DETECTION OF A WATER MOLECULE IN THE ACTIVE-SITE OF BACTERIORHODOPSIN - HYDROGEN-BONDING CHANGES DURING THE PRIMARY PHOTOREACTION
DETECTION OF A WATER MOLECULE IN THE ACTIVE-SITE OF BACTERIORHODOPSIN - HYDROGEN-BONDING CHANGES DURING THE PRIMARY PHOTOREACTION
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DOI：
10.1021/bi00209a005
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发表时间：
1994-11-01
期刊：
影响因子：
2.9
通讯作者：
ROTHSCHILD, KJ
中科院分区：
文献类型：
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作者：
FISCHER, WB;SONAR, S;ROTHSCHILD, KJ
FTIR-差分光谱结合定点突变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，其在初级bR--> K光跃迁期间经历H-键合的增加。由于水的带出现在bR-> K FTIR差谱的OH伸缩区，当样品与（H2O）-O-18水合时，该带下移约12 cm（-1）。与（H2O）-H-2相反，（H2O）-O-18-诱导的位移即使在水合24小时后也不完全。这表明，即使水仍然能够与外部介质交换质子，但它部分被困在蛋白质的内部。在突变体Y57D中，这些条带不存在，而一组新的条带出现在低得多的频率，经历（H2O）-O-18诱导的位移。由此推断，我们检测到的水分子位于bR活性位点内，并可能与Tyr-57相互作用。其氢键强度的变化很可能是由于在bR--> K跃迁期间，视网膜发色团的光诱导全反式-> 13-顺式异构化和带正电荷的席夫碱的相关运动。相比之下，第二个水分子，其红外差谱带不受Y57D突变的影响，似乎经历了减少氢键在K-> L和L-> M过渡。
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 (H2O)-O-18. In contrast to (H2O)-H-2, the (H2O)-O-18-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 (H2O)-O-18-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.