Identifying a key spot for electron mediator-interaction to tailor CO dehydrogenase's affinity

Industrial flue gases rich in CO, CO2, N2, and H2 are promising feedstocks for sustainable chemical and energy conversion. Gas-converting metalloenzymes such as CODH and FDH can catalyze transformations of CO and CO2, respectively, under mild conditions, but in vitro applications require artificial electron mediators. Viologens are widely used, versatile mediators for these enzymes; however, how they interact with metalloenzymes to mediate electron transfer is not well understood. Ni–Fe CODHs, with conserved homodimeric structures and characterized metal clusters (B, C, D), serve as ideal models. This study aims to identify and structurally validate the viologen interaction site in ChCODH2 (from Carboxydothermus hydrogenoformans), improve mediator affinity without compromising turnover, and demonstrate industrial relevance for waste-gas conversion, including under O2-containing conditions.

• Viologens (4,4'-bipyridinium salts) are cost-effective redox mediators with three reversible redox states and are widely used as alternatives to native cofactors in enzymatic and electro/photo-chemical systems. Many gas-converting enzymes relevant to climate applications commonly use viologens.
• Ni–Fe CODHs possess a unique [NiFe4S4OHx] C-cluster catalytic site and conserved B and D Fe–S clusters, with extensive structural knowledge available across species. Prior engineering has achieved oxygen-tolerant, high-efficiency CODH variants for syngas conversion but the mediator interaction site remained unresolved.
• Aromatic residues (Phe/Tyr/Trp) are known to facilitate electron transfer, suggesting possible roles in mediator binding and electron tunneling. Sequence/structure analyses show conserved aromatic positions near the exposed D-cluster across CODHs with either 4Fe-4S or 2Fe-2S D-clusters.
• Reported kinetic parameters indicate generally poor mediator (EV) affinities for CODHs (Km typically > 2 mM), motivating efforts to tailor mediator interaction sites.

• Rational residue selection: Identified surface-exposed aromatic (Phe, Tyr, Trp) and sulfur-containing (Cys, Met) residues on ChCODH2 (PDB 1SU7) as candidate mediator-interaction sites.
• Alanine scanning: Constructed E. coli–expressed alanine mutants of surface aromatic residues and assayed CO-dependent reduction of viologens (primarily ethyl viologen, EV; also MV, BV, DQ) at 30 °C, pH 8, 20 mM mediator.
• Cross-species validation: Generated equivalent phenylalanine mutants near the D-cluster in RrCODH (Rhodospirillum rubrum; PDB 1JQK) and DvCODH (Desulfovibrio vulgaris; PDB 6OND) and assayed with multiple viologens to test conservation.
• Electrochemistry (viologen-free): Protein-film cyclic voltammetry to confirm that mutations (e.g., F41 variants) did not alter D-cluster redox potential relative to WT, isolating effects to electron transfer rather than cluster energetics.
• Kinetics: Determined Michaelis-Menten parameters for EV across mutants (including R57 and N59 positions near F41) by varying EV (0.0625–32 mM) at 30 °C, pH 8; calculated kcat from Vmax.
• ITC: Measured EV binding to ChCODH2 WT and R57G/N59L under CO to obtain dissociation constants (Kd).
• Crystallography: Solved structures of R57G/N59L (apo; 8X9D), low-PEG form (8X9E), and complexes with EV (8X9F) and BV (8X9G) under anaerobic conditions; mapped electron density near F41 and characterized interactions (electrostatic/hydrophobic) and buried surface area; assessed surface charge changes and cavity formation.
• Bioinformatics: Analyzed conservation of aromatic residues around the D-cluster across CODHs (4Fe-4S and 2Fe-2S groups), confirming conservation of the F/Y residue corresponding to ChCODH2 F41.
• Industrial gas testing: Combined enhanced mediator-affinity (R57G/N59L) with previously engineered O2-tolerant tunnel mutation (A559W) to create triple mutant R57G/N59L/A559W; evaluated activity with real industrial gas mixtures (COG, BFG, LDG) and SRF-derived gas; assessed O2 inhibition tolerance.
• Controls and additional variants: Constructed and tested E43K/E43R, T44A, P60A, L583A, L612A to probe roles of surrounding residues in EV interaction.

