似Pfr的加合物，但发色团以非共价方式与蛋白质结合。Agp1 Pfr经历暗逆转回到Pr；非共价ZZE加合物也有相同的特征。暗逆转后，发色团与蛋白质共价结合。类似地，在用强远红光照射引发ZZE到ZZZ异构化后，PCB发色团与Cph1共价结合。农杆菌Agp2属于一小类光敏色素，它们也以Pr形式组装，但在黑暗中从Pr转化为Pfr。当Agp2脱辅基蛋白与胆绿素的ZZE形式组装时，与ZZZ对照相比，最终加合物的形成加速，表明ZZE发色团直接适合Agp2的发色团口袋。
