Mechanisms of CO₂ reduction in bacteria based on molecular studies of formate dehydrogenase (FDH) enzymes

1.1 Formate dehydrogenase (FDH)

Formate dehydrogenase is an enzyme that can catalyze the reduction of CO₂ to formate in certain microorganisms. The reaction involves the transfer of two electrons and two protons from a reduced cofactor, such as NADH or NADPH, to CO₂, which is then reduced to formate (Schlager et al., 2017). Formate dehydrogenase (FDH) is a stable enzyme, but hydrophobic interfaces can inactivate it. This is because FDH has a hydrophobic binding site sensitive to environmental changes, such as exposure to hydrophobic surfaces. The oxidoreductive interconversion of carbon dioxide and formic acid is catalyzed by FDH, a member of the D-specific 2-hydroxy acid dehydrogenase superfamily consisting of single subunits with four distinct domains. In many bacteria and eukaryotes, FDHs catalyze the final step of the catabolic process in which formate is oxidized to CO₂ (Deng et al., 2023). However, a certain type of FDH can also act as CO₂ reductase. Some redox enzymes also effectively catalyze reactions reversibly such as DMSO-reductase, succinate: quinine reductase, CO dehydrogenase, fumarate, menaquinone oxidoreductase, and some hydrogenases (Unden and Bongaerts 1997).

1.2 Type of formate dehydrogenase

Formate dehydrogenase (FDH) is a diverse family of enzymes found in various organisms, including bacteria, archaea, and eukaryotes. Several types of FDH enzymes differ in their structures, cofactor requirements, and catalytic activities. NAD-dependent FDH enzymes require the cofactor NAD⁺ or NADP⁺ for their activity, and are typically found in bacteria and some eukaryotes (Nielsen et al., 2019). They catalyze the interconversion of formate and CO₂ through an oxidoreductive process. Cytochrome c-dependent FDH enzymes require cytochrome c as a cofactor, and are found in certain bacteria and archaea (Makela et al., 2010). They are involved in the electron transport chain and play a role in the metabolism of various organic compounds. Molybdenum-dependent FDH enzymes require molybdenum as a cofactor, and are found in bacteria, archaea, and some eukaryotes. They are involved in the catabolism of various compounds, including formate, formaldehyde, and methanol. Tungsten-dependent FDH enzymes require tungsten as a cofactor, and are found in certain bacteria and archaea (Maia et al., 2015). They are involved in the catabolism of formate and other compounds and can also catalyze the reduction of CO₂ to formate. Iron-dependent FDH enzymes require iron as a cofactor and are found in some bacteria and archaea. They are involved in the catabolism of various compounds, and can catalyze the reduction of CO₂ to formate (Cotton et al., 2020).

1.3 Formate dehydrogenase – Metal-based classification

FDHs can be classified into two broad categories based on their cofactor requirements: metal-independent FDHs and metal-containing FDHs. Metal-independent FDHs enzymes do not require any metal cofactors for their activity. Instead, they utilize organic cofactors such as pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) to transfer electrons during the catalytic reaction (Yu et al., 2019, Alpdagtas et al., 2022). Metal-independent FDHs are found in some bacteria and archaea, and are involved in the catabolism of various compounds, including formate, formaldehyde, and methanol. Metal-containing FDHs enzymes require metal cofactors for their activity and can be further divided into several subtypes based on the specific metal involved. The most common metal cofactors in FDHs are molybdenum (Mo), tungsten (W), and iron (Fe). Molybdenum-containing FDHs utilize a molybdopterin cofactor to transfer electrons during the catalytic reaction. The molybdenum cofactor consists of a molybdenum ion coordinated with a pterin molecule and is involved in the oxidation or reduction of various substrates. Molybdenum-containing FDHs are found in bacteria, archaea, and some eukaryotes. They are involved in the catabolism of various compounds, including formate, formaldehyde, and methanol (Calzadiaz-Ramirez and Meyer 2022). Tungsten-containing FDHs utilize a tungsten cofactor to transfer electrons during the catalytic reaction. The tungsten cofactor consists of a tungsten ion coordinated with a dithiolene ligand and is involved in the oxidation or reduction of various substrates. Tungsten-containing FDHs are found in certain bacteria and archaea (Yang et al., 2022). They are involved in the catabolism of formate and other compounds and can also catalyze the reduction of CO₂ to formate (Yan et al., 2023). Both molybdenum and tungsten are transition metals with similar chemical properties, and they can substitute for each other in certain enzymes. However, molybdenum is more commonly found in nature and is more extensively studied than tungsten. Researchers continue to investigate the roles and mechanisms of molybdenum- and tungsten-containing FDHs in various biological processes and their potential applications in biotechnology (Schoepp-Cothenet et al., 2012).

