Determining complete electron flow in the cofactor photoreduction of oxidized photolyase
Determining complete electron flow in the cofactor photoreduction of oxidized photolyase
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DOI：
10.1073/pnas.1311073110
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发表时间：
2013-08-06
影响因子：
11.1
通讯作者：
Zhong, Dongping
中科院分区：
文献类型：
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作者：
Liu, Zheyun;Tan, Chuang;Zhong, Dongping
光酶、光解酶和光感受器隐色素中的黄素辅因子可能以氧化态存在，并应转化为还原态才能发挥生物学功能。这种氧化还原变化可以通过酶中的一系列芳香残基光诱导电子转移（ET）有效地实现。在这里，我们报告了我们的完整表征光裂解酶的光还原动力学与飞秒分辨率。通过各种位点定向突变，我们确定了酶中所有可能的电子供体，并确定了它们的ET时间尺度。被激发的辅因子就像一个电子汇一样，从中心的活性位点到蛋白质表面的三层芳香分子中吸引电子流。优势电子流跟随保守的色氨酸三联体，以跳跃的方式穿过具有多个隧道步骤的层。这些ET动力学发生在小于150ps的超快速度，并且与局部蛋白质和溶剂弛豫强烈耦合。黄素的反向电子流是缓慢的，在纳秒范围内，以确保高还原效率。通过实验确定的12个基本ET步骤和6个ET反应对，酶沿着相同的芳香残基表现出明显的还原电位梯度，具有有利的重组能，以驱动高度单向的电子流从蛋白质表面流向活性位点中心。
The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction-potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.