Yazawa Kenjiro, Furusawa Hiroyuki
Division of Biological and Medical Fibers, Interdisciplinary Cluster for Cutting Edge Research, Institute for Fiber Engineering, Shinshu University, 3-15-1 Tokida, Ueda City, Nagano 386-8567, Japan.
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
ACS Omega. 2019 May 9;4(5):8341-8349. doi: 10.1021/acsomega.9b00474. eCollection 2019 May 31.
A disulfide-bond formation system for nascent proteins in the periplasm contains efficient electron transfer systems for the catalysis of oxidation. This electrochemical system has interesting implications in vivo. Disulfide bonds are formed by disulfide-bond formation protein A (DsbA), which contains two reactive cysteines. DsbA is reoxidized by a membrane protein, disulfide-bond formation protein B (DsbB), which has four catalytic cysteines. The oxidation of DsbA by DsbB seems energetically unfavorable on the basis of the redox potential. The oxidizing power of ubiquinone (UQ), which endogenously binds with DsbB, is believed to promote this reaction. However, using UQ-deficient DsbB, it was found that the oxidation of DsbA by DsbB proceeds independently of UQ. Thus, the reaction mechanism of DsbA oxidation by DsbB is under debate. In this study, we used the quartz crystal microbalance technique, which detects the intermediate complex between DsbA and DsbB during DsbA oxidation as a change in mass, to obtain kinetic parameters of DsbA oxidation under both the oxidized and reduced states of UQ at acidic and basic pH. In addition, we utilized sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility shift assay technique to determine the p of the cysteine thiol groups in DsbA and DsbB. We found that DsbA oxidation proceeded independently of UQ and was greatly affected in kinetics by the shuffling of electrons among the four cysteine residues in DsbB, regardless of pH. These results suggest that DsbA oxidation is driven in an entropy-dependent manner, in which the electron-delocalized intermediate complex is stabilized by preventing a reverse reaction. These findings could contribute to the design of bio-inspired electrochemical systems for industrial applications.
周质中新生蛋白质的二硫键形成系统包含用于催化氧化的高效电子转移系统。这种电化学系统在体内具有有趣的意义。二硫键由含有两个反应性半胱氨酸的二硫键形成蛋白A（DsbA）形成。DsbA被具有四个催化性半胱氨酸的膜蛋白二硫键形成蛋白B（DsbB）重新氧化。基于氧化还原电位，DsbB对DsbA的氧化在能量上似乎是不利的。内源性与DsbB结合的泛醌（UQ）的氧化能力被认为促进了该反应。然而，使用缺乏UQ的DsbB，发现DsbB对DsbA的氧化独立于UQ进行。因此，DsbB氧化DsbA的反应机制存在争议。在本研究中，我们使用石英晶体微天平技术，该技术将DsbA氧化过程中DsbA与DsbB之间的中间复合物检测为质量变化，以获得在酸性和碱性pH下UQ氧化态和还原态下DsbA氧化的动力学参数。此外，我们利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳迁移率变动分析技术来确定DsbA和DsbB中半胱氨酸硫醇基团的pKa。我们发现DsbA氧化独立于UQ进行，并且在动力学上受到DsbB中四个半胱氨酸残基之间电子重排的极大影响，与pH无关。这些结果表明，DsbA氧化是以熵依赖的方式驱动的，其中电子离域的中间复合物通过防止逆向反应而稳定。这些发现可能有助于设计用于工业应用的仿生电化学系统。
