How a Formate Dehydrogenase Responds to Oxygen: Unexpected O₂ Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

Abstract

The reversible two-electron interconversion of formate and CO2 is catalyzed by both nonmetallo- 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 (Sec) 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. However, 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 O2. Here, we report the discovery and characterization of an O2-insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe–4S] clusters. 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 discloses 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 O2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O2 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 O2-insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O2-stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.

The Mechanism of Metal-Containing Formate Dehydrogenases Revisited: The Formation of Bicarbonate as Product Intermediate Provides Evidence for an Oxygen Atom Transfer Mechanism

“…The first product that was detected by NMR in substantial abundance was bicarbonate, the formation of which already began to decrease before the abundance of CO 2 increased up to its maximum reaching even an oversaturation of the solution. In a previous report, the same observations were made, detecting bicarbonate as the first intermediate by NMR [ 43 ]. The first data point, however, was drawn only after 25 min, which impedes disentangling the still quite fast enzymatic reaction and the subsequent secondary reaction of the CO 2 /HCO 3 – equilibrium at pH 9.0.…”

Reply to “Comment on ‘How a Formate Dehydrogenase Responds to Oxygen: Unexpected O2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters’”

“…Buckel's mechanistic basis is also unrealistic in that it takes no cognizance of a large body of experimental evidence 20,25 �some from his own laboratory 33,34 �which has yet to be challenged. If Buckel is correct, DvH-FDH2 must wait until most (if not all) of the O 2 has been reduced before commencing the reduction of Cyt c. Also, the enzymatic reaction profile would be akin to what we saw with aerobic reduction of benzyl viologen (see Supplementary Video to Graham et al 38 ). However, this is not what we observed.…”

“…EB is the process by which an unique electron donor, capable of 2e − /1e − "step-down" 12,14,15 (quinol, reduced flavin, Mo IV /W IV , or H 2 ), 31 simultaneously reduces two different 1e − acceptors. 20,47 such spatially separated electron acceptors: O 2 and Cyt c. 38 Since our enzyme harbors W co �and lacks both flavin and quinone�I have proposed that metal-based EB (MBEB) is at work, and I stand by this interpretation of our data. I also see no problems with drawing parallels between periplasmic MBEB and cytosolic FBEB.…”

“…−S31 in ref38). Since stoichiometric H 2 O 2 is concurrently produced during this reaction, it means that electrons derived from formate oxidation ended up on both Cyt c and O 2 at the same time.…”

Hydrogen-fueled CO2 reduction using oxygen-tolerant oxidoreductases

“…Under aerobic conditions, the inactivation of FDH occurs by substitution of oxo ligand for sulfide ligand at the active site by O 2 ( Duffus et al, 2020 ). It was proposed that the O 2 tolerance of W-containing FDH2 from Desulfovibrio vulgaris Hildenborough results from reduction of O 2 to hydrogen peroxide by formate oxidase activity ( Graham et al, 2022 ). The FDHs from Clostridium carboxidivorans strain P7T ( Alissandratos et al, 2013 ), Methylobacterium extorquens AM1 ( Laukel et al, 2003 ; Baccour et al, 2020 ), and Rhodobacter capsulatus ( Hartmann and Leimkühler, 2013 ) maintain high CO 2 -reducing activity under aerobic conditions.…”