Balashov S P, Imasheva E S, Govindjee R, Ebrey T G
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign 61801, USA.
Biophys J. 1996 Jan;70(1):473-81. doi: 10.1016/S0006-3495(96)79591-7.
Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.
在较宽的pH范围（2 - 11）内对细菌视紫红质中作为质子受体和反离子一部分的天冬氨酸85（Asp - 85）进行滴定，得出以下结论：1）Asp - 85具有复杂的滴定曲线，有两个pKa值；除了pKa = 2.6的主要转变外，它在高pH值时还显示出第二个拐点（在150 mM KCl中pKa = 9.7）。Asp - 85这种复杂的滴定行为是由Asp - 85与可电离残基X'的相互作用所解释的。从Asp - 85的滴定曲线拟合结果可知，X'的去质子化通过将其pKa从2.6变为7.5，增加了Asp - 85的质子亲和力。相反，Asp - 85的质子化使X'的pKa降低4.9个单位，从9.7降至4.8。Asp - 85与X'之间的相互作用对质子转移机制具有重要意义。在光循环中M中间体形成后（以及Asp - 85质子化），基团X'应释放一个质子。X'的这种去质子化状态将稳定Asp - 85的质子化状态。2）发色团的热异构化（暗适应）发生在Asp - 85的瞬时质子化和蓝色膜形成时。后一结论基于以下观察结果：在pH 2至11之间，暗适应的速率常数与蓝色膜（其中Asp - 85质子化）的比例成正比。异构化的速率常数在蓝色膜中比在紫色膜中至少快10^4倍。Asp - 85的质子化状态可能不仅对暗适应期间全反式⇔13 - 顺式热异构化的催化很重要，而且对光循环中N→O→bR转变期间发色团从13 - 顺式重新异构化为全反式构型也很重要。这可以解释为什么Asp - 85在N和O中间体中保持质子化状态。
