New insights into enzymatic CO₂ reduction using protein film electrochemistry
Keywords: carbon monoxide dehydrogenase
CO₂ reduction
reversible catalyst
protein film electrochemistry

Carbon monoxide dehydrogenase (CODH) is known to catalyze CO oxidation and CO₂ reduction reversibly with the minimal overpotential. A great advantage of protein film electrochemistry (PFE) is its ability to probe catalysis over a wide range of potentials, especially in the low potential region required for CO₂ reduction. CODH I and CODH II from Carboxydothermus hydrogenoformans(Ch) and the composite enzyme acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica(Mt) are intensively studied throughout this thesis.
The different catalytic redox-states in CODH, Cox (inactive state), Cred1 (for CO oxidation) and Cred2 (for CO₂ reduction) as characterized by spectroscopy, are studied by PFE in the presence of substrate-mimic inhibitors. Cyanide, isoelectronic with CO, mainly inhibits CO oxidation, whereas cyanate, isoelectronic with CO₂, mainly targets CO₂ reduction. Sulfide inhibits CODH rapidly when the potential is more positive than −50 mV, which suggests that sulfide reacts to form a state at the oxidation level of Cox in CODH and is not an activator for CODH catalysis as suggested before. Thiocyanate only partially inhibits CO oxidation. No inhibition of CODH by azide is detected, which is in contrast with previous studies with ACS/CODHMt. The main differences between CODH ICh and CODH IICh are the stronger CO product inhibition and inhibition of CODH IICh by cyanide. These discoveries might shed light on the possible role of CODH IICh,/sub> in biological systems.
In comparison with bidirectional (reversible) electrocatalysis by CODH ICh and CODH IICh, only unidirectional electrocatalysis for CO oxidation by ACS/CODHMt is observed with an overpotential of 0.1 V and the electrocatalytic current is much smaller. In order to identify whether ACS influences the performance of CODH, several chemical reagents, such as sodium dodecyl sulfate (which separates CODH and ACS partially), 1, 10-phenanthroline, (which inhibits the active site in ACS) and acetyl-CoA (the product of the reaction carried out by ACS/CODHMt) are added. However, we have yet to observe any electrocatalytic current from CO₂ reduction. Inhibition of ACS/CODHMt by cyanide, cyanate and azide is consistent with previous studies by spectroscopy.
Oxygen attack toward the active site in CODH is proved by cyanide protection. The inactive state, Cox can prevent oxygen attack and reductive reactivation restores CODH activity. In contrast, oxygen damages the active site irreversibly when CODH is in the Cred1 state. The new substrate, nitrous oxide (N₂O), isoelectronic with CO₂, is reduced by CODH and acts as the suicide substrate.
Finally, hydrogen formation in the direction of CO oxidation and formate formation in the direction of CO₂ reduction by CODH are detected. The small solvent kinetic isotope effect is observed in CO oxidation. These findings suggest metal-hydride should play a role in CODH catalysis, which might provide a new direction to design better catalysts for CO₂ reduction.