RAPID LONG-RANGE PROTON DIFFUSION ALONG THE SURFACE OF THE PURPLE MEMBRANE AND DELAYED PROTON-TRANSFER INTO THE BULK
RAPID LONG-RANGE PROTON DIFFUSION ALONG THE SURFACE OF THE PURPLE MEMBRANE AND DELAYED PROTON-TRANSFER INTO THE BULK
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DOI：
10.1073/pnas.92.2.372
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发表时间：
1995-01-17
影响因子：
11.1
通讯作者：
HEYN, MP
中科院分区：
文献类型：
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作者：
ALEXIEV, U;MOLLAAGHABABA, R;HEYN, MP
通过使溴甲基荧光素与通过定点诱变引入的半胱氨酸的巯基反应，将pH指示剂染料荧光素在细菌跳蛋白的细胞外侧的位置72和细胞质侧的位置101、105、160或231处共结合到紫膜的表面。在位置72，在细胞外表面上，在闪光后71 +/-4 μ s检测到光诱导的质子释放（条件：pH 7.3，22 ℃和150 mM KCl）。在101、105和160位染料的细胞质侧，相应值分别为77、76和74 +/- 5 μ s。在相同条件下，通过吡喃检测的本体介质中的质子释放时间为约880 μ s-即，在细胞外侧释放的质子在细胞质表面上比在含水体相中更快地被检测到的事实表明，它被保留在表面上并沿着紫膜迁移到另一侧。这些发现对生物能量学和局部质子耦合的支持模型具有有趣的意义，根据膜两侧标记的质子检测时间之间的微小差异，我们估计在22 ℃时质子表面扩散常数大于3 × 10 - 5 cm-2/s。在5 ℃下，位置72处的质子释放检测时间等于M中间体的两个主要上升时间中较快的一个（席夫碱的去质子化）。在较高温度下，这种相关性逐渐消失，但质子释放时间的曲线Arrhenius图与低温下M上升的线性Arrhenius图相切。这些观察结果与Schiff碱去质子化和质子释放之间的动力学耦合是相容的。
The pH-indicator dye fluorescein was co; valently bound to the surface of the purple membrane at position 72 on the extracellular side of bacteriorhopsin and at positions 101, 105, 160, or 231 on the cytoplasmic side by reacting bromomethylfluorescein with the sulfhydryl groups of cysteines introduced by site-directed mutagenesis. At position 72, on the extracellular surface, the light-induced proton release was detected 71 +/- 4 mu s after the flash (conditions: pH 7.3, 22 degrees C, and 150 mM KCl). On the cytoplasmic side with the dye at positions 101, 105, and 160, the corresponding values were 77, 76, and 74 +/- 5 mu s, respectively. Under the same conditions, the proton release time in the bulk medium as detected by pyranine was around 880 mu s-i.e., slower by a factor of more than 10, The fact that the proton that is released on the extracellular side is detected much faster on the cytoplasmic surface than in the aqueous bulk phase demonstrates that it is retained on the surface and migrates along the purple membrane to the other side, These findings have interesting implications for bioenergetics and support models of local proton coupling, From the small difference between the proton detection times by labels on opposite sides of the membrane, we estimate that at 22 degrees C the proton surface diffusion constant is greater than 3 x 10(-5) cm(2)/s. At 5 degrees C, the proton release detection time at position 72 equals the faster of the two main rise times of the M intermediate (deprotonation of the Schiff base). At higher temperatures this correlation is gradually lost, but the curved Arrhenius plot for the proton release time is tangential to the linear Arrhenius plot for the rise of M at low temperatures, These observations are compatible with kinetic coupling between Schiff base deprotonation and proton release.