Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227
Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227
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DOI：
10.1021/bi050438h
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发表时间：
2005-08-16
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：
Imasheva, ES;Shimono, K;Lanyi, JK
变形视紫红质是一种类似于古细菌的细菌视紫红质的海洋变形细菌的视网膜蛋白，是一种光驱动质子泵。光量子的吸收启动一个反应周期（大约50毫秒的转换时间），其中包括视网膜从全反式到13顺式的光异构化和视网膜席夫碱的短暂去质子化，随后恢复初始状态。我们在这里报道，除了这种快速循环转换，在高pH下的照明导致长寿命的光产物在362 nm吸收的积累。在D227N突变体中，阴离子Asp227（与Asp97一起构成希夫碱反离子）被中性残基取代，这种光转化效率更高。在pH 8.5的光照下，大部分D227N色素转化为362 nm的物质，量子效率约为0.2。在野生型中，这种转变的pK(a)为9.6，但在Asp227突变后降至7.5。光产物吸收最大值的短波长表明它具有去质子化的希夫碱。在黑暗中，该光产物以30分钟的时间常数（在D227N中，pH值为8.5）转化回初始色素，但它可以通过近紫外光照射更快地重新转化。用选择性排除C9=C10、C11=C12或C13=C14键旋转的“锁定”视网膜类似物进行的实验表明，362 nm物质的形成涉及C13=C14键周围的异构化。与此一致，视网膜提取表明，362 nm光产物是13-顺式的，而初始状态主要是全反式的。pH从8.5到4的快速变化极大地加速了362 nm物质向初始色素的热再转化，这表明它的恢复涉及发色团的热异构化是由可电离残基控制的，主要是席夫碱和Asp97。向长寿命的362 nm光产物的转变显然是光循环的副反应，是对高pH的响应，是由席夫碱的正常再繁殖和再异构化途径的改变引起的。
Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.