Guilherme Martins

Mo/W‐dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO ₂ to formate and are key biocatalysts with high potential for CO ₂ capture/conversion technologies. Although previous studies have suggested the presence of two substrate‐access tunnels in Fdhs, experimental evidence for CO ₂ ‐specific pathways has been lacking. Here, we present an integrated study of Nitratidesulfovibrio vulgaris FdhAB combining crystallography, molecular dynamics simulations, mutagenesis, and kinetic assays. Nv FdhAB crystals pressurized with Kr, O ₂ , and CO ₂ were used to map gas diffusion routes and uncovered a substrate‐retention site consistently occupied by small molecules in multiple crystal structures. Our results indicate that both substrates mostly use the main tunnel to reach this retention site, but H ₂ O and CO ₂ can also enter through a novel side branch before following a shared route to the buried W active site. The retention site, located at the junction of both tunnels, plays a synergistic role in enhancing CO ₂ reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO ₂ reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO ₂ ‐specific pathway and identify structural determinants underpinning efficient CO ₂ reduction in this enzyme family.

Mo/W‐dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO ₂ to formate and are key biocatalysts with high potential for CO ₂ capture/conversion technologies. Although previous studies have suggested the presence of two substrate‐access tunnels in Fdhs, experimental evidence for CO ₂ ‐specific pathways has been lacking. Here, we present an integrated study of Nitratidesulfovibrio vulgaris FdhAB combining crystallography, molecular dynamics simulations, mutagenesis, and kinetic assays. Nv FdhAB crystals pressurized with Kr, O ₂ , and CO ₂ were used to map gas diffusion routes and uncovered a substrate‐retention site consistently occupied by small molecules in multiple crystal structures. Our results indicate that both substrates mostly use the main tunnel to reach this retention site, but H ₂ O and CO ₂ can also enter through a novel side branch before following a shared route to the buried W active site. The retention site, located at the junction of both tunnels, plays a synergistic role in enhancing CO ₂ reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO ₂ reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO ₂ ‐specific pathway and identify structural determinants underpinning efficient CO ₂ reduction in this enzyme family.

Paired electrolysis enables the simultaneous coupling of the CO2 reduction reaction (CO2RR) with anodic waste valorisation to form valuable products, but achieving selective, efficient and stable product formation remains a challenge. In this study, W-containing formate dehydrogenase (FDH) from Nitratidesulfovibrio vulgaris Hildenborough is immobilised onto an electrode made from carbon felt coated with porous TiO2 and paired with commercial Ni foam to demonstrate the first enzymatic flow electrolyser for the simultaneous conversion of CO2 and waste (plastic and biomass) to the single product formate. The enzymatic flow electrolyser achieved a combined faradaic efficiency (FE) towards formate of almost 200% for both electrodes and can operate at an unprecedently low full-cell voltage of −1.5 V for 122 h. The aqueous formate produced in the semiartificial electrolyser was further utilised downstream as a C1 building block in the photocatalytic hydrocarboxylation of alkenes, providing a path for the domino valorisation of CO2 and waste towards bulk and fine chemical synthesis.

We report the formation of demethylation in 3-methyl-uracil (3meU) and 1-methyl-thymine (1meT), i.e. (3meU-CH₃)⁻ and (1meT-CH₃)⁻, through potassium-molecule collisions at different potassium kinetic energies. Study of the threshold of formation of this fragment can provide a value of threshold energy that can be compared with DEA studies.