首页> 外文OA文献 >Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate.
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Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate.

机译:细菌视紫红质的质子化转换的第一阶段的证据来自时间分辨的光电压和关于M-中间体光逆转的闪光光解实验。

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摘要

The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pK's of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.
机译:通过时间分辨光吸收光谱法和使用双闪激发(绿闪(532 nm)然后蓝闪(400 nm)的光电压测量)研究了细菌视紫红质(bR)的M中间体的光逆反应的动力学。可变延迟后)。光电压的符号和1H / 2H动力学同位素效应表明,席夫碱被席夫碱和细胞外表面之间的基团(可能是Asp85)质子化。动力学数据分析表明,在12摄氏度下于150 mM KCl中的电荷运动具有两个分量,分别具有约100 ns和约600 ns的时间常数,这与闪光灯和pH值之间的延迟时间无关。 。快速和慢速分量的幅度取决于延迟和pH。较慢的成分仅在延迟时间超过100微米之后才开始对电荷运动做出贡献,在低pH下不存在,并且振幅会以pKa约6增大。因为质子释放基团在70到100微米后会去质子并具有瞬态pKa值为5.8,这些结果表明以下分配:快速组分和快速组分与慢速组分的结合代表了两个M状态的光逆转,释放基团分别为质子化和去质子化。光逆转的缓慢阶段开始于释放基团去质子化且Asp85的pK升高的状态,这可能是由于Asp85的pK恢复到其初始低值所致。这为Asp85的pK与释放基团之间的偶联提供了进一步的证据,并表明质子释放是质子化转换的第一步。在碱性pH值下,来自背向光反应的电信号的振幅以8的pK降低,而M的量没有相应降低。在中性pH值下,Arg82带正电荷的胍基团从释放附近的位置移动表面上的Asp85基团对电光反向振幅起重要作用。在未光解状态下释放基团的pK之上(约8),Arg82停留在表面附近,导致相应的信号减少。

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  • 作者

    Dickopf, S; Heyn, M P;

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  • 年度 1997
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  • 原文格式 PDF
  • 正文语种 en
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