Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD.
Control of periplasmic interdomain thiol:disulfide exchange in the transmembrane oxidoreductase DsbD.
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DOI：
10.1074/jbc.m805963200
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发表时间：
2009-01-30
期刊：
影响因子：
--
通讯作者：
Redfield C
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文献类型：
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作者：
Mavridou DAI;Stevens JM;Goddard AD;Willis AC;Ferguson SJ;Redfield C
细菌蛋白DsbD将还原剂从细胞质转移到周质的氧化环境中。这种还原能力是几个基本途径所必需的，包括二硫键形成和细胞色素c成熟。DsbD包括一个跨膜结构域（tmDsbD），两侧是两个球状周质结构域（nDsbD/cDsbD）;每个结构域含有一个半胱氨酸对，通过二硫键交换级联参与电子转移。级联反应的最后一步涉及cDsbD的Cys 461还原nDsbD的Cys 103-Cys 109二硫键。在这里，我们表明，球状体周质域之间的复合物被困在体内只有当两者都是由tmDsbD连接。我们已经发现以前（Mavridou，D.一、史蒂文斯，J.M.，弗格森，S。J.，& Redfield，C.（），-658），分离的cDsbD中的攻击性半胱氨酸（Cys 461）具有高pKa值（10.5），这使得该硫醇相对不与nDsbD中的靶二硫化物反应。在这里，我们使用NMR表明，当cDsbD与nDsbD形成复合物时，活性位点的pKa值发生显着变化。pKa值的这种调节对于cDsbD的特异性和功能至关重要。未复合的cDsbD是一种较差的亲核试剂，使其能够避免非特异性再氧化;然而，在与nDsbD复合时，cDsbD的亲核性增加，允许还原剂转移。观察到的显着变化，在复杂的形成后，活性位点的pKa值有更广泛的影响，了解硫醇：二硫化物氧化还原酶的反应。
The bacterial protein DsbD transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm. This reducing power is required for several essential pathways, including disulfide bond formation and cytochrome c maturation. DsbD includes a transmembrane domain (tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade. The final step in the cascade involves reduction of the Cys103-Cys109 disulfide of nDsbD by Cys461 of cDsbD. Here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmDsbD. We have found previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J., & Redfield, C. () , -658) that the attacking cysteine (Cys461) in isolated cDsbD has a high pKa value (10.5) that makes this thiol relatively unreactive toward the target disulfide in nDsbD. Here we show using NMR that active-site pKa values change significantly when cDsbD forms a complex with nDsbD. This modulation of pKa values is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in complex with nDsbD, the nucleophilicity of cDsbD increases permitting reductant transfer. The observation of significant changes in active-site pKa values upon complex formation has wider implications for understanding reactivity in thiol:disulfide oxidoreductases.