Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase

Abstract

Protein film electrochemistry has been used to investigate reactions of highly active nickel-containing carbon monoxide dehydrogenases (CODHs). When attached to a pyrolytic graphite electrode, these enzymes behave as reversible electrocatalysts, displaying CO2 reduction or CO oxidation at minimal overpotential. The O2 sensitivity of CODH is suppressed by adding cyanide, a reversible inhibitor of CO oxidation, or by raising the electrode potential. Reduction of N2O, isoelectronic with CO2, is catalyzed by CODH, but the reaction is sluggish, despite a large overpotential, and results in inactivation. Production of H2 and formate under highly reducing conditions is consistent with calculations predicting that a nickel-hydrido species might be formed, but the very low rates suggest that such a species is not on the main catalytic pathway.

Figure 1

Structure of CODH II_Ch (PDB: 3B53) from Carboxydothermus hydrogenoformans. Two positions are shown for the “pendant” Fe atom that is thought to bind an oxygen atom during catalysis.

Figure 2

Effects on CODH II_Ch electrocatalysis of injecting O₂-saturated buffer (0.5 mL) into the electrochemical cell (3.0 mL) under different conditions. The black line indicates times during which the electrode potential was poised at (a) +140 mV or (b) −260 mV, and the red line indicates times during which the electrode potential was poised at −760 mV for reductive reactivation. Experimental conditions: 25 °C, 0.2 M MES buffer (pH 7.0), rotation rate 2500 rpm and 100% CO. In panel c, black, red, and blue lines indicate times during which the electrode potential was poised at −260, +140, and −560 mV, respectively, while the green dashed line indicates the film loss extrapolated from the first 10 min. The lower concentration of CO (5% CO in Ar in the white region) was used to accelerate formation of the inactive C_ox state in CODH at +140 mV. Oxygen-saturated buffer was injected at 1600 s. Experimental conditions: 10 °C, 0.2 M MES buffer (pH 7.0), rotation rate 2500 rpm, 100% CO (gray region), and 5% CO (white region).

Figure 3

Experiment showing that cyanide protects CODH II_Ch against irreversible degradation by O₂. The black line indicates when the potential was poised at −260 mV, and the red line refers to a poise at −760 mV. The gray windows refer to periods during which buffer is exchanged. The green dashed line shows an extrapolation for film loss. Experimental conditions: 25 °C, 0.2 M MES buffer (pH 7.0), rotation rate 2500 rpm, and 100% CO.

Figure 4

Cyclic voltammograms of the electrocatalytic activity of CODH I_Ch in the presence of different gases. The black, red, and blue lines indicate experiments carried out under 100% Ar, 100% N₂O, and 100% CO₂, respectively. (a) Cyclic voltammetry performed with an electrode rotated at 1000 rpm. (b) Cyclic voltammetry performed with a stationary electrode. Experimental conditions: 25 °C, 0.2 M MES buffer (pH 7.0), scan rate 20 mV s⁻¹.

Figure 5

Experiments in which CODH I_Ch on the PGE electrode was poised at (a) −209 mV, (b) −439 mV, and (c) −760 mV in the presence of N₂O. The white region indicates the presence of 100% CO (a, b) or 100% CO₂ (c); the blue and gray regions indicate the presence of 100% N₂ and 100% Ar, respectively; the red region shows when the atmosphere is 100% N₂O. The inset in Figure 5b shows an enlargement of the region of N₂O reduction at −439 mV. Experimental conditions: 25 °C, 0.2 M MES buffer (pH 7.0), and rotation rate 1500 rpm.

Figure 6

Formation of formate by CODH I_Ch adsorbed on a MWCT-modified PGE electrode at −760 mV. The upper panel shows the NMR spectrum of the solution after flowing CO₂ (40 scc/m) through the headspace for 3 days. Experimental conditions: 25 °C, 0.2 M MES (pH 7.0 and 45.5% D₂O and 55.5% H₂O), electrode rotation rate 400 rpm. The middle panel shows the spectrum obtained for the control experiment conducted under N₂. The lower panel shows the spectrum of 1 mM potassium formate in 100% D₂O under the same conditions.

Figure 7

Catalysis of H₂ evolution by CODH I_Ch and CODH II_Ch in the presence of 96% CO/4% CH₄. Aliquots of enzyme, 30 µL of CODH I_Ch (15 mg/mL) and 20 µL of CODH II_Ch (18 mg/mL) were added to 0.2 M MES buffer (pH 7.0) to give a final volume of 0.3 mL. Reactions were performed at 20 °C. See text for details.

Scheme 1

Two Possible Mechanisms of Action of Ni–CODH Proposed by (a) the Groups of Dobbek and Lindahl^, (Involving a Ni(0) Subsite for “Book-Keeping” Purposes) or (b) Fontecilla-Camps and Coworkers (in Which a Ni(II)–H Intermediate State Is Proposed)^{a a}B refers to the amino acid that provides or accepts a proton, possibly histidine or lysine. The substrate analogue CN⁻ inhibits CO oxidation, whereas NCO⁻ inhibits CO₂ reduction.