How a Formate Dehydrogenase Responds to Oxygen: Unexpected O-2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe-4S] Clusters

The reversible two-electron interconversion of formate and CO2 is catalyzed by both nonmetallo- and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenumor tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl (Sec) ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe-4S] dusters for facilitating long-distance electron transfer. However, an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O-2. Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O-2 lability of their metal centers, studies dating as far back as the 1930s emphasize that some of these systems exhibit formate oxidase (FOX) activity, coupling formate oxidation to O-2 reduction. Therefore, to reconcile these conflicting views, we explored context-dependent functional linkages between metallo-FDHs and their cognate electron acceptors within the same organism vis-a-vis catalysis under atmospheric O-2. Here, we report the discovery and characterization of an O-2-insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe-4S] dusters. By advancing a robust expression platform for its recombinant production, we eliminate both the requirement of nitrate or azide during purification and reductive activation with thiols and/or formate prior to catalysis. Because the distinctive spectral signatures of formate-reduced DvH-FDH2 remain invariant under anaerobic and aerobic conditions, we benchmarked the enzyme activity in air, identifying CO2 as the catalytic product. Full reaction progress curve analysis disdoses a high catalytic efficiency when probed with a high-potential artificial electron acceptor. Furthermore, we show that DvH-FDH2 enables near-stoichiometric hydrogen peroxide production without superoxide release to achieve O-2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O-2 reveals that metal-based electron bifurcation is operational in this system. Taken together, our work proves the co-occurrence of redox bifurcated FDH and FOX activities within a metalloenzyme scaffold. These findings set the stage for uncovering previously unknown O-2-insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O-2-stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.