Ames J B, Raap J, Lugtenburg J, Mathies R A
Department of Chemistry, University of California, Berkeley 94720.
Biochemistry. 1992 Dec 22;31(50):12546-54. doi: 10.1021/bi00165a002.
Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.
利用在0.3M硝酸根、0.3M氯离子和3M氯离子中从5微秒到10毫秒的时间延迟，获得了嗜盐菌视紫红质光循环中HR520、HR640和HR578物种的动力学共振拉曼光谱。通过使用分子守恒约束将拉曼强度转换为绝对浓度。能令人满意地模拟数据的最简单动力学方案是HR578→HR520与HR640→HR578处于平衡状态。HR640→HR578转变的速率常数随氯离子浓度增加，表明氯离子在HR640和HR578之间被吸收。连接HR520和HR640的正向与反向速率常数之比随氯离子浓度的倒数增加，表明氯离子在HR520→HR640步骤中被释放。通过用[12,14 - 2H2]视黄醛再生蛋白质，研究了HR640中视黄醛发色团C13 = C14键的构型。在943厘米-1处观察到HR640的C12 - 2H + C14 - 2H摇摆振动，表明发色团是13 - 顺式的。HR640的共振拉曼光谱响应2H2O悬浮液的变化表明与蛋白质的席夫碱连接被质子化。HR640的指纹振动在2H2O中均无明显位移，表明席夫碱采用C = N反式构型；这一归属得到C15 - 2H摇摆模式频率（1002厘米-1）的支持。HR640中发色团的13 - 顺式结构要求在HR640→HR578转变中热异构化为全反式。这些结构和动力学结果被纳入氯离子泵浦的双态C - T模型。
