THE 2 PK(A) OF ASPARTATE-85 AND CONTROL OF THERMAL-ISOMERIZATION AND PROTON RELEASE IN THE ARGININE-82 TO LYSINE MUTANT OF BACTERIORHODOPSIN
THE 2 PK(A) OF ASPARTATE-85 AND CONTROL OF THERMAL-ISOMERIZATION AND PROTON RELEASE IN THE ARGININE-82 TO LYSINE MUTANT OF BACTERIORHODOPSIN
复制标题
DOI：
10.1021/bi00027a034
复制
发表时间：
1995-07-11
期刊：
影响因子：
2.9
通讯作者：
MENICK, DR
中科院分区：
文献类型：
--
作者：
BALASHOV, SP;GOVINDJEE, R;MENICK, DR
为了探索Arg 82在细菌视紫红质中催化质子转移的作用，我们用Lys代替Arg 82，Lys在中性pH下也带正电荷，但具有比Arg低约1.7个pH单位的固有pK（a）。在盐生盐杆菌中表达的R82 K突变体中，我们发现以下：（1）低pH下紫-蓝转变的pK（a）（反映pK;，Asp 85）为3.6 +/-0.1，在高pH值下，pK（a）= 8.0时出现蓝紫转变的第二个拐点。Asp 85的络合滴定行为表明，pK（a）= 8.0时，Asp 85的质子化依赖于另一个氨基酸残基X '的质子化状态，X'在R82 K中具有pK（a）= 8.0。实验数据与两个相互作用残基的模型的拟合表明，在高pH下X'的去质子化导致Asp 85的pK（a）从3.7移动到6.0。反过来，Asp 85的质子化使X'的pK（a）降低2.3个pH单位，这表明X'在光循环中形成M中间体和伴随的Asp 85质子化时可以释放质子。(2)暗适应速率常数k（da）在pH 2 ~ 10范围内与蓝膜的比例成正比，表明热异构化是通过Asp 85的瞬时质子化进行的。k（da）的pH依赖性表明pK（a1）= 3.9和pK（a2）= 8.0的两个基团控制着R82 K的暗适应速率。与野生型（WT）（pK（a2）= 9.7）相比，R82 K中pK（a2）的1.7个pH单位的偏移支持了X'在WT中是Arg 82而在R82 K中是Lys 82的假设（或：至少这些基团是构成X ′的残基簇的主要部分），（3）在稳态光照下，掺入磷脂酰胆碱囊泡中的R82 K中的质子转运效率至少是WT中的40%，闪光诱导的pH敏感染料吡喃的瞬态信号与WT中的信号相似（质子释放先于摄取），但在R82 K中的幅度较小（约为WT中发现的幅度的15%），表明仅释放一小部分质子。在R82 K中快速。这支持了Arg 82与质子释放途径相关的建议（4）R82 K显示出不依赖于pH的M形成速率，缺乏O中间体的积累，以及13-顺式颜料K-c的电解质产物的快速衰减在低pH和中性pH下，R82 K含有大量处于光适应状态的13-cis-bR，因为trans-bR在pH < 9的光照下被转化回13-cis-bR。光适应的pH依赖性导致M中间体的pi-I依赖性产量（pK（a）= 8.0）。还观察到两个具有类似pK（a）的可逆跃迁：发色团吸收带红移2nm和色氨酸残基吸收带红移。（5）Lys 82可被乙酸酐修饰。Lys 82的乙酰化使Asp 85的pK（a）移动到6.5，这与我们的模型对当X'去质子化时Asp 85的pK（a）的预测一致。
TO explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pK(a) of about 1.7 pH units lower than that of Arg, In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pK(a) of the purple-to-blue transition at low pH (which reflects the pK;, of Asp85) is 3.6 +/- 0.1, At high pH a second inflection in the blue-to-purple transition with pK(a) = 8.0 is found. The complex titration behavior of Asp85 indicates that the pK, of Asp85 depends on the protonation state of another amino acid residue, X', which has a pK(a) = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pK(a) of Asp85 from 3.7 to 6.0, In turn, protonation of Asp85 decreases the pK(a) of X' by 2.3 pH units, This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, k(da), is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85, The pH dependence of k(da) shows that two groups with pK(a1) = 3.9 and pK(a2) = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pK(a2) in R82K compared to the wild type (WT) (pK(a2) = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or:lt least that these groups are the principal part of a cluster of residues that constitute X'), (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT, A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released. fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arl in this process, (4) R82K shows a pH-independent rate of M formation, lack of accumulation of the O intermediate, and fast decay of the bathoproduct of the 13-cis pigment, K-c (0.2 ms), At low and neutral pH, R82K contains a significant fraction of 13-cis-bR in the light-adapted state since trans-bR is convened back into 13-cis-bR under illumination at pH < 9, The pH dependence of light adaptation results in a pi-I-dependent yield of the M intermediate (pK(a) = 8.0). Two other reversible transitions with similar pK(a)'s are observed: a 2 nm red shift of the chromophore absorption band and a red shift of the absorption band of a Trp residue, (5) Lys82 is accessible to modification by acetic anhydride. Acetylation of Lys82 shifts the pK(a) of Asp85 to 6.5, which agrees with the prediction of our model for the pK(a) of Asp85 when X' is deprotonated.