ASPARTIC ACID-96 IS THE INTERNAL PROTON DONOR IN THE REPROTONATION OF THE SCHIFF-BASE OF BACTERIORHODOPSIN
ASPARTIC ACID-96 IS THE INTERNAL PROTON DONOR IN THE REPROTONATION OF THE SCHIFF-BASE OF BACTERIORHODOPSIN
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DOI：
10.1073/pnas.86.23.9228
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
HEYN, MP
中科院分区：
文献类型：
--
作者：
OTTO, H;MARTI, T;HEYN, MP
在pH 8以上，细菌视紫红质的光循环中间体M的衰变分成两个部分：通常的毫秒pH无关的部分和另外的较慢的部分，其速率常数与H+的摩尔浓度成比例，[H+]。与此同时，与席夫碱的再质子化相关的电荷易位信号也产生了类似的慢分量。这些观察结果解释了一个两步reprotonation机制。内部供体首先在M衰变为N时使席夫碱再质子化，然后在N → N中从细胞质再质子化。O过渡。N的衰变率与[H+]成正比。通过假设从N到M的逆反应，M的衰变分为两个部分，较慢的部分与N的衰变具有相同的pH依赖性。光循环，光电压，和pH指示剂的突变体中，天冬氨酸-96被天冬酰胺或丙氨酸，我们称之为D96 N和D96 A取代的实验表明，天冬氨酸-96是参与再摄取途径的内部质子供体。在这两种突变体中，质子泵的化学计量与野生型相同。然而，M衰减是双相的，衰减时间的对数[log（τ）]线性依赖于pH值，这表明内部供体是不存在的，希夫碱是直接从细胞质中reprotonated。像H+一样，叠氮化物增加了D96 N中的M衰变速率。速率常数与叠氮化物浓度成比例，并且可以变得比野生型大> 100倍。因此，叠氮化物作为移动的质子供体在双分子反应中直接重质子化席夫碱。质子和叠氮化物的影响，这是不存在的野生型，表明内部供体被删除，并在这些突变体中的reprotonation途径是不同于野生型。
Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N .fwdarw. O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (.tau.)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become > 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.