Proton transfers in the photochemical reaction cycle of proteorhodopsin
Proton transfers in the photochemical reaction cycle of proteorhodopsin
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DOI：
10.1021/bi025563x
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发表时间：
2002-04-30
期刊：
影响因子：
2.9
通讯作者：
Lanyi, JK
中科院分区：
文献类型：
--
作者：
Dioumaev, AK;Brown, LS;Lanyi, JK
测定了变形紫质（PR）的光谱和光化学性质，比较了其与细菌紫质（BR）的质子传递步骤。在可见光和红外（FTIR和FT-Raman）中对野生型PR和几个突变体进行了静态和时间分辨测量。观察到的C=O拉伸带的分配表明，Asp-97和Glu-108分别作为视网膜希夫碱的质子受体和供体，BR中相应位置的残基也是如此，但两种蛋白质之间存在许多光谱和动力学差异。在PR中没有可检测到的黑暗适应，并且发色团几乎完全包含全反式视网膜。由于Asp97的pK(a)相对较高（7.1），质子传递光循环仅在碱性ph下产生。它包含至少7个瞬态，衰减时间在10 ~ 200 ms之间，但分析显示只有三种不同的光谱形式。第一个是红移的类k态。质子释放不会发生在第二个m样中间体非常缓慢（几毫秒）的上升过程中，这与BR中缺乏促进细胞外质子释放的残基一致。质子从主体摄取，可能是在细胞质侧，发生在释放之前（tau类似于2 ms），并与视网膜希夫碱的再生相一致。与BR的N态一样，该过程产生的中间体也含有13-顺式视网膜，但其吸收最大值相对于PR发生了红移（如BR的0态）。这种n -样态的衰变与视网膜的全径再异构化相耦合，并在其C-C拉伸带中产生o -样态，但其吸收最大值明显接近未光解的PR。
The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 mus to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau similar to 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the 0 state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trails, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that Of unphotolyzed PR.