During the last decade the number of characterized F₄₂₀-dependent enzymes has significantly increased. Many of these deazaflavoproteins share a TIM-barrel fold and are structurally related to FMN-dependent luciferases and monooxygenases. In this work, we traced the origin and evolutionary history of the F₄₂₀-dependent enzymes within the luciferase-like superfamily. By a thorough phylogenetic analysis we inferred that the F₄₂₀-dependent enzymes emerged from a FMN-dependent common ancestor. Furthermore, the data show that during evolution, the family of deazaflavoproteins split into two well-defined groups of enzymes: the F₄₂₀-dependent dehydrogenases and the F₄₂₀-dependent reductases. By such event, the dehydrogenases specialized in generating the reduced deazaflavin cofactor, while the reductases employ the reduced F₄₂₀ for catalysis. Particularly, we focused on investigating the dehydrogenase subfamily and demonstrated that this group diversified into three types of dehydrogenases: the already known F₄₂₀-dependent glucose-6-phosphate dehydrogenases, the F₄₂₀-dependent alcohol dehydrogenases, and the sugar-6-phosphate dehydrogenases that were identified in this study. By reconstructing and experimentally characterizing ancestral and extant representatives of F₄₂₀-dependent dehydrogenases, their biochemical properties were investigated and compared. We propose an evolutionary path for the emergence and diversification of the TIM-barrel fold F₄₂₀-dependent dehydrogenases subfamily.

Mo/W-containing formate dehydrogenases (FDH) catalyzed the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active sites. While in the reaction of formate oxidation, the product is CO₂, which exits the active site via a hydrophobic channel; bicarbonate is formed as the first intermediate during the reaction at the active site. Other than what has been previously reported, bicarbonate is formed after an oxygen atom transfer reaction, transferring the oxygen from water to formate and a subsequent proton-coupled electron transfer or hydride transfer reaction involving the sulfido ligand as acceptor.

A minimal light‐driven approach was established for studying enzymatic CO₂conversion spectroscopically. The system consists of a photosensitizer Eosin Y, EDTA as a sacrificial electron donor and substrate source, and formate dehydrogenase fromRhodobacter capsulatus(RcFDH) as a biocatalyst. This simplified three‐component system provides a photo‐triggered control forin situcharacterization of the entire catalytic reaction. Direct reduction ofRcFDH by the photosensitizer without additional electron carriers was confirmedviaUV‐Vis spectroscopy, while GC‐MS and IR spectroscopy were used to follow photoinduced CO₂generation from EDTA and its subsequent enzymatic reduction, yielding the product formate. Photo‐driven andin vitro, dye‐based CO₂reduction was inhibited by azide under a mixed (competitive‐non‐competitive) inhibition mode. IR spectroscopy reveals displacement of the competitively‐bound azide by CO₂, reflecting an interaction of both with the active site cofactor. This work comprises a proof‐of‐concept for a new approach to employ light for regulating the reaction of formate dehydrogenases and other CO₂reductases.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO₂ reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO₂-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO₂ capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD⁺ vs. NADP⁺ reduction. Starting from the NAD⁺-specific wild-type RcFDH, key residues were exchanged to enable NADP⁺ binding on the basis of the NAD⁺-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys¹⁵⁷) in the β-subunit of the enzyme is essential for the binding of NAD⁺. RcFDH variants that had Glu²⁵⁹ exchanged for either a positively charged or uncharged amino acid had additional activity with NADP⁺. The FdsB^L279R and FdsB^K276A variants also showed activity with NADP⁺. Kinetic parameters for all the variants were determined and tested for activity in CO₂ reduction. The variants were able to reduce CO₂ using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.