Fig. 2: EV response of surface aromatic residues in CODHs.

a Comparison of the relative activities of the ChCODH2 variants. Enzyme activities were assayed using 20 mM EV (ethyl viologen) at 30 °C, pH 8. The dashed line indicates the relative activity of WT (wild type). White dots for each point overlay bar charts. The data represent the mean ± standard deviation (S.D.), as determined from n = 3 independent experiments. b Relative F41 activity for various viologens. The activity of the WT and the F41A mutant towards viologen homologs was observed. The dotted line indicates WT activity toward EV. The F41A mutant showed no activity towards any of the viologen homologs, whereas the WT enzyme showed activity. White dots for each point overlay bar charts. The data represent the mean ± S.D., as determined from n = 3 independent experiments. Abbreviation: MV, methyl viologen (R₁ = R₂ = CH₃); EV, ethyl viologen (R₁ = R₂ = CH₂CH₃); BV, benzyl viologen (R₁ = R₂ = CH₂C₆H₅); DQ, diquat. c Conserved coordination of D-cluster in CODHs. The D-cluster environments of both 4Fe4S and 2Fe2S CODHs were analyzed by examining their sequences and structures. While the cysteine residues differ between 4Fe4S and 2Fe2S CODHs, the position of aromatic residues remains unchanged. The PDB IDs are ChCODH2 (1SU7)¹⁴, RrCODH (1JQK)¹³, ChCODH4 (6ELQ)¹⁵, DvCODH (6OND)²², and DsCODH (AF-A0A0D2HSF2) is a predicted structure from the AlphaFold database⁴⁸. d Relative activities of RrCODH F43A and DvCODH F44A for various viologens. The standard redox potential (E₀), under standard conditions (pH 7 and 25 °C), is derived from the references^55,56. MV reduction (MV²⁺ + e^‒ → MV^+•) of ‒446 mV, EV reduction (EV²⁺ + e^‒ → EV^+•) of ‒449 mV, BV reduction (BV²⁺ + e^‒ → BV^+•) of ‒359 mV, DQ reduction (DQ²⁺ + e^‒ → DQ^+•) of ‒370 mV. White dots for each point overlay bar charts. The data represent the mean ± S.D., as determined from n = 3 independent experiments.