Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)–glutamic acid (E)–tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN^·–/E-COOH/WH^·+, the proton first rapidly jumps from the bridging E-COOH to FMN^– (τ_fPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH^·+ to E-COO^– (τ_fPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W^· (τ_rPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH^· to E-COO^– (τ_rPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN^·– and WH^·+ to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.

中文翻译：

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剖析蓝光感光器中的超快逐步双向质子中继

通过氢键网络的质子中继对于实现光合作用和光感受器等蛋白质机器的功能至关重要。剖析质子中继过程中各个质子转移步骤的速率和能量一直具有挑战性。在这里，我们使用黄素 (BLUF) 结构域和黄素单核苷酸 (FMN)–谷氨酸 (E)–色氨酸 (W) 三联体设计了质子摇摆蓝光，并使用超快光谱动力学解决了四个单独的质子转移步骤. 我们发现，在形成 FMN ^{· –} /E-COOH/WH ^{· +}的光致电荷分离之后，质子首先从桥接 E-COOH 快速跃迁到 FMN ^– (τ _fPT2= 3.8 皮秒；KIE = 1.0)，随后是第二次质子从 WH ^{· +}转移到 E-COO ^– (τ _fPT1 = 336 ps; KIE = 2.6)，立即摇回 W ^· (τ _rPT1 = 85 ps; KIE = 6.7)，随后质子从 FMNH ^·返回到 E-COO ^– (τ _rPT2 = 34 ps; KIE = 3.3)，最终电荷在 FMN ^{· –}和 WH ^{· +}之间重新组合以结束反应循环。我们的结果使用 BLUF 结构域作为范例蛋白在超快时间尺度上重新审视了 Grotthuss 机制。