How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters
التفاصيل البيبلوغرافية
العنوان:
How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters
The reversible two-electron interconversion of formate and CO2 is catalyzed by both non-metallo and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenum-or tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe-4S] clusters for facilitating long-distance electron transfer. But an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O2. Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O2 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 O2 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-à-vis catalysis under atmospheric conditions. Here, we report the discovery and characterization of an O2-insensitive FDH2 from the sulfate-reducing bacterium Desulfovibiro vulgaris Hildenborough that ligates tungsten, selenocysteine, and four [4Fe-4S] clusters. Notably, we advance a robust expression platform for its recombinant production, eliminating 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 bona fide product of catalysis. Full reaction progress curve analysis uncovers a high catalytic efficiency when probed with an artificial electron acceptor pair. Furthermore, we show that DvH-FDH2 enables hydrogen peroxide production sans superoxide release to achieve O2 insensitivity. Direct electron transfer to cytochrome c in air also reveals that electron bifurcation is operational in this system. Taken together, our work unambiguously proves for the first time the coexistence of redox bifurcated FDH and FOX activities within a metallo-FDH scaffold. These findings have important implications for engineering O2-tolerant FDHs and bio-inspired artificial metallocatalysts, as well as for the development of authentic formate/air biofuel cells, modulation of catalytic bias, assessing the limits of reversible catalysis, understanding directional electron transfer, and discerning formate bioenergetics of gut microbiota.