Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues
Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues
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DOI：
10.1021/jacs.7b07849
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发表时间：
2017-10-18
影响因子：
15
通讯作者：
Tonge, Peter J.
中科院分区：
文献类型：
--
作者：
Gil, Agnieszka A.;Laptenok, Sergey P.;Tonge, Peter J.
使用蓝光的 FAD (BLUF) 光感受器中的黄素发色团被氢键网络包围，该网络能够在超快时间尺度上感知黄素电子结构的变化并做出响应。氢键网络包括严格保守的 Tyr 残基，之前我们通过引入氟酪氨酸 (F-Tyr) 类似物，分别将 Y21 的 pKa 和还原电位调节 3.5 pH 单位和 200 mV，探索了该残基 Y21 在 BLUF 蛋白 AppAnup 的光活化机制中的作用。尽管观察到生命对前向（暗到光适应形式）光反应的影响； Y21 pKa 的变化导致暗态恢复率提高了 4000 倍。在目前的工作中，我们将这些研究扩展到 BLUF 蛋白 PixD，与 AppAnuF 相比，Tyr (Y8) plc 中的调节对正向光反应具有深远的影响。特别是，Y8 pKa 降低 2 个或更多 pH 单位可防止形成稳定的光态，这与涉及从 Y8 到电子激发的 FAD 的质子转移或质子耦合电子转移的光活化机制一致。相反，PixD 中 pKa 对暗恢复率的影响显着降低。这些观察结果凸显了 PixD 和 AppABLuF 的光周期之间非常显着的差异，尽管它们共享高度保守的 FAD 结合结构。
The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechahism of the BLUF protein AppAnup by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although life impact on the forward (dark-to light-adapted form) photoreaction was obServed; the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppAnuF, Modulation in the Tyr (Y8) plc has a profound impact on the forward photoreaction. In particular, a decrease in. Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photo cycles of PixD and AppABLuF, despite their sharing highly conserved FAD binding architectures.