No competitive inhibition of bicarbonate or carbonate for formate dehydrogenase from Candida boidinii-catalyzed CO2 reduction

Abstract

Formate dehydrogenase from Candida boidinii (CbFDH) reversibly catalyzes formate to CO₂ with the redox couple NAD⁺/NADH. While many studies on CbFDH-catalyzed formate oxidation in the presence of NAD⁺ are reported, there are few studies on the detailed mechanism of the CbFDH-catalyzed CO₂ reduction in the presence of NADH. In this study, the detailed mechanism of formate production by CbFDH with NADH in GTA buffer solution (consisting of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane and 2-amino-2-methyl-1,3-propanediol) with different proportions of CO₂, bicarbonate and carbonate was studied. A good correlation was found between the pH of the buffer solution, the CO₂ concentration and the apparent initial rate of formate production with CbFDH. Under constant conditions of total carbonate concentration in the buffer solution, the process of the CbFDH-catalyzed reduction of CO₂ to formate followed the Michaelis–Menten kinetics. Particularly, the reaction rate of formate production with CbFDH increased with increasing CO₂ concentration under constant conditions of total carbonate concentration in the solution. As the apparent formate production rate with CbFDH increases under higher total carbonate concentration, bicarbonate or carbonate does not act as a competitive inhibitor of CO₂, and it is predicted that the CO₂ concentration equivalent consumed in the CbFDH-catalyzed formate production is supplied by bicarbonate or carbonate by the pH control function of the GTA buffer solution. Thus, we have clarified for the first time that these carbonates do not function as any inhibitor for the CbFDH-catalyzed CO₂ reduction.