Abstract: Oxygen-resistant carbon monoxide dehydrogenases (CODHs) fall into two categories: those derived from aerobic CO-metabolizing bacteria and the engineered oxygen-resistant variants from anaerobic CO-metabolizing bacteria. The former, containing molybdenum (Mo) and copper (Cu) cofactors, are termed as Mo/Cu-CODHs. They specifically catalyze the oxidation of carbon monoxide (CO) to carbon dioxide (CO2), generating two protons (H+) and two electrons (e-) in the process, and are insensitive to O2. The latter, Ni/Fe-CODHs, featuring nickel (Ni) and iron (Fe) cofactors, originate from anaerobic CO-metabolizing bacteria. Most Ni/Fe-CODHs are O2-sensitive, though some variants exhibit limited O2 tolerance at the expense of reduced activity. Nevertheless, high-activity Ni/Fe-CODHs can be engineered for oxygen tolerance. Unaffected by O2 concentration, oxygen-tolerant CODHs hold significant application potential in biomanufacturing and electrochemistry under aerobic conditions. This work first reviews the structure, function, and catalytic mechanisms of Mo/Cu-CODHs. Mo/Cu-CODHs comprise three subunits (coxL, coxS, coxM), with CO oxidation primarily occurring at the active site of the L subunit. The catalytic mechanism of CO oxidation by this enzyme has been largely elucidated: CO initially binds to Cu, followed by nucleophilic attack by the O ligand of Mo=O to form five-membered ring intermediates. A sequence of subsequent intermediates, including thiocyclic carbonate, then ensues, ultimately leading to CO2 release and completion of the catalytic cycle. Additionally, this work explores the factors influencing the oxygen tolerance of Ni/Fe-CODHs and methods to enhance this trait. It also summarizes progress in the in vitro heterologous expression and purification of oxygen-tolerant CODHs, reviews their applications in the bioconversion of synthetic chemicals and in vitro catalysis, and provides an outlook on future development trends. These contents will offer references for further research and development of oxygen-tolerant CODHs.

Key words: carbon monoxide dehydrogenase, enzyme structure, catalytic mechanism, biomanufacturing

摘要： 耐氧一氧化碳脱氢酶(Carbon Monoxide Dehydrogenases, CODHs)包括好氧一氧化碳(CO)代谢菌中的CODHs及厌氧CO代谢菌中经改造后具备耐氧能力的CODHs。好氧CO代谢菌中的CODHs含有钼(Mo)和铜(Cu)金属辅助因子，缩写为Mo/Cu-CODHs。该酶可专一性催化CO氧化为二氧化碳(CO2)，同时产生2个质子(H+)和2个电子(e-)，且对O2不敏感。厌氧CO代谢菌中的CODHs含有镍(Ni)和铁(Fe)金属辅助因子，缩写为Ni/Fe-CODHs。大多数Ni/Fe-CODHs对O2敏感，虽有少数种类可轻微耐受O2，但其酶活较对O2敏感的Ni/Fe-CODHs更低。通过工程手段可提高高酶活Ni/Fe-CODHs的耐氧性，使其成为耐氧CODHs。耐氧CODHs不受环境O2浓度限制，在生物制造和电化学领域展现出重要的应用价值。本工作首先综述了Mo/Cu-CODHs的结构、功能及催化机制。Mo/Cu-CODHs由3个亚基(coxL, coxS, coxM)组成，CO氧化反应主要发生在L亚基的活性位点上。目前，该酶催化CO氧化机制已基本明确：CO首先与Cu相结合，随后受到Mo=O的O配体亲核攻击，进而形成五元环中间体，再通过硫代碳酸酯等一系列中间体的转化，最终释放CO2完成催化循环。此外，本工作还介绍了影响Ni/Fe-CODHs耐氧性的因素及提高耐氧性的改造方法，总结了耐氧CODHs异源表达与纯化的研究进展，梳理了该酶在生物转化合成化学品及体外催化应用方面的进展，并对其未来发展趋势进行了展望，为耐氧CODHs的进一步研究与开发提供参考。

关键词: 一氧化碳脱氢酶, 酶结构, 催化机制, 生物制造