Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct.
Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct.
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细菌视紫红质突变体 Tyr-185-->Phe 的傅里叶变换拉曼光谱：在光适应过程中形成稳定的 O 类物质并检测其瞬态 N 类光产物。
DOI：
10.1021/bi00060a020
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Rothschild,KJ
中科院分区：
文献类型：
--
作者：
Rath,P;Krebs,MP;He,Y;Khorana,HG;Rothschild,KJ
摘要：近红外FT-拉曼光谱可用于测量细菌视紫红质（bR）发色团的振动，而没有传统可见光共振拉曼光谱的缺点，其中可见光激发驱动bR光反应。我们利用这种技术研究了室温下细菌视紫红质和突变体Tyr-185-^ Phe（Y185 F）在溶液中的光暗适应。与野生型bR相比，光适应Y185 F的FT-拉曼和共振拉曼光谱显示了O中间体振动的新特征。发现Y185 F的光适应涉及视网膜发色团的13-顺式，C= N顺式-全反式异构化，其产生类似于bRsvo的物质和第二类O物质。暗适应，这是慢得多的Y185 F相比，野生型bR，涉及一个平行的衰减的bRsvo和O-样的物种，并导致在一个降低的全反式：13-顺式比相比，野生型。Y185 F中存在类O物质的进一步证据来自泵浦-探测拉曼差谱，其中发现红色泵浦光束产生与光循环中的N中间体非常相似的物质。通过频闪拉曼测量表明，该物种即使在高pH下也短暂存在。我们假设，当Y 185 F发色团具有全反式结构时，由于Asp-212/Tyr-185氢键的破坏，Y 185 F中Asp-85和Asp-212的有效pKa升高，从而解释了O样物质中这些残基质子化的增加。1是在极端嗜盐杆菌（Halobacterium halobium）的紫色膜中发现的一种整合膜蛋白（Oesterhelt & Stoeckenius，1971; Stoeckenius & Bogomolni，1982）。在吸收一个光子后，bR经历一系列的光循环，包括一系列的中间体：Ké 30、L550、M412、N550和C> 640，每一个都有其特征吸收最大值。在这个过程中，一个净质子穿过紫膜从细胞内部运输到细胞外部。虽然这些中间体的动力学和可见光吸收已被很好地表征，但它们在质子传输机制中的确切作用尚未完全了解。阐明bR中质子泵机制的一种方法是应用振动光谱学。共振拉曼光谱（RRS）提供了一种通过选择性地增强它们的振动来研究各种亚视黄基发色团（包括bR及其光中间体的发色团）的结构的手段[关于综述，参见例如Callender和Honig（1977）、Warshel（1977）和Mathies等人（2005）]。（1991）和其中引用的参考文献]。这项工作产生了一个
Revised Manuscript Received December 18, 1992 abstract: Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visibleexcitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-^ Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adaptedY185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C= N syn—’• all-trans isomerization of the retinal chromophore which produces a species similar to bRsvo and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bRsvo and O-like species and resulted in a decreased all-trans: 13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shownby stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated inY185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.Bacteriorhodopsin (bR) 1 is an integral membrane protein found in the purple membrane of the extremely halophilic Halobacterium halobium (Oesterhelt & Stoeckenius, 1971; Stoeckenius & Bogomolni, 1982). Upon absorption of a photon, bR undergoes a photocycle involving a series of intermediates: Ké30, L550, M412, N550, and C> 640, each with its characteristic absorption maximum. In this process, a net proton is transported across purple membrane from the inside to the outside of the cell. Although the kinetics and visible absorption of each of these intermediates havebeen well characterized, their exact role in the proton transport mech-anism is not yet completely understood. One approach to elucidating the mechanism of proton pumping in bR is the application of vibrational spectroscopy. Resonance Raman spectroscopy (RRS) provides a means to study the structure of various retinylidene chromophores including those of bR and itsphotointermediates by selectively enhancing their vibrations [for reviews, see, eg, Callender and Honig (1977), Warshel (1977), and Mathies et al.(1991) and references citedtherein]. This work has resulted in a