Fe-S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), utilize artificial electron mediators like viologens for efficient biocatalysis in industrial off-gas cleanup. This study identifies the electron mediator-interaction site on ChCODHs (*Carboxydothermus hydrogenoformans*) using a systematic approach targeting viologen-reactive aromatic residues. Engineered variants (R57G/N59L) near the D-cluster show a ten-fold increase in ethyl viologen affinity without impacting turnover rate. Structural analysis reveals the crucial roles of surface phenylalanine residues as electron conduits. A variant (R57G/N59L/A559W) effectively treats diverse waste gases, including oxygen-containing ones. This work advances the design of highly efficient biocatalysts for industrial gas utilization.

Suk Min Kim, Sung Heuck Kang, Jinhee Lee, Yoonyoung Heo, Eleni G. Poloniataki, Jingu Kang, Hye-Jin Yoon, So Yeon Kong, Yaejin Yun, Hyunwoo Kim, Jungki Ryu, Hyung Ho Lee, Yong Hwan Kim