On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain.
On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain.
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关于 AppA BLUF 结构域中的信号传导机制和光可逆性的缺失。
DOI：
10.1529/biophysj.107.117788
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发表时间：
2008
影响因子：
3.4
通讯作者：
J. Kennis
中科院分区：
文献类型：
--
作者：
K. Toh;I. V. Stokkum;J. Hendriks;M. Alexandre;J. Arents;M. A. Perez;R. Grondelle;K. Hellingwerf;J. Kennis
来自球形红细菌的黄素蛋白AppA含有N-末端的FAD结合BLUF光感受器结构域。在光照下，AppA BLUF结构域形成信号传导状态，其特征在于吸收红移10 nm，这种状态称为AppA（RED）。我们应用超快光谱的光累积AppA（RED）状态，调查的AppA BLUF域的光可逆特性。在AppA（RED）的光吸收下，FAD单重激发态FAD（RED）* 在7 ps内单指数衰减，形成中性半醌自由基FADH（*），其随后在60 ps内衰减到原始AppA（RED）分子基态。因此，FAD（RED）* 通过电子和质子转移迅速失活，可能是从保守的酪氨酸Tyr-21到FAD，随后是自由基对重组。我们的结论是，与许多其他光感受器相反，AppA BLUF结构域不是光可逆的，并且在吸收第二个光子后不会进入替代反应途径。为了解释这些特性，我们提出，一个分子配置是在激发AppA（RED），对应于一个正向反应中间体先前确定的暗态BLUF光反应。在AppA（RED）激发时，BLUF结构域因此进入其正向反应坐标，容易重新形成AppA（RED）基态并抑制反向或副反应。FAD* 的单指数衰减表明AppA（RED）中的FAD结合口袋比暗态AppA中的FAD结合口袋显著更刚性。野生型，W104 F，和W 64 F突变体BLUF域的稳态荧光实验显示色氨酸荧光最大值，对应于一个掩埋的构象的色氨酸-104在黑暗和光明的状态。我们得出结论，色氨酸-104不会成为暴露于溶剂在BLUF光循环。
The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.
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