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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk.
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Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk.

机译：远距离质子沿紫色膜表面快速扩散，并延迟质子转移到主体中。

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摘要

The pH-indicator dye fluorescein was covalently 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 microseconds 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 microseconds, respectively. Under the same conditions, the proton release time in the bulk medium as detected by pyranine was around 880 microseconds--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) cm2/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.
机译：通过使溴甲基荧光素与引入的半胱氨酸的巯基反应，将pH指示剂染料荧光素共价结合到紫色膜表面的细菌视紫红质细胞外侧的72位和细胞质侧的101、105、160或231位。通过定点诱变。在细胞外表面上的位置72处，在闪光（条件：pH 7.3、22摄氏度和150 mM KCl）后71 +/- 4微秒内检测到光诱导的质子释放。在染料位于位置101、105和160的细胞质侧，相应的值分别为77、76和74 +/- 5微秒。在相同条件下，通过吡喃检测到的质子在散装介质中的释放时间约为880微秒，即慢了10倍以上。事实上，检测到在细胞外释放的质子要快得多。在细胞质表面上的水相比在本体相上的水相表明，它保留在表面上并沿着紫色膜迁移到另一侧。这些发现对生物能学和局部质子耦合的支持模型具有有趣的意义。从质膜相对侧的标记检测质子的时间之间的微小差异，我们估计在22摄氏度时质子表面扩散常数大于3 x 10（-5）cm2 / s。在5摄氏度下，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（席夫碱的去质子）中的较快时间。在较高温度下，这种相关性逐渐消失，但是质子释放时间的曲线阿伦尼乌斯图与低温下M升高的线性阿伦尼乌斯图成正切。这些观察结果与席夫碱去质子化和质子释放之间的动力学耦合是相容的。

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