Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha)

Abstract

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO₂ to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction with a k_cat of 11 ± 0.4 s^-1 We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reactions are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO₂-utilizing enzyme to catalyze the reverse direction to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme and on the basis of the present work, we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalyzing the reversible interconversion of CO₂ and formate under the appropriate experimental conditions.

Keywords: biofuel; carbon dioxide; enzyme catalysis; molybdenum; multifunctional enzyme.

Figure 1.

The proposed hydride transfer mechanism for the formate dehydrogenases. As the second carbon–oxygen double bond of the product forms, the displaced hydride attacks the Mo=S moiety, resulting in the formal two-electron reduction of the metal.

Figure 2.

The dependence of k_cat for FdsABG on pH. Kinetic experiments were performed using an overlapping buffer system (75 mm each of malate, potassium phosphate, and Tris base) containing 200 μm NADH and saturated with CO₂(aq). The fit to the data (black solid line) yielded two pK_a values of pK_a1 = 5.5 and pK_a2^eff = 8.3. The plot in blue represents the corresponding conductivity, a consequence of increasing concentrations of bicarbonate/carbonate as well as KOH. All reactions were performed at 30 °C under anaerobic conditions. mS, millisiemens. Error bars represent standard deviations.

Figure 3.

Demonstration of the ability of FdsABG to catalyze both CO₂ reduction and formate oxidation. The reaction was initiated by addition of FdsABG (arrow 1) via an argon-purged Gastight syringe to 200 μm NADH in 100 mm phosphate saturated with CO₂(aq), final pH 6.3. At ∼200 s, 400 μm NAD⁺ was injected into the cuvette (arrow 2), and the reaction was allowed to proceed. At ∼400 s, 40 mm formate was further injected into the cuvette (arrow 3). The reaction was performed at 30 °C under anaerobic conditions.

Figure 4.

A, reaction of FdsABG with saturated CO₂(aq) and 300 μm NADH in 20 mm Bis-Tris propane (final pH 6.3) performed at 30 °C under anaerobic conditions. The arrow indicates addition of enzyme. B, ion chromatography analysis of the product of the reaction in A as described under “Experimental procedures.” Retention times are indicated with arrows for formate (5.9 min), bicarbonate (7.4 min), and NADH/NAD⁺ (11.2 min). C, ¹³C NMR spectrum for [¹³C]formate generated enzymatically with dissolved [¹³C]bicarbonate at 161.08 ppm. D, ¹³C NMR standard spectrum with 100 mm natural abundance formic acid brought to pH ∼6.3 with sodium bicarbonate. μS, microsiemens.

Figure 5.

A, hyperbolic plots for the reaction of FdsABG with CO₂(aq) in the presence of 200 μm NADH. B, hyperbolic plots for the reaction of FdsABG with NADH in the presence of saturated CO₂(aq). Plots in A and B yielded a k_cat of 11 ± 0.37 s⁻¹, a K_m(CO2) of 2.7 ± 0.34 mm, and a K_m(NADH) of 46 ± 4.3 μm, respectively. All reactions were performed in 100 mm potassium phosphate, pH 7.0, at 30 °C under anaerobic conditions. Error bars represent standard deviations.

Figure 6.

Active site structures for formate dehydrogenases and formylmethanofuran dehydrogenases (oxidized and reduced forms, respectively).

Figure 7.

The active site of the FdhF formate dehydrogenase (Protein Data Bank code 1FDO) from E. coli. The highly conserved Arg and His residues implicated in formate binding are indicated.