Computational framework for analyzing inhibitory gas access in carbon monoxide dehydrogenases for industrial biocatalysis

Carbon monoxide dehydrogenases (CODHs) are emerging as promising biocatalysts for industrial gas fermentation and sustainable C1 bioconversion. However, their practical application is frequently limited by inhibitory gases present in complex industrial gas streams. Here, we used computational analysis to characterize molecular transport pathways in four ChCODH isoforms from Carboxydothermus hydrogenoformans (Ch) and compare ligand transport profiles across the identified pathways. Comparison of the substrate carbon monoxide (CO) and representative inhibitor-associated species, including hydrogen cyanide (HCN), nitrite (NO2-), nitrate (NO3-), and hydrosulfide (HS-), revealed that although multiple tunnels are present in each enzyme, both CO and the smaller inhibitor-associated species were predicted to favor a limited subset of pathways. While ChCODH-II and ChCODH-III exhibited higher calculated transport resistance toward bulky species such as nitrate, they showed limited transport discrimination between CO and smaller inhibitors that showed overlapping favorable transport pathways. Based on these findings, we propose a tunnel-guided asymmetric pathway-modulation strategy via in silico mutagenesis. Computational analyses predict that introducing a localized steric constriction into the secondary transport pathway (T6) of ChCODH-II increases transport resistance along T6 while retaining T1 as an important pathway for CO transport. Experimental evaluation of V472F showed comparable basal CO oxidation activity to WT but delayed cyanide-induced activity loss during early exposure. The combined computational and experimental results support pathway-specific tunnel modulation as a practical strategy for identifying functionally relevant tunnel-engineering candidates.