Fischer W B, Sonar S, Marti T, Khorana H G, Rothschild K J
Physics Department, Boston University, Massachusetts 02215.
Biochemistry. 1994 Nov 1;33(43):12757-62. doi: 10.1021/bi00209a005.
FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.
傅里叶变换红外差示光谱法结合定点诱变技术已被用于研究细菌视紫红质光循环过程中水的作用。检测到至少一个水分子，在初级bR→K光转变过程中其氢键作用增强。在bR→K傅里叶变换红外差示光谱的OH伸缩区域出现了归因于水的谱带，当样品用H₂¹⁸O水合时，这些谱带向下位移约12 cm⁻¹。与2H₂O不同，即使水合24小时后，H₂¹⁸O诱导的位移也不完全。这表明，尽管水仍能与外部介质交换质子，但它部分被困在蛋白质内部。在突变体Y57D中，这些谱带不存在，而出现了一组频率低得多的新谱带，它们会发生H₂¹⁸O诱导的位移。得出的结论是，我们检测到的水分子位于bR活性位点内部，可能与Tyr-57相互作用。其氢键强度的变化很可能是由于视黄醛发色团的光诱导全反式→13-顺式异构化以及bR→K转变过程中带正电的席夫碱的相关移动。相比之下，另一个水分子的红外差示谱带不受Y57D突变的影响，在K→L和L→M转变过程中其氢键作用似乎减弱。
