Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA
Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA
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DOI：
10.1093/emboj/21.10.2354
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发表时间：
2002-05-15
期刊：
影响因子：
11.4
通讯作者：
Beckwith, J
中科院分区：
文献类型：
--
作者：
Kadokura, H;Beckwith, J
在大肠杆菌中蛋白质二硫键的形成是由周质蛋白DsbA催化的。细胞质膜蛋白DsbB通过将电子从DsbA转移到呼吸链上的苯二酚来维持DsbA的氧化状态。在这里，我们证明了DsbB的活性可以通过蛋白质的N-末端和C-末端片段的共表达来重组，每个片段都含有一个氧化还原活性的二硫键。这个系统使我们(I)证明了两个DsbB氧化还原中心直接通过两个DsbB结构域之间形成的二硫键相互作用，以及(Ii)识别参与这种共价相互作用的特定半胱氨酸残基。此外，我们能够捕捉到电子从一个氧化还原中心到另一个氧化还原中心转移过程中的中间体。这些结果导致我们提出了一个模型，描述半胱氨酸在DsbA氧化的早期阶段是如何合作的。DsbB似乎采用了一种新的机制来氧化DsbA，利用它的两对半胱氨酸在配位反应中接受来自DsbA中活性半胱氨酸的电子。
Protein disulfide bond formation in Escherichia coli is catalyzed by the periplasmic protein DsbA. A cytoplasmic membrane protein DsbB maintains DsbA in the oxidized state by transferring electrons from DsbA to quinones in the respiratory chain. Here we show that DsbB activity can be reconstituted by co-expression of N- and C-terminal fragments of the protein, each containing one of its redox-active disulfide bonds. This system has allowed us (i) to demonstrate that the two DsbB redox centers interact directly through a disulfide bond formed between the two DsbB domains and (ii) to identify the specific cysteine residues involved in this covalent interaction. Moreover, we are able to capture an intermediate in the process of electron transfer from one redox center to the other. These results lead us to propose a model that describes how the cysteines cooperate in the early stages of oxidation of DsbA. DsbB appears to adopt a novel mechanism to oxidize DsbA, using its two pairs of cysteines in a coordinated reaction to accept electrons from the active cysteines in DsbA.