Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

中文翻译：

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光驱动离子泵细菌视紫红质中的主要转移步骤：DNP 增强 MAS NMR 揭示的不可逆 U 形转弯

尽管受到了很多关注，但光驱动离子泵细菌视紫红质 (bR) 中重要的初级质子转移的路径仍然是个谜。在这里，我们使用 DNP 增强魔角旋转 (MAS) NMR 来研究在席夫碱 (SB) 去质子化之前（在 L 中间体中），即在 SB 去质子化和 Asp85 质子化之后（在 L 中间体中）活性位点的关键元素Mo 中间体），并且就在 SB 重质子化和 Asp96 去质子化之后（在 N 中间体中）。使这些实验成为可能的一个基本特征是通过 DNP 将信号增强 75 倍。15N(SB)-1H 相关性表明新去质子化的 SB 接受来自醇的氢键，13C-13C 相关性表明 Asp85 在初级质子转移之前接近 Thr89。同时，SB 和 Asp85 之间的 15N-13C 相关性表明螺旋 C 和 G 在质子转移之前靠得更近，然后放松。总之，这些结果表明 Thr89 用于将 SB 质子传递给 Asp85，并且创建该途径涉及 C 和 G 螺旋之间的和解以及发色团扭转。