Photoinduced proton release in proteorhodopsin at low pH: The possibility of a decrease in the pKa of Asp227
Photoinduced proton release in proteorhodopsin at low pH: The possibility of a decrease in the pKa of Asp227
复制标题
低 pH 条件下蛋白视紫红质光诱导质子释放：Asp227 pKa 降低的可能性
DOI：
10.1021/bi300940p
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Kamo Naoki
中科院分区：
文献类型：
--
作者：
Tamogami Jun;Kikukawa Takashi;NaraToshifumi ;Shimono Kazumi;Demura Makoto;Kamo Naoki
前视紫红质（PR）是海洋真细菌中发现的微生物视紫红质之一，可能起着向外光驱动质子泵的作用。此前，我们[Tamogami，J.，等人（2009）Photochem. Photobiol.85，578-589]报道了使用透明ITO（氧化铟锡）或SnO 2电极（其用作时间分辨pH电极）在pH 5和10之间的PR中发生光诱导质子转移。在本文所述的研究中，研究了在低pH（<4）下的质子转移。在这些条件下，Asp 97，质子化席夫碱的主要抗衡物，被质子化。我们观察到的第一个质子释放，其次是吸收，在这个过程中，然而，M中间体没有形成。通过使用几个PR突变体的实验，我们发现Asp 227在质子释放中起着至关重要的作用。该残基对应于细菌视紫红质的Asp 212残基，即所谓的二级席夫碱。我们估计这个残基在黑暗和释放质子的光产物中的pKa分别为10.3.0和10.2.3。还通过光谱法估算了Asp 227在黑暗中的pKa值，其值约等于ITO实验测定的值，这可能意味着Asp 227可能释放质子。在无Cl-存在的情况下，我们观察到D227 N的质子释放，并发现主抗衡离子Asp 97起着关键作用。据推测，负电荷是需要通过Asp 227（第一选择），Cl-（第二选择）的结合，或Asp 97的去质子化的光产物的稳定。除鱼腥藻属（Anabaenasensory）视紫红质（BR）和盐视紫红质（halorhodopsin）的Asp 212或Asp 227位上缺少可解离残基外，其它微生物视紫红质在酸性介质中也观察到光诱导质子释放（可能是由于次级电荷的pKa降低）。本文还讨论了这种pKadrease的意义。
Proteorhodopsin (PR) is one of the microbial rhodopsins that are found in marine eubacteria and likely functions as an outward light-driven proton pump. Previously, we [Tamogami, J., et al. (2009)Photochem. Photobiol.85, 578–589] reported the occurrence of a photoinduced proton transfer in PR between pH 5 and 10 using a transparent ITO (indium–tin oxide) or SnO2electrode that works as a time-resolving pH electrode. In the study presented here, the proton transfer at low pH (<4) was investigated. Under these conditions, Asp97, the primary counterion to the protonated Schiff base, is protonated. We observed a first proton release that was followed by an uptake; during this process, however, the M intermediate did not form. Through the use of experiments with several PR mutants, we found that Asp227 played an essential role in proton release. This residue corresponds to the Asp212 residue of bacteriorhodopsin, the so-called secondary Schiff base counterion. We estimated the pKaof this residue in both the dark and the proton-releasing photoproduct to be ∼3.0 and ∼2.3, respectively. The pKavalue of Asp227 in the dark was also estimated spectroscopically and was approximately equal to that determined with the ITO experiments, which may imply the possibility of the release of a proton from Asp227. In the absence of Cl–, we observed the proton release in D227N and found that Asp97, the primary counterion, played a key role. It is inferred that the negative charge is required to stabilize the photoproducts through the deprotonation of Asp227 (first choice), the binding of Cl–(second choice), or the deprotonation of Asp97. The photoinduced proton release (possibly by the decrease in the pKaof the secondary counterion) in acidic media was also observed in other microbial rhodopsins with the exception of theAnabaenasensory rhodopsin, which lacks the dissociable residue at the position of Asp212 of BR or Asp227 of PR and halorhodopsin. The implication of this pKadecrease is discussed.