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对细菌视紫红质光诱导质子泵的共振拉曼和光学瞬态研究揭示了平行的光循环。

Resonance Raman and optical transient studies on the light-induced proton pump of bacteriorhodopsin reveal parallel photocycles.

作者信息

Eisfeld W, Pusch C, Diller R, Lohrmann R, Stockburger M

机构信息

Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany.

出版信息

Biochemistry. 1993 Jul 20;32(28):7196-215. doi: 10.1021/bi00079a017.

Abstract

The photocycle of bacteriorhodopsin (bR) was studied at ambient temperature in aqueous suspensions of purple membranes using time-resolved resonance Raman (RR) and optical transient spectroscopy (OTS). The samples were photolyzed, and the fractional concentrations of the retinylidene chromophore in its parent state, BR570, and in the intermediate states L550, M412, N560, and O640 were determined in the time domain 20 microseconds-1 s and in the pH range 4-10.5. Two kinetically different L components could be identified. At pH 7 one fraction of L (approximately 65%) decays in 80 microseconds to M (deprotonation of the Schiff base), whereas the residual part is converted in approximately 0.5 ms to N. The RR spectra reveal only minor structural changes of the chromophore in the L-->N transition. These were attributed to a conformational change of the protein backbone [Ormos, P., Chu, K., & Mourant, J. (1992) Biochemistry 31, 6933]. With decreasing pH the L-->N transition is delayed to > 2 ms following a titration-like function with pKa approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (Mf, tau f; Ms, tau s; f = fast, s = slow). Both Mf and Ms consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). Mf occurs in the reaction sequences L-->Mf-->N-->BR and L-->Mf-->O-->BR. The population of the first sequence, in which N is formed with the time constant tau f (approximately 2-4 ms, pH 6-10.5), increases with pH. Ms is also found in two different reaction sequences of the form L-->Ms-->BR. The quantitative analysis reveals that each "titration effect" can be related to a certain fraction of bR. It is proposed that each fraction can be identified with a "subspecies" of bR which undergoes an independent and individual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from the pH dependence of the lifetimes of Ms and N that the reconstitution of BR570 in the reaction steps Ms-->BR and N-->BR requires the uptake of a proton from the external phase. It is argued that this proton catalyzes the reisomerization of retinal, whereas the Schiff base is internally reprotonated from Asp-85. A model for proton pumping is proposed in which the proton taken up from the external phase to catalyze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.

摘要

利用时间分辨共振拉曼光谱(RR)和光学瞬态光谱(OTS),在室温下对紫色膜水悬浮液中的细菌视紫红质(bR)的光循环进行了研究。对样品进行光解,并在20微秒至1秒的时域以及pH值4 - 10.5的范围内,测定视黄叉发色团处于其母体状态BR570以及中间状态L550、M412、N560和O640时的分数浓度。可以识别出两种动力学上不同的L成分。在pH 7时,一部分L(约65%)在80微秒内衰减为M(席夫碱去质子化),而剩余部分在约0.5毫秒内转化为N。RR光谱显示发色团在L→N转变过程中只有微小的结构变化。这些变化归因于蛋白质主链的构象变化[奥尔莫斯,P.,朱,K.,& 穆兰特,J.(1992年)《生物化学》31,6933]。随着pH值降低,L→N转变延迟至> 2毫秒,遵循pKa约为6.2的类似滴定的函数关系。通过OTS监测的M412的衰减,对于每个pH值都可以用两个不同的幅度和时间常数(Mf,τf;Ms,τs;f = 快,s = 慢)进行拟合。Mf和Ms都由亚成分组成,这些亚成分可以通过它们不同的反应途径区分(但不能通过OTS区分)。Mf出现在反应序列L→Mf→N→BR和L→Mf→O→BR中。第一个序列中N以时间常数τf(约2 - 4毫秒,pH 6 - 10.5)形成,其数量随pH值增加。Ms也存在于两种不同形式的反应序列L→Ms→BR中。定量分析表明,每个“滴定效应”都可以与一定比例的bR相关。有人提出,每个比例都可以与bR的一个“亚类”相识别,该亚类经历独立且独特的循环反应。建立了一个完整的反应方案,该方案代表了所观察到的现象的多样性。从Ms和N寿命的pH依赖性可以得出结论,在反应步骤Ms→BR和N→BR中BR570的重构需要从外部相中摄取一个质子。有人认为,这个质子催化视黄醛的重新异构化,而席夫碱则从Asp - 85进行内部质子化。提出了一种质子泵浦模型,其中从外部相中摄取以催化视黄醛重新异构化的质子,就是在bR光循环过程中通过膜泵出的质子。

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