The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.

中文翻译：

---

调节视紫红质 ReaChR 中从发色团到蛋白质的光能转移

光感受器的功能依赖于从发色团到蛋白质的吸收光能的有效转移，以驱动构象变化，最终产生输出信号。在视网膜结合蛋白中，主要存在两种机制来存储光异构化后的光子能量：1) 视网膜假体的构象畸变，以及 2) 质子化视网膜希夫碱 (RSBH+) 与其反离子复合物之间的电荷分离。因此，通过生色团弛豫和/或RSBH+-抗衡离子复合物中电荷分离的减少来实现向蛋白质的能量转移。结合 FTIR 和 UV-Vis 光谱以及分子动力学模拟，我们在这里展示了广泛使用的、红色可激活的Volvox careri channelrhodopsin-1 衍生ReaChR，能量储存和转移到蛋白质中取决于谷氨酸E163 (Ci1) 的质子化状态，谷氨酸E163 是RSBH+ 的抗衡离子之一。Ci1 保持 7.6 的 pKa，因此其质子化和去质子化形式在生理条件下达到平衡。Ci1 的质子化导致活性位点区域中的刚性氢键网络。这稳定了光活化后视网膜的扭曲构象，并通过削弱应变能的释放来减缓能量转移到蛋白质中。相比之下，通过去质子化 Ci1 或去除 Ci1 谷氨酸侧链，氢键系统的刚性降低，并且发色团弛豫引起的能量转移加速。基于氢平面外 (HOOP) 带衰减动力学，