A linkage of the pK(a)'s of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin
A linkage of the pK(a)'s of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin
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DOI：
10.1021/bi952883q
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发表时间：
1996-04-02
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：
Richter, HT;Brown, LS;Lanyi, JK
由于 asp-85 在细菌视紫红质光驱动质子运输过程中是视网膜席夫碱质子的受体，因此预计光循环中其 pK(a) 的调节，建议对未光解蛋白中的 asp-85 进行复杂滴定 [Balashov, S. P., Govindjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch, R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] 反映该残基对另一个未识别基团的质子化状态的依赖性，从视网膜异构态热平衡速率常数的 pH 依赖性（暗适应）和希夫碱在光循环过程中的去质子化动力学 E204Q和E204D突变体，我们鉴定残基为glu-204。它与 asp-85 相互作用的本质是，在中性 pH 值下，任一残基都可以是阴离子，但不能同时是阴离子。这与我们最近的发现一致，即 glu-204 是在运输过程中 asp-85 质子化时释放到细胞外表面的质子的来源。因此，我们建议光周期中发生以下一系列事件。在与光异构化视网膜的席夫碱的质子平衡中，asp-85 的质子化导致 glu-204 解离，并将质子释放到细胞外表面。 glu-204 的去质子化反过来又提高了 asp-85 的 pK，并且与希夫碱的平衡转向完全质子转移。这构成：重质子化转换的第一阶段，因为它排除了 asp-85 作为随后希夫碱重质子化的供体。随后通过衍射检测到的蛋白质的连续结构变化表明，作为转换的第二阶段，促进席夫碱向细胞质侧的进入的变化，并降低 asp-96 的 pK，从而使其成为质子供体，作为第三阶段。
Because asp-85 is the acceptor of the retinal Schiff base proton during light-driven proton transport by bacteriorhodopsin, modulation of its pK(a) in the photocycle is to be expected, The complex titration of asp-85 in the unphotolyzed protein was suggested [Balashov, S. P., Govindjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch, R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] to reflect the dependence of this residue on the protonation state of another, unidentified group, From the pH dependencies of the rate constant for the thermal equilibration of retinal isomeric states (dark adaptation) and the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. The nature of its interaction with asp-85 is that at neutral pH either residue can be anionic but not both. This is consistent with our recent finding that glu-204 is the origin of the proton released to the extracellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface. The deprotonation of glu-204, in turn, raises the pK, of asp-85, and the equilibrium with the Schiff base shifts toward complete proton transfer. This constitutes: the first phase of the reprotonation switch because it excludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the change of the access of the Schiff base toward the cytoplasmic side as the second phase of the switch, and the lowering the pK, of asp-96, so as to make it a proton donor, as the third phase.