Fortification of FeS Clusters Reshapes Anaerobic CO Dehydrogenase into an Air-Viable Enzyme Through Multilayered Sealing of O2 Tunnels

Abstract

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 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/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.

Keywords: CO dehydrogenase; Enzyme engineering; FeS clusters; O2 sensitivity; Tunnel engineering.