Decarbonizing the steel industry, a major CO₂ emitter, is crucial for achieving carbon neutrality. Escaping the grip of CO combustion methods, a key contributor to CO₂ discharge, is a seemingly simple yet formidable challenge on the path to industry-wide net-zero carbon emissions. Here we suggest enzymatic CO hydration (enCOH) inspired by the biological Wood‒Ljungdahl pathway, enabling efficient CO₂ fixation. By employing the highly efficient, inhibitor-robust CO dehydrogenase (ChCODH2) and formate dehydrogenase (MeFDH1), we achieved spontaneous enCOH to convert industrial off-gases into formate with 100% selectivity. This process operates seamlessly under mild conditions (room temperature, neutral pH), regardless of the CO/CO₂ ratio. Notably, the direct utilization of flue gas without pretreatment yielded various formate salts, including ammonium formate, at concentrations nearing two molar. Operating a 10-liter-scale immobilized enzyme reactor feeding live off-gas at a steel mill resulted in the production of high-purity formate powder after facile purification, thus demonstrating the potential for decarbonizing the steel industry.

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (k_cat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O₂. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

Carbon monoxide dehydrogenase (CODH), formate dehydrogenase (FDH), hydrogenase (H2ase), and nitrogenase (N2ase) are crucial enzymatic catalysts that facilitate the conversion of industrially significant gases such as CO, CO₂, H₂, and N₂. The tunnels in the gas-converting enzymes serve as conduits for these low molecular weight gases to access deeply buried catalytic sites. The identification of the substrate tunnels is imperative for comprehending the substrate selectivity mechanism underlying these gas-converting enzymes. This knowledge also holds substantial value for industrial applications, particularly in addressing the challenges associated with separation and utilization of byproduct gases. In this comprehensive review, we delve into the emerging field of tunnel engineering, presenting a range of approaches and analyses. Additionally, we propose methodologies for the systematic design of enzymes, with the ultimate goal of advancing protein engineering strategies.

The inherent O ₂ sensitivity of Ni─Fe carbon monoxide dehydrogenases (CODHs), crucial for rapid CO to CO ₂ interconversion, presents substantial challenges for industrial application. Transforming CO/CO ₂ , 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 ( Ch CODH2) with dual blocking at both the O ₂ entrance and near the active site, effectively sealing the tunnel against atmospheric O ₂ levels (20%). The O ₂ ‐tunnel engineered A559W/V610H variant demonstrated a marked improvement in air stability, with a half‐life of 24.6 h compared to the wild type's 2.4 h. Crystallographic snapshots of this air‐viable variant after 24 h of exposure revealed the robust integrity of the fortified FeS and NiFeS clusters. Additionally, electro‐enzymatic reactions corroborated its CO/CO ₂ conversion capability even in ubiquitous air. These findings, which address the O ₂ sensitivity of anaerobic enzymes caused by O ₂ ‐induced metal cluster collapse, enhance their potential for biological CO/CO ₂ transformations in O ₂ ‐rich environments, thereby broadening their industrial viability and applicability.

The inherent O ₂ sensitivity of Ni─Fe carbon monoxide dehydrogenases (CODHs), crucial for rapid CO to CO ₂ interconversion, presents substantial challenges for industrial application. Transforming CO/CO ₂ , 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 ( Ch CODH2) with dual blocking at both the O ₂ entrance and near the active site, effectively sealing the tunnel against atmospheric O ₂ levels (20%). The O ₂ ‐tunnel engineered A559W/V610H variant demonstrated a marked improvement in air stability, with a half‐life of 24.6 h compared to the wild type's 2.4 h. Crystallographic snapshots of this air‐viable variant after 24 h of exposure revealed the robust integrity of the fortified FeS and NiFeS clusters. Additionally, electro‐enzymatic reactions corroborated its CO/CO ₂ conversion capability even in ubiquitous air. These findings, which address the O ₂ sensitivity of anaerobic enzymes caused by O ₂ ‐induced metal cluster collapse, enhance their potential for biological CO/CO ₂ transformations in O ₂ ‐rich environments, thereby broadening their industrial viability and applicability.