Pan Duohai, Ganim Ziad, Kim Judy E, Verhoeven Michiel A, Lugtenburg Johan, Mathies Richard A
Department of Chemistry, University of California, Berkeley, California 94720, USA.
J Am Chem Soc. 2002 May 1;124(17):4857-64. doi: 10.1021/ja012666e.
Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.
进行了时间分辨共振拉曼微芯片流动实验,以获取视紫红质的BSI中间体中生色团的振动光谱,并探测从嗜热视紫红质到BSI以及从BSI到发光视紫红质转变过程中的结构变化。从250纳秒到3微秒的动力学拉曼光谱确定了BSI的关键振动特征。BSI在886和945厘米⁻¹处表现出相对较强的HOOP模式,分别归属于C(14)H和C(11)H = C(12)H A(u)摆动。这一结果表明,在从嗜热视紫红质到BSI的转变中,高度应变的全反式生色团在C(10)-C(11)=C(12)-C(13)区域已松弛,但在C(14)附近仍有扭曲。与发光视紫红质和变视紫红质I相比,BSI中11,12 A(u) HOOP模式的低频表明,由于BSI生色团在C(11)=C(12)附近的残余扭曲,11H和12H摆动之间的耦合较弱。BSI中1653厘米⁻¹处的C=NH⁺伸缩模式表现出正常的氘化诱导下移23厘米⁻¹,这意味着在从嗜热视紫红质到BSI的转变中,席夫碱抗衡离子区域没有明显的结构重排。然而,在从BSI到发光视紫红质的转变中,席夫碱环境发生了显著变化,因为发光视紫红质中1638厘米⁻¹处的C=NH⁺伸缩模式异常低,在D₂O中仅移动7厘米⁻¹,这表明它基本上没有氢键受体。有了这些数据,我们首次能够比较和讨论视觉激发中所有关键中间体的室温共振拉曼振动结构。