BLUF domain function does not require a metastable radical intermediate state.
BLUF domain function does not require a metastable radical intermediate state.
复制标题
BLUF结构域功能不需要亚稳组织中间状态。
DOI：
10.1021/ja4121082
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发表时间：
2014-03-26
影响因子：
15
通讯作者：
Meech SR
中科院分区：
文献类型：
--
作者：
Lukacs A;Brust R;Haigney A;Laptenok SP;Addison K;Gil A;Towrie M;Greetham GM;Tonge PJ;Meech SR
BLUF(Blue Light Using Flavin)结构域蛋白是一类重要的蓝光敏感蛋白家族，在细胞中控制着多种功能。BLUF域中的主要光激活步骤尚未建立。许多实验和理论研究表明，高度保守的酪氨酸和黄素生色团之间的光致电子转移(PET)可以形成自由基中间态。在这里，我们使用超快宽带瞬变红外光谱研究了PET在三种不同的BLUF蛋白质中的作用。我们表征和识别了激发态和基态物种的红外活性标记模式，并用它们来记录蛋白质中的光化学动力学。我们还产生了明确显示PET的突变体，并通过对蛋白质和生色团的同位素标记，能够指定黄素和蛋白质自由基态的模式特征。我们发现，在所研究的三个BLUF区域中的两个区域中没有观察到这些自由基中间体，这使得人们对BLUF光循环中形成一组自由基中间体的重要性产生了怀疑。此外，非天然氨基酸的突变被用氟酪氨酸取代保守的酪氨酸，从而改变了所提出的电子转移反应的驱动力；观察到的速率变化也与PET的机制不一致。因此，虽然在BLUF蛋白中可以观察到PET反应的中间体，但它们与光活性无关，这表明自由基中间体对它们的操作不是中心。考虑了其他非自由基途径，包括由黄素环的电子激发引起的酮烯醇互变异构化。
BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered.
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影响因子：
15
作者：
Brust R;Lukacs A;Haigney A;Addison K;Gil A;Towrie M;Clark IP;Greetham GM;Tonge PJ;Meech SR
通讯作者：
Meech SR
DOI：
10.1021/jz4023738
发表时间：
2014-01-02
期刊：
The journal of physical chemistry letters
影响因子：
--
作者：
Brust R;Haigney A;Lukacs A;Gil A;Hossain S;Addison K;Lai CT;Towrie M;Greetham GM;Clark IP;Illarionov B;Bacher A;Kim RR;Fischer M;Simmerling C;Meech SR;Tonge PJ
通讯作者：
Tonge PJ
影响因子：
2.9
作者：
Haigney, Allison;Lukacs, Andras;Tonge, Peter J.
通讯作者：
Tonge, Peter J.
影响因子：
2.9
作者：
Laan, W;Gauden, M;Hellingwerf, KJ
通讯作者：
Hellingwerf, KJ
影响因子：
3.3
作者：
Khrenova, M. G.;Nemukhin, A. V.;Domratcheva, T.
通讯作者：
Domratcheva, T.