Kinetic and thermodynamic analysis of Cu2+-dependent reductive inactivation in direct electron transfer-type bioelectrocatalysis by copper efflux oxidase
Kinetic and thermodynamic analysis of Cu2+-dependent reductive inactivation in direct electron transfer-type bioelectrocatalysis by copper efflux oxidase
复制标题
铜外流氧化酶直接电子转移型生物电催化中 Cu2+ 依赖性还原失活的动力学和热力学分析
DOI：
10.1016/j.electacta.2022.140987
复制
发表时间：
2022
影响因子：
6.6
通讯作者：
Lojou Elisabeth
中科院分区：
文献类型：
--
作者：
Adachi Taiki;Mazurenko Ievgen;Mano Nicolas;Kitazumi Yuki;Kataoka Kunishige;Kano Kenji;Sowa Keisei;Lojou Elisabeth
铜流出氧化酶 (CueOs) 是铜稳态系统中的关键酶。然而，所涉及的机制在很大程度上尚不清楚。 CueO 型酶具有由富含蛋氨酸（富含 Met）结构域组成的典型结构特征，这些结构域被认为参与铜稳态。在 Cu2+ 存在下使用 CueO 型酶的生物电催化最近强调了与亚铜氧化酶活性相关的新的 Cu2+ 依赖性催化途径。在这项工作中，我们进一步研究了Cu2+对NH2功能化多壁碳纳米管上CueO直接电子转移（DET）型生物电催化还原O2的影响。在 Cu2+ 存在的情况下，CueO 的 DET 型生物电催化活性在低电位下降低，显示出独特的峰形伏安图，我们将其归因于失活和再激活过程。使用计时电流分析法对这些过程进行动力学分析，结果表明失活/再激活速率常数与电极电势之间存在线性自由能关系。伪稳态分析还表明Cu2+非竞争性地抑制酶活性。提出了 CueO 依赖于 Cu2+ 的还原失活的详细模型来解释电化学数据以及相关的热力学和动力学参数。具有截短的铜结合 α 螺旋和不含富含 Met 结构域的胆红素氧化酶的 CueO 变体也显示出这种还原失活过程，这表明多铜氧化酶含有导致失活的铜结合位点。
Copper efflux oxidases (CueOs) are key enzymes in copper homeostasis systems. The mechanisms involved are however largely unknown. CueO-type enzymes share a typical structural feature composed of Methionine-rich (Met-rich) domains that are proposed to be involved in copper homeostasis. Bioelectrocatalysis using CueO-type enzymes in the presence of Cu2+recently highlighted a new Cu2+-dependent catalytic pathway related to a cuprous oxidase activity. In this work, we further investigated the effects of Cu2+on direct electron transfer (DET)-type bioelectrocatalytic reduction of O2by CueO at NH2-functionalized multi-walled carbon nanotubes. The DET-type bioelectrocatalytic activity of CueO decreased at low potential in the presence of Cu2+, showing unique peak-shaped voltammograms that we attribute to inactivation and reactivation processes. Chronoamperometry was used to kinetically analyze these processes, and the results suggested linear free energy relationships between the inactivation/reactivation rate constant and the electrode potential. Pseudo-steady-state analysis also indicated that Cu2+uncompetitively inhibited the enzymatic activity. A detailed model for the Cu2+-dependent reductive inactivation of CueO was proposed to explain the electrochemical data, and the related thermodynamic and kinetic parameters. A CueO variant with truncated copper-binding α helices and bilirubin oxidase free of Met-rich domains also showed such reductive inactivation process, which suggests that multicopper oxidases contain copper-binding sites that lead to inactivation.