The inherent O2 sensitivity of Ni–Fe carbon monoxide dehydrogenases (CODHs), crucial for rapid CO to CO2 interconversion, presents substantial challenges for industrial application. Transforming CO/CO2, a prevalent anthropogenic air pollutant, into valuable carbon chemicals either directly or through intermediate steps via biocatalytic methods offers a promising pathway to achieve net-zero emissions across industries and the environment. However, completely eliminating oxygen from industrial biotransformations, especially under ambient conditions, is exceedingly onerous. Here, we engineered variants of the CODH2 from Carboxydothermus hydrogenoformans (ChCODH2) with dual blocking at both the O2 entrance and near the active site, effectively sealing the tunnel against atmospheric O2 levels (20%). The O2-tunnel engineered A559W/V610H variant demonstrated a marked improvement in air stability, with a half-life of 24.6 hours compared to the wild type’s 2.4 hours. Crystallographic snapshots of this air-viable variant after 24 hours of exposure revealed the robust integrity of the fortified FeS and NiFeS clusters. Additionally, electro-enzymatic reactions corroborated its CO/CO2 conversion capability even in ubiquitous air. These findings, which address the O2 sensitivity of anaerobic enzymes caused by O2-induced metal cluster collapse, enhance their potential for biological CO/CO2 transformations in O2-rich environments, thereby broadening their industrial viability and applicability.

中文翻译：

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FeS 簇的强化通过多层密封 O2 隧道将厌氧 CO 脱氢酶重塑为空气活性酶

Ni-Fe 一氧化碳脱氢酶 （CODH） 固有的 O2 敏感性对于 CO 到 CO2 的快速相互转化至关重要，这给工业应用带来了重大挑战。将 CO/CO2（一种普遍的人为空气污染物）直接或通过生物催化方法通过中间步骤转化为有价值的碳化学品，为实现跨行业和环境的净零排放提供了一条有前途的途径。然而，从工业生物转化中完全消除氧气，尤其是在环境条件下，是极其繁重的。在这里，我们从氢双羧基低温菌 （ChCODH2） 中设计了 CODH2 的变体，在 O 2 入口和活性部位附近都有双重封闭，有效地密封了隧道，防止大气 O 2 水平 （20%）。O2 隧道设计的 A559W/V610H 变体在空气稳定性方面表现出显着改善，半衰期为 24.6 小时，而野生型为 2.4 小时。暴露 24 小时后，这种空气活性变体的晶体学快照揭示了强化 FeS 和 NiFeS 簇的稳健完整性。此外，电酶反应证实了其 CO/CO2 转化能力，即使在无处不在的空气中也是如此。这些发现解决了由 O2 诱导的金属簇塌陷引起的厌氧酶的 O2 敏感性，增强了它们在富含 O2 的环境中进行生物 CO/CO2 转化的潜力，从而拓宽了它们的工业可行性和适用性。