Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB
Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB
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DOI：
10.1073/pnas.0507570103
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发表时间：
2006-01-10
影响因子：
11.1
通讯作者：
Hayashi, S
中科院分区：
文献类型：
--
作者：
Inaba, K;Takahashi, Y;Hayashi, S
最近的研究已经揭示了许多例子，其中蛋白质中半胱氨酸的氧化和还原被整合到生物调节系统的特定级联中。通常，这些反应作为硫醇-二硫化物交换事件进行。然而，人们并不完全理解二硫键是如何从头产生的。DsbB是一种大肠杆菌质膜蛋白，是在其自身和DsbA中产生新的二硫键的酶之一，DsbA是周质空间中蛋白质二硫键形成的直接催化剂。DsbB与辅因子（泛醌或甲基萘醌）相关，作为氧化等价物的来源。在DsbB酶促反应的过程中，DsbB结合的醌经历转变为粉红色（λ（max）约为500 nm，泛醌）或紫色（λ（max）约为550 nm，甲萘醌）的状态。在这里，我们表明，不仅巯基形式的Cys-44先前建议，但也Arg-48在a-螺旋排列是必不可少的醌过渡。量子化学模拟表明，硫醇盐阴离子和泛醌与精氨酸的带正电荷的胍基部分结合的适当定位允许形成具有在约500 nm处的吸收峰的硫醇盐-泛醌电荷转移复合物以及半胱氨酰醌共价加合物。我们认为电荷转移态导致过渡态的形成，它接受另一个半胱氨酸的亲核攻击，从头生成一个二硫键。对于一类具有FAD辅因子的真核二硫醇氧化酶，类似的机制是可以想象的。
Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created de novo. DsbB, an Escherichia coli plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink (lambda(max) approximate to 500 nm, ubiquinone) or violet (lambda(max)approximate to 550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the a-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanicliniurn moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at -500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adcluctthat accepts a nucleophilic attack from another cysteine to generate a disulficle bond de novo. A similar mechanism is conceivable for a class of eukaryotic dithiol oxiclases having a FAD cofactor.