1.4 Biological pathway of FDH in microbes

FDH, or formate dehydrogenase, is an enzyme that plays an essential role in the metabolism of microbes. It is involved in the oxidation of formate to carbon dioxide, which is an important step in various biological pathways such as carbon fixation, energy production, and anaerobic respiration (Appel et al., 2013). The biological pathway of FDH in microbes involves several steps. (i) Formate uptake: Microbes can take up formate from the environment through specific transporters. (ii) Formate oxidation: Once inside the cell, formate is oxidized by FDH, catalyses formate conversion to carbon dioxide and electrons. The electrons produced in this reaction are transferred to the electron transport chain, where they are used to generate ATP. (iii) Electron transport: The electrons produced by FDH are transferred to various electron carriers, such as NAD⁺ or FAD. These electron carriers then donate their electrons to the electron transport chain, which generates a proton gradient across the cell membrane. This gradient is used to drive ATP synthesis. (iv) Carbon fixation: In some microbes, the carbon dioxide produced by FDH is used in the carbon fixation process, where it is converted into organic compounds such as sugars or amino acids. (v) Anaerobic respiration: In anaerobic conditions, some microbes can use FDH to generate energy by using alternative electron acceptors such as nitrate or sulfate (Parkin et al., 2007).

1.5 Proteins involved in FDH pathway

Formate utilization begins with oxidation to carbon dioxide and then enters the CO2 reduction pathway, which is catalysed by formate dehydrogenase. Molybdenum as a molybdoprotein cofactor, flavin adenine dinucleotide (FAD), zinc, iron, inorganic sulphur, and two different subunits in a configuration may be present (Burgdorf et al., 2001). Anaerobic bacteria can metabolize C1 carbon molecules. As intermediates in this metabolism, formaldehyde, formyltetrahydrofolate, formate, and a methyl corrinoid are assumed. Four moles of formate results in three moles of carbon dioxide and one mole of methane when used as a substrate (Yang et al., 2022). For energy, anaerobic bacteria like Clostridium thermoaceticum and others that feed on carbohydrates or purines use carbon dioxide (CO₂) as an electron acceptor and produce acetate. Formyltetrahydrofolate is synthesized from CO₂ by formate dehydrogenase, with formyl tetrahydrofolate synthetase serving as an intermediary (Berrios-Rivera et al., 2002). Enterobacteriaceae break down carbohydrates anaerobically to pyruvate, which is further broken down to acetyl-CoA or acetyl phosphate and formate. Pyruvate is broken down into formate by the enzyme pyruvate formate lyase. Primarily, formate is a precursor for the 2- and 8-carbon positions of purines, the S-methyl group of methionine, and the methyl group of thymidine. Small amounts of formate are generated during the production of D-erythro-dihydroneopterin triphosphate from GTP (Haussmann et al., 1998).

1.6 Function of formate dehydrogenase

The enzyme formate dehydrogenase (FDH) converts formate to carbon dioxide while reducing a cofactor like nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide adenine dinucleotide (NAD⁺). FDH's main job in living things is to produce energy. In bacteria, for example, FDH is involved in the anaerobic metabolism of formate as an energy source (Sanchez et al., 2005). In this process, formate is oxidized to carbon dioxide, releasing electrons that are used to produce ATP through the electron transport chain. FDH also plays a role in carbon dioxide fixation in certain organisms, such as methanogens, which use formate as a precursor for methane biosynthesis (Niu et al., 2023). In these organisms, FDH is involved in the reduction of carbon dioxide to formate, which is then used as a substrate for methanogenesis. Additionally, FDH has been found to play a role in the detoxification of formate in certain organisms, such as mammals. In humans, for example, FDH is involved in the metabolism of formate, which is a toxic byproduct of methanol metabolism (Steiniger et al., 2022). In this process, formate is oxidized to carbon dioxide and water, which the body can safely excrete. Formate dehydrogenases, a broad family of enzymes in both prokaryotes and eukaryotes, catalyse formate oxidation to carbon dioxide. Formate may be used efficiently by many hydrogenotrophic methanogens in place of H2, while the latter cannot. Formate dehydrogenases are enzymes that catalyze the reaction in methanogens that convert formate to reduced coenzymes during oxidation (Kurt and Ordu 2021).

