Existence of a proton transfer chain in bacteriorhodopsin: Participation of Glu-194 in the release of protons to the extracellular surface
Existence of a proton transfer chain in bacteriorhodopsin: Participation of Glu-194 in the release of protons to the extracellular surface
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DOI：
10.1021/bi971842m
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发表时间：
1998-02-24
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：
Dioumaev, AK;Richter, HT;Lanyi, JK
Glu-194在细菌视紫红质的细胞外表面附近是光驱动运输过程中Asp-SS质子化后质子释放到介质中不可或缺的。至于Glu-204，用谷氨酰胺（而不是天冬氨酸）取代它，消除了质子释放和Asp-85的异常滴定，这种异常滴定源于这种隐藏的天冬氨酸和其他酸性基团的pK（a）之间的偶联。然而，与Glu-204的情况不同，替换。Glu-194与天冬氨酸的结合提高了质子释放的pK。在E194D突变体的傅里叶变换红外光谱中，在1720 cm（-1）处观察到一个显著的正带。根据[4-（13）C]天冬氨酸和D（2）O同位素位移，可将其归属于质子化Asp-194的C = O伸缩。其升高与质子从视网膜席夫碱转移到Asp-85相关。它的衰变与表面质子的出现相一致，在类似的条件下用共价结合到Lys-129和吡喃的荧光素检测到。其振幅随着pH值的增加而减小，pK（a）约为9。我们表明，该pK（a）可能是光激发前Asp-194（Glu-204位点）的内部质子供体的pK（a）：而（13）C NMR滴定表明Asp-194的初始pK（a）约为3。我们建议，有一个链的相互作用残基之间的视网膜席夫碱和细胞外表面。在视黄醛的光异构化后，pK（a）的变化使得（i）Asp-85被席夫碱质子化，（ii）Glu-204位点将其质子转移到E194 D中的Asp-194，并因此转移到野生型中的Glu-194，以及（iii）残基194将质子释放到培养基中。
Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protanation of Asp-SS during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pK(a) of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement. of Glu-194 with aspartate raises the pK, for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm(-1). It can be assigned from [4-(13)C]aspartate and D(2)O isotope shifts to the C=O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pK(a) of about 9. We show that this pK(a) is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation: while (13)C NMR titration indicates that Asp-194 has an initial pK(a) of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pK(a)'s change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.