Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.
Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.
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盐视紫红质光循环动力学、发色团结构和氯离子泵机制的共振拉曼研究。
DOI：
10.1021/bi00165a002
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Mathies,RA
中科院分区：
文献类型：
--
作者：
Ames,JB;Raap,J;Lugtenburg,J;Mathies,RA
1992年10月6日收到的修订版摘要：在0.3 M NO3-、0.3 M Cl-和3 M Cl-中，使用5 qs至10 ms范围内的时间延迟获得了盐视紫红质光循环中HR 520、HR 640和HR 578物质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。令人满意地模拟数据的最简单的动力学方案是HR 578-HR 520 ** HR 640-* HR 578。HR 640-* HR 578转变的速率常数随Cl-浓度增加，表明Cl-在HR 640和HR 578之间被吸收。连接HR 520和HR 640的正向速率常数与反向速率常数的比率随着Cl-浓度的倒数而增加，表明Cl-在HR 520-* HR 640步骤期间释放。通过用[12，14- 2H_2]视黄醛再生蛋白质，检查了HR_（64）中关于视黄醛发色团的C_（13）= C_（14）键的构型。在943 cm-1处观察到HR 640的C12 - 2 H + C [4- 2 H]摇摆振动，表明发色团是13-顺式的。HR 640的共振拉曼光谱响应于2 H20悬浮液的变化表明与蛋白质连接的席夫碱被质子化。HR 640指纹振动在2 H2O中没有显著偏移，表明席夫碱采用C= N反构型;这种分配得到C = 5- 2 H摇摆模式（1002 cm-1）的频率的支持。HR 640中发色团的13-顺式结构要求在HR 640-*·HR 578跃迁中发生热异构化回到全反式。这些结构和动力学结果被并入Cl-泵送的双态CT模型中。盐视紫红质（HR）是在盐生盐杆菌（Halobacterium halobium）的质膜中发现的一种含视网膜的蛋白质，其利用光能来主动运输氯离子（Lanyi，1986，1988; Oesterhelt & Tittor，1989）。这种氯离子转运调节细胞质离子强度并产生用于驱动ATP合成的膜电位。HR 378中的全反式视网膜修复基团的光吸收启动了图1所示的循环光化学反应。
Revised Manuscript Received October 6, 1992 abstract: Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 qs to 10 ms in 0.3 M N03", 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** HR640-* HR578, The rate constant for the HR640-*• HR578 transition increases with Cl" concentration, suggesting that Cl" is taken up between HR64o and HR578. The ratio of the forward to the reverse rate constants connecting HR52o and HR640 increases as the inverse of the Cl" concentration, suggesting that Cl" is released during the HR52o-* HR640 step. The configuration about the Ci3= Ci4 bond of the retinal chromophore in HR64o is examined by regenerating the proteinwith [12, 14-2H2] retinal. The Ci2-2H+ C [4-2H rocking vibration for HR640 is observed at 943 cm"’, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to2H20 suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H20, suggesting that the Schiff base adopts a C= N anti configuration; this assignment is supported by the frequency of the C) 5-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR64o requires that thermal isomerization back to all-trans occurs in the HR64o—*• HR578 transition. These structural and kinetic results are incorporated into a two-state CT model forCl" pumping.Halorhodopsin (HR), 1 a retinal-containing protein found in the plasma membrane of Halobacterium halobium, utilizes light energy to activelytransport chloride ions (Lanyi, 1986, 1988; Oesterhelt & Tittor, 1989). This chloride transport regulates the cytoplasmic ionic strength and generates a membrane potential that is used to drive ATP synthesis. Light absorption by the all-trans-retinal prostheticgroup in HR378 initiates the cyclic photochemical reaction depicted in Figure