Tokonami Shunrou, Onose Morihiko, Nakasone Yusuke, Terazima Masahide
Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
J Am Chem Soc. 2022 Mar 9;144(9):4080-4090. doi: 10.1021/jacs.1c13121. Epub 2022 Feb 23.
Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ- and τ-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.
使用黄素的蓝光传感器（BLUF）蛋白由黄素结合BLUF结构域和功能结构域组成。在蓝光激发下，黄素发色团周围的氢键网络发生变化，可见光区域的吸收光谱出现红移。最终，在BLUF结构域接收到的光信息被传递到功能区域。人们一直认为这种红移在纳秒内就会完成。在本研究中，发现所有检测的BLUF蛋白（AppA、OaPAC、BlrP1、YcgF、PapB、SyPixD和TePixD）在毫秒级（τ相和τ相）存在缓慢的反应动力学。尽管关于BLUF有大量报道，但这是首次在毫秒时间区域内清晰观察到BLUF蛋白吸收随时间的变化。通过对OaPAC的一些结构域缺失突变体和PixD蛋白的两个嵌合突变体的测量，发现较慢的动力学（τ相）受到与BLUF结构域相邻的C末端区域的大小和性质的强烈影响。因此，这种毫秒级反应是C末端区域构象变化的重要指标，而C末端区域对生物学功能至关重要。另一方面，τ相普遍存在于所有BLUF蛋白中，包括任何突变体。使用位点特异性突变体研究了缓慢动力学的起源。这些结果清楚地表明了色氨酸在BLUF结构域中的重要性。基于此，提出了BLUF反应的反应方案。
