Hessling B, Herbst J, Rammelsberg R, Gerwert K
Lehrstuhl für Biophysik, Fakultät Biologie, Ruhr Universität Bochum, Germany.
Biophys J. 1997 Oct;73(4):2071-80. doi: 10.1016/S0006-3495(97)78237-7.
The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M1 to M2 transition. To resolve the molecular events in this M1 to M2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M1 intermediate, and for longer delay times most of the reset M molecules are in the M2 intermediate. The BR-M1 and BR-M2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is kM1 = 0.8 x 10(7) s(-1) in the light-induced M1 back-reaction and kM2 = 0.36 x 10(7) s(-1) in the M2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M1 to M2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M1 to M2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.
中心质子结合位点（质子化席夫碱）从质子释放侧到质子摄取侧的取向对于质子泵细菌视紫红质的方向性至关重要。有人提出，这种被称为再质子化开关的运动发生在M1到M2的转变过程中。为了解决这一M1到M2转变中的分子事件，我们进行了双闪光实验。在这些实验中，第一个脉冲启动光循环，第二个脉冲选择性地将处于M中间体的细菌视紫红质分子驱动回到BR基态。对于启动脉冲和重置脉冲之间的短延迟时间，大多数被重置的M分子处于M1中间体，而对于较长的延迟时间，大多数被重置的M分子处于M2中间体。通过纳秒级步进扫描傅里叶变换红外光谱监测BR-M1和BR-M2的差光谱。由于在光诱导的M1反向反应中席夫碱的再质子化速率为kM1 = 0.8×10^7 s^(-1)，在M2反向反应中为kM2 = 0.36×10^7 s^(-1)，这两种不同的M中间体代表了席夫碱的两种不同的质子可及性构型。结果表明，在M1到M2的转变中，只有一两个肽键发生了微小的移动，这改变了席夫碱与Y185的相互作用。这种主链运动与随后的M到N转变中的较大运动不同。在M1到M2的转变中没有观察到发色团异构化的证据。此外，结果表明，在M光反向反应中，席夫碱是从D85进行时间分辨再质子化的，而不是像传统循环那样从D96进行再质子化。
