Deng H, Huang L, Callender R, Ebrey T
Department of Physics, City College of the City University of New York, New York 10031.
Biophys J. 1994 Apr;66(4):1129-36. doi: 10.1016/S0006-3495(94)80893-8.
The retinal chromophores of both rhodopsin and bacteriorhodopsin are bound to their apoproteins via a protonated Schiff base. We have employed continuous-flow resonance Raman experiments on both pigments to determine that the exchange of a deuteron on the Schiff base with a proton is very fast, with half-times of 6.9 +/- 0.9 and 1.3 +/- 0.3 ms for rhodopsin and bacteriorhodopsin, respectively. When these results are analyzed using standard hydrogen-deuteron exchange mechanisms, i.e., acid-, base-, or water-catalyzed schemes, it is found that none of these can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can not be hydroxyl (or acid-)-catalyzed. Moreover, the deuterium-hydrogen exchange of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicted to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa values of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodopsin (pKa approximately 13.5). We have also measured the deuterium-hydrogen exchange of a protonated Schiff base model compound in aqueous solution. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-catalyzed schemes. Remarkably, the water-catalyzed exchange, which has a half-time of 16 +/- 2 ms and which dominates at pH 3.0 and below, is slower than the exchange rate of the Schiff base in rhodopsin and bacteriorhodopsin. Thus, there are two anomalous results, the inconsistency of the observed hydrogen exchange rates of retinal Schiff base in the two pigments with those predicted from the standard exchange schemes and the enhancement of the rate of hydrogen exchange in the two proteins over the model Schiff base in aqueous solution. We suggest that these results are explained by the presence of a structural water molecule (or molecules) at the retinal binding sites of the two pigments, quite close, probably-hydrogen bonded, to the Schiff base proton. In this case, the rate of exchange can be faster than that found for the model compound due to an "effective water concentration" near the Schiff base that is increased from that found in aqueous solution.
视紫红质和细菌视紫红质的视网膜发色团均通过质子化席夫碱与它们的脱辅基蛋白结合。我们对这两种色素进行了连续流动共振拉曼实验，以确定席夫碱上的氘核与质子的交换非常快，视紫红质和细菌视紫红质的半衰期分别为6.9±0.9毫秒和1.3±0.3毫秒。当使用标准的氢-氘交换机制（即酸催化、碱催化或水催化方案）分析这些结果时，发现这些机制均无法解释实验结果。由于发现交换速率与pH无关，所以氘-氢交换不可能是由羟基（或酸）催化的。此外，视网膜席夫碱的氘-氢交换也不可能由作为碱的水催化，因为在那种情况下，预计的交换速率要比观察到的慢几个数量级。计算得到的相对较慢的交换速率主要是由于视紫红质（pKa>17）和细菌视紫红质（pKa约为13.5）中席夫碱的高pKa值。我们还测量了质子化席夫碱模型化合物在水溶液中的氘-氢交换。与色素的席夫碱不同，其交换特性依赖于pH，并且与标准碱催化方案一致。值得注意的是，水催化交换的半衰期为16±2毫秒，在pH 3.0及以下占主导，但其比视紫红质和细菌视紫红质中席夫碱的交换速率要慢。因此，出现了两个异常结果，即两种色素中视网膜席夫碱的观察到的氢交换速率与从标准交换方案预测的速率不一致，以及两种蛋白质中的氢交换速率比水溶液中的模型席夫碱有所提高。我们认为，这些结果可以通过两种色素的视网膜结合位点处存在一个（或多个）结构水分子来解释，该水分子与席夫碱质子非常接近，可能通过氢键相连。在这种情况下，由于席夫碱附近的“有效水浓度”比水溶液中的有所增加，交换速率可能比模型化合物的更快。
