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

Abstract

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.

The Reversible Electrochemical Interconversion of Formate and CO2 by Formate Dehydrogenase from Cupriavidus necator

“…12,13 At issue is whether CO 2 (eq 1) or HCO 3 − (eq 3) is the immediate product of enzymatic formate oxidation. 13,14 There are also structural studies that have revealed alternative active site structures to those shown in Figure 1 where dissociation of the protein ligand has occurred, at least under reducing conditions and dependent on the reductant. 15,16 Regardless of the specific chemical mechanism, the reversible catalytic oxidation of formate/reduction of CO 2 continues to receive the attention of researchers across diverse fields due to its relevance to global warming and also to future energy storage.…”

“…The physiological function of all formate dehydrogenases is the two-electron oxidation of formate (HCOO – ) to carbon dioxide (CO 2 ) (eq ) using various electron acceptors including NAD(P) + , cytochromes, quinones, and Fe–S proteins to mention but a few. ,, It has emerged that most, if not all, formate dehydrogenases are in fact bidirectional and may catalyze the reverse reduction of CO 2 to HCOO – . , The preponderance of evidence is that the reaction occurs via hydride transfer from the C–H group of formate to form CO 2 directly , consistent with the earlier conclusion that CO 2 is the direct product of the reaction − and that the reaction occurs without oxygen atom transfer. Still, mechanisms involving oxygen atom transfer have been proposed. , At issue is whether CO 2 (eq ) or HCO 3 – (eq ) is the immediate product of enzymatic formate oxidation. , There are also structural studies that have revealed alternative active site structures to those shown in Figure where dissociation of the protein ligand has occurred, at least under reducing conditions and dependent on the reductant. , Regardless of the specific chemical mechanism, the reversible catalytic oxidation of formate/reduction of CO 2 continues to receive the attention of researchers across diverse fields due to its relevance to global warming and also to future energy storage HCOO − ⇌ CO 2 + H + + 2 e − CO 2 + H 2 O ⇌ normalHCO 3 − + H + …”

“…Still, mechanisms involving oxygen atom transfer have been proposed. 12,13 At issue is whether CO 2 (eq 1) or HCO 3 − (eq 3) is the immediate product of enzymatic formate oxidation. 13,14 There are also structural studies that have revealed alternative active site structures to those shown in Figure 1 where dissociation of the protein ligand has occurred, at least under reducing conditions and dependent on the reductant.…”

Metal-Containing Formate Dehydrogenases, a Personal View

“…Our first results clearly showed that after a reaction time of 10 s with 13 C-labeled formate, labelled 13 C 18 O 16 O was detected, demonstrating that the oxygen of H 2 18 O water is indeed inserted into the product, which is rather bicarbonate and not CO 2 . We showed that the enzyme-catalyzed reaction was much faster as compared to the secondary hydration of CO 2 under our experimental conditions [ 40 ]. In contrast, in the azide-inhibited reaction, which we performed as a control, we obtained the same results as reported by Khangulov, showing that azide interferes with the reaction and with product formation.…”

“…When 13 C-labelled thioformate was used in the NMR experiment with low azide concentrations, thiocarbonate was clearly detected as the first intermediate before COS abundance began to increase steadily. Here, thiocarbonate is detected instead of thiobicarbonate, based on the fact that thiobicarbonate is more acidic than bicarbonate, less stable at this pH and is, in fact, easily deprotonated [ 40 ]. This clearly confirmed the oxygen atom transfer with bicarbonate/thiocarbonate as first reaction intermediate of the reaction, which are released from the enzyme before CO 2 or COS are formed in the slower non-enzymatic secondary reaction outside the enzyme.…”