Understanding the reversibility of formate/carbon dioxide redox reaction catalyzed by formate dehydrogenases.

SLABOCH, MARIA V.; ELIZALDE, HILEN; BRONDINO, CARLOS D.; RIVAS, MARÍA G.; GONZALEZ, PABLO J.

Encuentro; XLIX Reunión Anual de la SAB.; 2021

Formate dehydrogenase (Fdh) is an enzyme whose physiological role is catalyzing the oxidation of formate (HCOO-) to carbon dioxide (CO2) to obtain reducing equivalents required for several metabolic routes in a living cell. Fdhs are also able to reduce CO2 to HCOO- under a reducing environment, either in solution (with a physiological or artificial electron donor) or adsorbed to an electrode under reducing overpotentials. In this work, we present the isolation and biochemical/kinetic characterization of Fdh from Thiobacillus sp. (TspFdh), a metal−free, O2−tolerant Fdh. TspFdh is a monomeric protein of ~45 kDa that uses NAD+ as the redox cofactor. The catalytic (bi−substrate) mechanism for NAD−dependent Fdhs (NAD−Fdh) implies that both HCOO- and NAD+ must interact in the enzyme active site for hydride transfer to occur, releasing CO2 and NADH as products. It was reported elsewhere that TspFdh catalyzes the reverse reaction more efficiently than other NAD−Fdhs, therefore, we investigated the reason behind this difference. Kinetic assays using several concentrations of both formate and NAD+ showed that TspFdh follows a sequential rather than a Ping−Pong (substituted) mechanism. Previous reports pointed out this, and also showed through inhibition studies that NAD-Fdhs from plants would follow a compulsory ordered mechanism with a negligible accumulation of ternary complex (Theorell−Chance), while NAD−Fdhs from prokaryotes fit well to a rapid−equilibrium sequentially random mechanism. The latter means that HCOO- and NAD+ have improved affinity for Fdh−NAD+ and Fdh− HCOO- complexes, respectively, compared to free enzyme. A variable temperature study of the kinetic isotope effect (KIE) showed that in TspFdh would occur H−tunneling, which is detectable in the lowtemperature limit. Nevertheless, we only used protio-1H− and 2H−formate, so we were unable to determine the intrinsic KIE. NAD−Fdhs from Candida boidinii and other sources do not present detectable tunneling. This observation together with studies being performed in Mo/W−dependent Fdhs might be important to understand why some Fdhs catalyze more efficiently the reverse reaction.References- Choe H et al. (2014) PLoS ONE 9(7): e103111.- Choe H et al (2015) Acta Cryst. D71, 313–323.- Tishkov VI and Popov VO (2004) Biochemistry (Mosc.) 69: 1252–1267.