An unexpected alternative viologen electron mediator site in tungsten-containing formate dehydrogenase

The molecular mechanisms by which artificial electron mediators, such as ethyl viologen (EV), interact with tungsten-containing formate dehydrogenases (FDHs) during reversible CO₂ reduction remain poorly understood. Here, we reveal an unexpected alternative mediator interaction site in FDH1 of Methylorubrum extorquens AM1. Removing the native flavin mononucleotide cofactor abolishes NAD⁺-dependent activity but preserves EV-driven catalysis. Through mutagenesis, kinetic analysis, and molecular docking, we identified a cooperative aromatic network-comprising residues F232, F471, and Y329-that stabilizes EV via stacking interactions near the proximal B1 iron-sulfur cluster. Disrupting these residues impairs EV-mediated electron transfer without destabilizing global structure. These findings reveal a dual interaction strategy for artificial mediators in tungsten FDHs, offering a structural framework to rationally engineer biocatalysts for CO₂ conversion.