Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at -250 mV (up to 6.13 mA cm-2 ). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.

中文翻译：

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通过半定量设计对漆酶CueO进行工程设计，以改善生物电催化中的电子转移。

大肠杆菌的铜外排氧化酶（CueO）由于具有第五个铜结合位点，因此是一种特殊的细菌漆酶。在本文中，发现第五占有率在生物电催化氧还原中起电子中继的关键和有利作用。通过将残基替换为第五个Cu的四个协调位置，可以鉴定出11个有益变体，在-250 mV（最大6.13 mA cm-2）下，电流增加了2.5倍以上。详细的电催化特性表明，第五个Cu结合位点的微环境控制着CueO的电催化电流。另外，借助分子动力学（MD）模拟进行的进一步电子转移分析表明，局部结构稳定性的提高以及第五Cu与T1 Cu之间距离的减小是有助于改善CueO变体动力学的两个主要因素。它可以指导为生物酶催化应用定制漆酶和其他氧化还原酶的新方法。