THE RETINAL SCHIFF BASE-COUNTERION COMPLEX OF BACTERIORHODOPSIN - CHANGED GEOMETRY DURING THE PHOTOCYCLE IS A CAUSE OF PROTON-TRANSFER TO ASPARTATE-85
THE RETINAL SCHIFF BASE-COUNTERION COMPLEX OF BACTERIORHODOPSIN - CHANGED GEOMETRY DURING THE PHOTOCYCLE IS A CAUSE OF PROTON-TRANSFER TO ASPARTATE-85
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DOI：
10.1021/bi00206a001
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发表时间：
1994-10-11
期刊：
影响因子：
2.9
通讯作者：
LANYI, JK
中科院分区：
文献类型：
--
作者：
BROWN, LS;GAT, Y;LANYI, JK
细菌视紫红质含有通过质子化的席夫碱连接到K216的全反式视黄醇。在该泵中的质子传输是由视黄醛的全反式到13-顺式光异构化和随后的席夫碱质子到D85的转移引发的。光异构化后席夫碱与D85的几何关系发生了变化，这可能是导致质子转移的原因。我们引入了小的体积/形状变化，对与K216侧链接触的残基V49和A53进行位点特异性诱变，以迫使席夫碱相对于D85进入稍微不同的位置。早些时候[Zimanyi，L.，瓦尔普，G.，张，M.，Ni，B.，尼德曼河，和Lanyi，J.K.（1992）Biochemistry 31，8535-8543]我们已经用方案L双左右箭头M（1）双左右箭头M（2）+ H+（其中第一平衡是内部质子转移，第二平衡是细胞外表面上的质子释放）描述了光激发后微秒至毫秒时间范围内吸光度变化的动力学。在不同的pH值与突变体，其中选定的速率常数发生变化的测试，现在证实了这一计划的有效性。因此，M态的动力学允许检查在L双左右箭头M（1）反应中发展的瞬态平衡，并且代表了席夫碱和D85之间质子的再分配。根据蛋白质的结构，预测V49 A和V49 M残基替换均会导致席夫碱和D85的对齐减少，并且确实如此。我们发现它们都将平衡向质子化的席夫碱方向改变。相比之下，预测残基置换A53 V和A53 G以相反方向移动席夫碱，分别远离和更接近与D85的比对。前者确实使平衡向质子化Schiff碱方向改变，后者向去质子化Schiff碱方向改变。此外，在L状态的结合水的羟基拉伸带的影响，不利于质子转移到D85的所有突变。我们得出结论，在希夫碱-D85对，介导的束缚水的质子供体和受体的几何形状，是一个决定因素的质子转移平衡。
Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimanyi, L., Varp, G., Chang, M., Ni, B., Needleman, R., and Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L double left right arrow M(1) double left right arrow M(2) + H+ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L double left right arrow M(1) reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed. we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.