Birge R R, Einterz C M, Knapp H M, Murray L P
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Biophys J. 1988 Mar;53(3):367-85. doi: 10.1016/S0006-3495(88)83114-X.
The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B. (b) The presence of a small activation barrier (~0.2 kcal mol-1) in the 9-cis - 9-trans excited-state surface is associated with the location of the counterion as well as the intrinsic photophysical properties of the 9-cis chromophore. The principal difference between the 1 1-cis -c 1 -transphoto reaction surface and the 9-cis - 9-trans photoreaction surface is the lack of effective electrostatic stabilization of distorted 9 = 10 conformations due to incomplete charge polarization. (c) Hydrogen bonding to the imine proton, ifpresent, does not involve the counterion. We conclude that water in the active site, or secondary interactions with the protein (not involving the CTN), are responsible. (d) All photochemical transformations involve one-bond photoisomerizations.This prediction is based on the observation of a very small excited state barrier for the I -- B photoreaction and a negative barrier for the R - B phototransformation, coupled with the theoretical prediction that all two-bond photoisomerizations have significant S, barriers while one-bond photoisomerizations have small to negative S, barriers.(e) Rhodopsin is energetically stabilized relative to isorhodopsin due to both electrostatic interactions and conformational distortion, both favoring stabilization of R. The INDO-PSDCI calculations suggest that rhodopsin chromophore-CTN electrostatic interactions provide an enhanced stabilization of -2 kcal mol-1 relative to I. Conformational distortion of the 9-cis chromophore-lysine system accounts for -3 kcal mol-1. (f) Energy storage in bathorhodopsin is-60% conformational distortion and 40% charge separation. Our model predicts that the majority of the chromophore protein conformational distortion energy involves interaction of the C,3(-CH3)=CI4--C,5=N-lysine moiety with nearby (unknown) protein residues. (g) Strong interactions between the counterion and the chromophore in R and I will generate weak, but potentially observable charge-transfer bands in the near infrared. The key predictions are the presence of an observable charge-transfer transition at 859 nm (1 1,640 cm- 1) in I and an analogous, but slightly weaker band at 897 nm (11,150 cm-1) in R. Both transitions involve the transfer of an electron from the counterion into low-lying l theta* molecular orbitals.
通过低温光量测定法、低温吸收光谱法以及含部分单双构型相互作用的间忽略微分重叠（INDO - PSDCI）分子轨道理论，研究了视紫红质和异视紫红质中初级光化学事件的性质。主要目标是更好地理解蛋白质结合位点如何影响结合发色团的能量、光化学和光谱性质。通过将视紫红质（R）到视紫红质的光转化作为内标（φR→B = 0.67），在77 K下测定了异视紫红质（I）到视紫红质（B）光转化的绝对量子产率。与视紫红质光化学不同，异视紫红质在77 K下光化学生成视紫红质的量子产率表现出波长依赖性。在七个波长处的测量值范围从565 nm处的0.089±0.021到440 nm处的0.168±0.012。基于各种动力学模型对这些数据的分析表明，I→B光转化遇到与9 - 顺式→9 - 反式激发态势能面相关的小活化能垒（约0.2 kcal mol⁻¹）。溶液（EPA，77 K）中的9 - 顺式视黄醛发色团相对于其他异构体具有最小的振子强度：1.17（全反式）、0.98（9 - 顺式）、1.04（11 - 顺式）和1.06（13 - 顺式）。对于视蛋白结合的发色团，构象的影响差异很大。观察到I的λmax吸收带的振子强度相对于R（0.98）和B（1.07）的λmax吸收带异常大（1.11）。与异视紫红质相关的波长依赖性光异构化量子产率和异常振子强度提供了有关视蛋白结合位点性质的重要信息。通过使用INDO - PSDCI分子轨道理论测试了结合位点的各种模型，以预测R、B和I的振子强度，并计算每个模型中与R和I的光化学相关的能垒和能量存储。我们的实验和理论研究得出以下结论：（a）抗衡离子（缩写为CTN）在R、B或I中与亚胺质子没有紧密关联。抗衡离子位于R和I中发色团平面下方，主要的发色团 - 抗衡离子静电相互作用涉及C15 - CTN和C13 - CTN。这些相互作用导致I相对于R和B的异常振子强度。（b）9 - 顺式 - 9 - 反式激发态表面存在小活化能垒（约0.2 kcal mol⁻¹）与抗衡离子的位置以及9 - 顺式发色团的固有光物理性质有关。11 - 顺式 - c1 - 反式光反应表面与9 - 顺式 - 9 - 反式光反应表面之间的主要差异是由于电荷极化不完全导致扭曲的9 = 10构象缺乏有效的静电稳定作用。（c）与亚胺质子的氢键（如果存在）不涉及抗衡离子。我们得出结论，活性位点中的水或与蛋白质的二级相互作用（不涉及CTN）起作用。（d）所有光化学转化都涉及单键光异构化。该预测基于对I - B光反应非常小的激发态势垒和R - B光转化的负能垒的观察，以及所有双键光异构化具有显著S，能垒而单键光异构化具有小到负的S，能垒的理论预测。（e）由于静电相互作用和构象扭曲，视紫红质相对于异视紫红质在能量上更稳定，两者都有利于R的稳定。INDO - PSDCI计算表明，视紫红质发色团 - CTN静电相互作用相对于I提供了 - 2 kcal mol⁻¹的增强稳定性。9 - 顺式发色团 - 赖氨酸系统的构象扭曲占 - 3 kcal mol⁻¹。（f）视紫红质中的能量存储为 - 60％的构象扭曲和40％的电荷分离。我们的模型预测，发色团与蛋白质构象扭曲能量的大部分涉及C，3（ - CH3）= CI4 - - C，5 = N - 赖氨酸部分与附近（未知）蛋白质残基的相互作用。（g）R和I中抗衡离子与发色团之间的强相互作用将在近红外区域产生弱但可能可观察到的电荷转移带。关键预测是I在859 nm（11,640 cm⁻¹）处存在可观察到的电荷转移跃迁，R在897 nm（11,150 cm⁻¹）处存在类似但稍弱的带。两个跃迁都涉及电子从抗衡离子转移到低能lθ*分子轨道。
