Dickopf S, Heyn M P
Biophysics Group, Department of Physics, Freie Universität Berlin, Germany.
Biophys J. 1997 Dec;73(6):3171-81. doi: 10.1016/S0006-3495(97)78343-7.
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.
利用双闪光激发（一个绿色闪光（532纳米），随后在可变延迟后施加一个蓝色闪光（400纳米））的时间分辨光吸收光谱和光电压测量，研究了细菌视紫红质（bR）的M中间体的光逆转反应动力学。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一个基团重新质子化，可能是天冬氨酸85。动力学数据分析表明，在12摄氏度的150 mM KCl中，电荷移动的特征是两个分量，时间常数分别约为100纳秒和约600纳秒，这两个分量与闪光之间的延迟时间和pH无关。快速和慢速分量的幅度取决于延迟时间和pH。较慢的分量仅在延迟超过100微秒后才开始对电荷移动有贡献，在低pH时不存在，并且幅度随着约为6的pKa增加。由于质子释放基团在70 - 100微秒后去质子化，并且具有5.8的瞬态pKa，这些结果表明以下归属：快速以及快速和慢速分量的组合分别代表来自两个M状态的光逆转，其中释放基团分别被质子化和去质子化。光逆转的慢相从释放基团去质子化且天冬氨酸85的pK升高的状态开始，并且可能是由于天冬氨酸85的pK恢复到其初始低值。这为天冬氨酸85和释放基团的pK之间的耦合提供了进一步的证据，并表明质子释放是重新质子化开关的第一步。在碱性pH下，来自反向光反应的电信号幅度以约为8的表观pK降低，而M的量没有相应减少。在中性pH下，精氨酸82带正电荷的胍基从表面上靠近释放基团的位置移动到天冬氨酸85，对光电逆转幅度有很大贡献。在未光解状态下释放基团的pK（约为8）以上，精氨酸82停留在表面附近，导致相应的信号降低。
