Health, Safety & Environment

Formate dehydrogenase

Molybdenum and tungsten-containing formate dehydrogenases: Aiming to inspire a catalyst for carbon dioxide utilization

The global energy demand and the present high dependence on fossil fuels have caused an unprecedented increase in the Earth's atmosphere carbon dioxide concentration. Its exponential and uncontrollable rise is responsible for large and unpredictable impacts on the world climate and for ocean acidification, thus, being a major concern for the ecosystems and human's daily life. On the other hand, the carbon dioxide abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. Yet, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its utilization a laboratorial/industrially challenging task. In this Review, we propose to use the molybdenum and tungsten-containing formate dehydrogenase (FDH) enzymes as a model to understand the mechanistic strategies and key chemical features needed to reduce carbon dioxide to formate. We will highlight the present knowledge about the structure of FDHs, with particular emphasis on active site features, reaction mechanism and ability to reduce carbon dioxide to formate. The information gathered aims to inspire the development of new efficient (bio)catalysts for the atmospheric carbon dioxide utilization, to produce energy and chemical feedstocks, while reducing an important environmental pollutant. (C) 2016 Elsevier B.V. All rights reserved.

Maia, L. B., Moura, I., and Moura, J. J. G., Molybdenum and tungsten-containing formate dehydrogenases: Aiming to inspire a catalyst for carbon dioxide utilization, Inorganica Chimica Acta, 2017, 455, 350-363.

Spectroscopic and kinetic properties of the molybdenum-containing, NAD(+) - dependent formate dehydrogenase from ralstonia eutropha

We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+) -dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo-V state and demonstrated the direct transfer of the substrate C alpha hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

Niks, D., Duvvuru, J., Escalona, M., and Hille, R.,Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD(+) - dependent Formate Dehydrogenase from Ralstonia eutropha, Journal of Biological Chemistry, 2016, 291, 1162-1174.

Formate and nitrate. The sulfur shift: An activation mechanism for periplasmic nitrate reductase and formate dehydrogenase

The sulfur-shift mechanism is characterized by the displacement of the coordinating cysteine (in Nap or SeCys in Fdh) side chain to a second shell of the Mo-coordination sphere. This rearrangement enables a free coordination position for substrate binding to Mo ion and provides an efficient mechanism to maintaining a constant coordination number throughout the entire catalytic pathway. This type of mechanism is very similar to the carboxylate shift observed in other enzymes, and it has been recently detected by experimental means.

A structural rearrangement known as sulfur shift occurs in some Mo-containing enzymes of the DMSO reductase family. This mechanism is characterized by the displacement of a coordinating cysteine thiol (or SeCys in Fdh) from the first to the second shell of the Mo-coordination sphere metal. The hexa-coordinated Mo ion found in the as-isolated state cannot bind directly any exogenous ligand (substrate or inhibitors), while the penta-coordinated ion, attained upon sulfur shift, has a free binding site for direct coordination of the substrate. This rearrangement provides an efficient mechanism to keep a constant coordination number throughout an entire catalytic pathway. This mechanism is very similar to the carboxylate shift observed in Zn-dependent enzymes, and it has been recently detected by experimental means. In the present paper, we calculated the geometries and energies involved in the sulfur-shift mechanism using QM-methods (M06/(6-311++G(3df,2pd),SDD)//B3LYP/(6-31G(d),SDD)). The results indicated that the sulfur-shift mechanism provides an efficient way to enable the metal ion for substrate coordination.

Cerqueira, N. M., Fernandes, P. A., Gonzalez, P. J., Moura, J. J., and Ramos, M. J. The Sulfur Shift: An Activation Mechanism for Periplasmic Nitrate Reductase and Formate Dehydrogenase. Inorg. Chem., 2013, 52 , 10766–10772.

Formate and nitrate. periplasmic nitrate reductase and formate dehydrogenase: Similar molecular architectures with very different enzymatic activities

It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both enzymes; however, Met and His have different roles. His participates directly on catalysis, and it is therefore detrimental for the catalytic cycle of FdH. Met only participates in substrate binding. We concluded that this small but key difference dictates the type of reaction that is catalyzed by each enzyme. In addition, it allows explaining why formate can bind in the Nap active site in the same way as the natural substrate (nitrate), but the reaction becomes stalled afterward.

Cerqueira, N. M., Gonzalez, P. J., Fernandes, P. A., Moura, J. J., and Ramos, M. J., Periplasmic Nitrate Reductase and Formate Dehydrogenase: Similar Molecular Architectures with Very Different Enzymatic Activities, Accounts of chemical research, 2015.

[Periplasmic: The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria. Cytoplasm is the fluid that fills a cell. Wikipedia.]

Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria

The global carbon cycle depends on the biological transformations of C1 compounds, which include the reductive incorporation of CO2 into organic molecules (e.g. in photosynthesis and other autotrophic pathways), in addition to the production of CO2 from formate, a reaction that is catalyzed by formate dehydrogenases (FDHs). FDHs catalyze, in general, the oxidation of formate to CO2 and H+. However, selected enzymes were identified to act as CO2 reductases, which are able to reduce CO2 to formate under physiological conditions. This reaction is of interest for the generation of formate as a convenient storage form of H2 for future applications. Cofactor-containing FDHs are found in anaerobic bacteria and archaea, in addition to facultative anaerobic or aerobic bacteria. These enzymes are highly diverse and employ different cofactors such as the molybdenum cofactor (Moco), FeS clusters and flavins, or cytochromes. Some enzymes include tungsten (W) in place of molybdenum (Mo) at the active site. For catalytic activity, a selenocysteine (SeCys) or cysteine (Cys) ligand at the Mo atom in the active site is essential for the reaction. This review will focus on the characterization of Mo- and W-containing FDHs from bacteria, their active site structure, subunit compositions and its proposed catalytic mechanism. We will give an overview on the different mechanisms of substrate conversion available so far, in addition to providing an outlook on bio-applications of FDHs. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Hartmann, T., Schwanhold, N., and Leimkuhler, S.,Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria, Biochimica et biophysica acta, 2015, 1854, 1090-100.


The prokaryotic formate metabolism is considerably diversified. Prokaryotes use formate in the C1 metabolism, but also evolved to exploit the low reduction potential of formate to derive energy, by coupling its oxidation to the reduction of numerous electron acceptors. To fulfil these varied physiological roles, different types of formate dehydrogenase (FDH) enzymes have evolved to catalyse the reversible 2-electron oxidation of formate to carbon dioxide. This review will highlight our present knowledge about the diverse physiological roles of FDH in prokaryotes, their modular structural organisation and active site structures  and the mechanistic strategies followed to accomplish the formate oxidation. In addition, the ability of FDH to catalyse the reverse reaction of carbon dioxide reduction, a potentially relevant reaction for carbon dioxide sequestration, will also be addressed.

Maia LB, Moura JJ, Moura I. Molybdenum and tungsten-dependent formate dehydrogenases. J Biol Inorg Chem. 2015 Mar;20(2):287-309. doi: 10.1007/s00775-014-1218-2. Epub 2014 Dec 5.

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO2 and H2. The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein, FdhE. E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation

Luke, I., Butland, G., Moore, K., Buchanan, G., Lyall, V., Fairhurst, S., Greenblatt, J., Emili, A., Palmer, T., and Sargent, F., Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein, Archives of Microbiology, 2008, 190, 685-696.

The mechanism of formate oxidation by metal-dependent formate dehydrogenases

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate [formate] to the active site.

The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools.

The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species.

Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle.

The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results

Mota, Cristiano S., Rivas, Maria G., Brondino, Carlos D., Moura, Isabel, Moura, Jose J. G., Gonzalez, Pablo J., and Cerqueira, Nuno M. F. S., The mechanism of formate oxidation by metal-dependent formate dehydrogenases, Journal of Biological Inorganic Chemistry, 2011, 16, 1255-1268.

Clostridium carboxidivorans strain P7T recombinant formate dehydrogenase catalyzes reduction of CO2 to formate

Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7(T), expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4 degrees C under oxic conditions

Alissandratos, A., Kim, H. K., Matthews, H., Hennessy, J. E., Philbrook, A., and Easton, C. J., Clostridium carboxidivorans Strain P7T Recombinant Formate Dehydrogenase Catalyzes Reduction of CO2 to Formate, Applied and Environmental Microbiology, 2013, 79, 741-744.

Periplasmic nitrate reductases and formate dehydrogenases: Biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role

Mo- and W-enzymes are widely distributed in biology as they can be found in all domains of life. They perform key roles in several metabolic pathways catalyzing important reactions of the biogeochemical cycles of the more abundant elements of the earth. These reactions are usually redox processes involving the transfer of an atom from the substrate to the metal ion or vice versa. The Mo or W reactivity and specificity toward a substrate is determined by the polypeptide chain of the enzyme, which tunes the chemical properties of the metal ion. Two enzymes sharing almost identical active sites but catalyzing very different reactions are periplasmic nitrate reductase and formate dehydrogenase from bacteria. They represent a good example of how key changes in the amino acid sequence tune the properties of an enzyme. In order to analyze the chemistry of Mo and W in these enzymes, structural, kinetic and spectroscopic data are reviewed, along with the role of these enzymes in cell metabolism. In addition, the features that govern selectivity of metal uptake into the cell and Mo/W-cofactor biosynthesis are revised. (C) 2012 Published by Elsevier B.V

Gonzalez, P. J., Rivas, M. G., Mota, C. S., Brondino, C. D., Moura, I., and Moura, J. J. G., Periplasmic nitrate reductases and formate dehydrogenases: Biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role, Coordination Chemistry Reviews, 2013, 257, 315-331.