Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors
Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors
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DOI：
10.1074/jbc.m112.391896
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发表时间：
2012-09-14
影响因子：
4.8
通讯作者：
Kennis, John T. M.
中科院分区：
文献类型：
--
作者：
Mathes, Tilo;van Stokkum, Ivo H. M.;Kennis, John T. M.
生物系统中，尤其是蛋白质中的光诱导电子转移是一个非常有趣的问题。其机械细节不能仅通过晶体学获得的结构数据来解决，因为这仅提供给定氧化还原系统的静态信息。然而，结合瞬态光谱和蛋白质的定点操作，可以获得 ET 过程的动态分子图像。在 BLUF（使用 FAD 的蓝光传感器）光感受器中，酪氨酸和黄素辅因子之间的质子耦合电子转移是从暗适应状态切换到光适应状态的关键反应，这对应于生物信号状态。特别令人费解的是，尽管各种天然存在的 BLUF 结构域在黄素结合袋的氨基酸组成方面几乎没有差异，但正向反应的反应速率差异很大，从几皮秒到几百皮秒。在本研究中，我们分别通过接近黄素 C2 羰基的定点诱变和酪氨酸的氟化来改变黄素/酪氨酸氧化还原对的氧化还原电位。我们提供了有关任一反应伙伴的氧化还原电位的变化如何显着影响光诱导质子耦合电子转移的信息。改变的氧化还原电位使我们能够进一步通过实验描述 BLUF 光循环中电子转移之前的激发态电荷转移。此外，我们表明电子转移速率与信号状态形成的量子产率直接相关。
Photoinduced electron transfer in biological systems, especially in proteins, is a highly intriguing matter. Its mechanistic details cannot be addressed by structural data obtained by crystallography alone because this provides only static information on a given redox system. In combination with transient spectroscopy and site-directed manipulation of the protein, however, a dynamic molecular picture of the ET process may be obtained. In BLUF (blue light sensors using FAD) photoreceptors, proton-coupled electron transfer between a tyrosine and the flavin cofactor is the key reaction to switch from a dark-adapted to a light-adapted state, which corresponds to the biological signaling state. Particularly puzzling is the fact that, although the various naturally occurring BLUF domains show little difference in the amino acid composition of the flavin binding pocket, the reaction rates of the forward reaction differ quite largely from a few ps up to several hundred ps. In this study, we modified the redox potential of the flavin/tyrosine redox pair by site-directed mutagenesis close to the flavin C2 carbonyl and fluorination of the tyrosine, respectively. We provide information on how changes in the redox potential of either reaction partner significantly influence photoinduced proton-coupled electron transfer. The altered redox potentials allowed us furthermore to experimentally describe an excited state charge transfer intermediately prior to electron transfer in the BLUF photocycle. Additionally, we show that the electron transfer rate directly correlates with the quantum yield of signaling state formation.