• A single surface phenylalanine near the D-cluster is critical for viologen-mediated activity in ChCODH2: F41A reduced activity to <0.01% of WT with EV at 20 mM; similarly reduced or abolished activity across MV, EV, BV, DQ.
• Cross-species conservation: Equivalent residues in RrCODH (F43) and DvCODH (F44) are essential for viologen reduction across multiple viologens, indicating a conserved mediator-interaction site in the 40–44 region near the D-cluster in CODHs with 4Fe-4S or 2Fe-2S D-clusters.
• Electrochemistry showed no significant change in D-cluster redox potential between WT and F41 variants (~0.28 V vs RHE), indicating mutations affect electron transfer rather than cluster energetics.
• Enhanced mediator affinity via neighboring residues: ChCODH2 R57G/N59L showed ~10-fold improved EV affinity (Km ≈ 0.2 mM at 30 °C) relative to WT (Km ~2.3–2.4 mM), with similar turnover (kcat ~2,100 s−1 vs WT ~2,200 s−1). R57G/N59F and R57G/N59K also improved affinity (Km 0.2–0.5 mM) with high catalytic efficiencies.
• Diminished affinity when disrupting the key Phe: ChCODH2 F41A and RrCODH F43A displayed markedly worse EV affinity (Km ~7.0–7.1 mM and ~11.9 mM, respectively) and specific activities ~0.3–0.5 U mg−1.
• ITC under CO: R57G/N59L binds EV with higher affinity (Kd ~144 µM) than WT (Kd ~449 µM), consistent with kinetic improvements.
• Structural basis: R57G/N59L apo structure revealed a new cavity (~38.45 Å3) near position 57 and reduced electrostatic repulsion, facilitating viologen access. Viologen-bound structures showed EV/BV electron density near F41, with interactions involving E43, T44, L583, L612; buried surface area ~160.5 Å2 (EV) and ~230.3 Å2 (BV). Distances support electron transfer: EV to F41 ~9.9 Å; EV to D-cluster ~12.6 Å; EV to B′ ~17.2 Å; EV to C ~24.8 Å.
• Electrostatics at E43: E43K/E43R increased Km 2.5–3.2-fold vs WT, indicating the negative charge of E43 stabilizes binding to positively charged oxidized viologens.
• Industrial relevance and O2 tolerance: Triple mutant R57G/N59L/A559W maintained high EV catalytic efficiency and exhibited improved O2 tolerance. With real gases, relative activities vs pure CO: low CO gas 100%, COG 98%, BFG 110%, LDG 112%, SRF-derived gas 87%.
• Overall, mediator affinity can be tuned by targeted mutations near the conserved surface phenylalanine and D-cluster without sacrificing catalytic turnover, enabling efficient conversion of real waste gases.

The study resolves a longstanding gap by locating and validating a conserved viologen interaction site near a surface phenylalanine (F41 region) adjacent to the D-cluster in Ni–Fe CODHs. Data indicate that electron transfer to viologens can proceed via a dual pathway through F41 and/or the D-cluster, with distance-dependent tunneling favoring the proximal F41 site. Structural and kinetic analyses show that reducing electrostatic repulsion (e.g., removing Arg57 by R57G and adjusting N59) creates a cavity and surface environment conducive to binding the positively charged oxidized viologen, greatly lowering Km while preserving kcat. Electrostatic interactions at E43 further stabilize mediator binding. Conservation of the key aromatic residue across CODHs suggests a generalizable mediator-interaction motif, informing engineering of CODHs and other viologen-using metalloenzymes (e.g., FDH, hydrogenases). Enhanced mediator affinity improves cost-effectiveness and mediator balance in multi-enzyme systems and supports performance with complex, O2-containing industrial gases when combined with tunnel-engineered O2-tolerant variants.

This work identifies and structurally validates a conserved mediator-interaction hotspot at surface phenylalanine residues near the D-cluster (F41 region) in Ni–Fe CODHs. Rational mutations of neighboring residues (R57G/N59L) enhance ethyl viologen affinity by ~10-fold without compromising turnover, and structural complexes with EV/BV elucidate charge and hydrophobic interactions and electron transfer distances. Combining enhanced mediator affinity with tunnel engineering (A559W) yields enzymes with both high efficiency and improved O2 tolerance that perform robustly on real industrial gas mixtures. These insights provide a framework to locate and tailor mediator interaction sites in CODHs and related metalloenzymes and suggest routes for designing efficient bioelectrocatalysts for industrial gas cleaning and coupled enzymatic conversions. Future work could further dissect native mediator interactions, optimize electrostatics and surface residues for other mediators, and develop site-specific immobilization strategies for direct electron transfer systems.

• Crystallization conditions: High PEG concentrations initially interfered with viologen access to the D-cluster; reduced PEG was required to resolve mediator binding, potentially biasing binding site visualization.
• Structural ambiguity: Conformational changes observed at E43 that influence surface charge remain mechanistically unresolved.
• Mutant structural constraints: F41A yielded only low-resolution crystals (7–8 Å), limiting structural interpretation of this critical site.
• Mediator scope: Focus on artificial mediators (viologens) rather than native electron carriers may limit direct physiological extrapolation.
• In vitro conditions: Kinetics and ITC under controlled conditions (CO-saturated buffers, anaerobic environments) may not fully capture behavior in complex operational settings.