College of Life Sciences, Peking University, Beijing, 100871, China.
Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing, 100871, China.
Sci Rep. 2018 Oct 18;8(1):15448. doi: 10.1038/s41598-018-33766-4.
The thioredoxin (Trx)-coupled arsenate reductase (ArsC) is a family of enzymes that catalyzes the reduction of arsenate to arsenite in the arsenic detoxification pathway. The catalytic cycle involves a series of relayed intramolecular and intermolecular thiol-disulfide exchange reactions. Structures at different reaction stages have been determined, suggesting significant conformational fluctuations along the reaction pathway. Herein, we use two state-of-the-art NMR methods, the chemical exchange saturation transfer (CEST) and the CPMG-based relaxation dispersion (CPMG RD) experiments, to probe the conformational dynamics of B. subtilis ArsC in all reaction stages, namely the enzymatic active reduced state, the intra-molecular C10-C82 disulfide-bonded intermediate state, the inactive oxidized state, and the inter-molecular disulfide-bonded protein complex with Trx. Our results reveal highly rugged energy landscapes in the active reduced state, and suggest global collective motions in both the C10-C82 disulfide-bonded intermediate and the mixed-disulfide Trx-ArsC complex.
硫氧还蛋白（Trx）偶联砷酸盐还原酶（ArsC）是一类酶，可在砷解毒途径中将砷酸盐还原为亚砷酸盐。催化循环涉及一系列中继的分子内和分子间巯基-二硫键交换反应。已确定了不同反应阶段的结构，表明在反应途径中存在显着的构象波动。在此，我们使用两种最先进的 NMR 方法，化学交换饱和转移（CEST）和基于 CPMG 的弛豫分散（CPMG RD）实验，来探测枯草芽孢杆菌 ArsC 在所有反应阶段的构象动力学，即酶活性还原态、分子内 C10-C82 二硫键结合的中间态、非活性氧化态以及与 Trx 形成的分子间二硫键结合的蛋白质复合物。我们的结果揭示了活性还原态中高度崎岖的能量景观，并表明 C10-C82 二硫键结合的中间态和混合二硫键 Trx-ArsC 复合物中存在整体集体运动。