1.7 Structure of FDH

Formate dehydrogenase (FDH) is a large enzyme typically consisting of several subunits. The exact structure of FDH varies among different organisms, but generally, it consists of a catalytic subunit, a cofactor-binding subunit, and often a membrane-spanning subunit. The catalytic subunit contains the active site where formate oxidation takes place, and it typically has a molybdenum cofactor (Moco) or tungsten cofactor (Wco) bound to it. The cofactor-binding subunit binds the electron-transfer cofactor (e.g., NAD⁺ or NADP⁺) and transfers the electrons to the catalytic subunit (Sosa Torres and Kroneck 2020). The membrane-spanning subunit anchors the enzyme to the cell membrane, as FDH is often located on the cytoplasmic membrane. Overall, the structure of FDH is highly complex and can vary greatly depending on the organism in which it is found. The enzyme may exist in different conformations depending on whether it is in an active or inactive state, and it may undergo conformational changes during the catalytic cycle (Jormakka et al., 2008). FDH is used to regenerate NADH in enzymatic syntheses of optically active compounds. This enzyme belongs to the D-specific 2-hydroxy acid dehydrogenase superfamily. The catalytic mechanism of the dehydrogenase enzyme is defined by the direct transfer of a hydride ion from the substrate to the C-4 atom of the nicotinamide moiety of NAD⁺ (Schirwitz et al., 2007). Numerous studies have demonstrated the importance of FDH in enzymatic processes, particularly those involving the replenishment and decrease of cofactors. The amino acid residues responsible for catalysis in the active site of each of these enzymes are extremely well conserved. Alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are the most used enzymes in mechanistic investigations, despite NAD⁺ dependent dehydrogenase being one of the most researched proteins (Xie et al., 2020). The catalytic domain is the region of a protein that performs the actual catalytic reaction. The subunit interface is a coenzyme-binding region that is centrally placed along the polypeptide chain. The dimer's two catalytic domain pieces are spatially separated and do not interact with one another. The two domains are linked by two long α-helices, αA and α8. In the secondary structure of NAD⁺-dependent FDH, each domain has the same type of structural organization, showing a left-handed twisted parallel β-sheet surrounded by α-helices. The coenzyme binding domain consists of seven standard β-sheets and the catalytic domain consists of five standard β-sheets with two short antiparallel strands (Lim et al., 2022). Formate is oxidized to carbon dioxide (CO₂) by molybdenum-containing NAD⁺-dependent formate dehydrogenases found in bacteria like Ralstonia eutropha, which also reduce NAD⁺ to NADH and other members of the NADH dehydrogenase superfamily of enzymes. Its spatial arrangement of many redox-active centers and structural similarity to a comparable subunit of NADH dehydrogenase's matrix- or cytosol-exposed region are striking (Niks et al., 2016).

1.8 Role of metal ions in FDH

FDH is an enzyme that catalyses the conversion of formate to carbon dioxide, coupled with the reduction of an electron acceptor such as NAD⁺ or a quinone. Metal ions play a crucial role in the catalytic activity of FDH by assisting in the transfer of electrons and in stabilizing the enzyme structure. One important metal ion in FDH is molybdenum, which is a part of the enzyme's active site (Alekseeva et al., 2011). Molybdenum interacts with the formate substrate, assisting in its deprotonation and subsequent oxidation to CO₂. Molybdenum also plays a role in the transfer of electrons from formate to the electron acceptor. Another metal ion that can be involved in FDH is iron-sulfur clusters. These clusters help to transfer electrons during the catalytic reaction and can also contribute to stabilizing the enzyme structure (Bulut et al., 2020). Other metal ions that have been shown to play a role in FDH activity include calcium, magnesium, and zinc. Calcium and magnesium are involved in stabilizing the enzyme structure, while zinc can transfer electrons. In FDH, metal ions play important roles in the catalytic activity and stability of FDH (Talekar et al., 2022). Molybdenum is an essential metal ion in the enzyme's active site, while iron-sulfur clusters, calcium, magnesium, and zinc can also contribute to the enzyme's function. Formate dehydrogenases (FDHs) in prokaryotes are metalloproteins that catalyze the oxidation of formate anion to carbon dioxide via a redox mechanism involving the transfer of two electrons from the substrate to the active site. Four sulfur atoms from two pyranoprotein molecules, one selenium atom from selenocysteine (SeCys), and one inorganic sulfur atom form a deformed trigonal prismatic geometry around a hexacoordinated molybdenum or tungsten ion in the active site of this metal-dependent dehydrogenase. Many bacteria that thrive in sulfide-rich environments have been found to have molybdenum FDHS as well as tungsten FDHS, albeit the molybdenum level is likely to be lower than that of tungsten due to the very poor solubility of molybdenum sulphides (Dong and Ryde 2018). Thermophilic bacteria and hyperthermophilic archaebacteria both have enzymes containing tungsten. Tungsten enzymes, like molybdenum enzymes, fall into one of three families; each of these families contains two pterin cofactor molecules for every molybdenum atom. They share similarities with the molybdenum enzyme family that reduces DMSO. The first two classes catalyze different types of redox reactions. The redox processes are catalyzed by the first two members of this family. Before transferring reducing equivalents to a [4Fe-4S] center, the aldehyde oxidoreductase family catalyzes the oxidation of aldehydes to carboxylic acid (Kisker et al., 1997).

1.9 Catalytic mechanism of formate dehydrogenase

The catalytic mechanism of formate dehydrogenase (FDH) involves the transfer of electrons from formate to an electron acceptor, such as NAD⁺ or a quinone, with the concomitant conversion of formate to carbon dioxide. The active site of FDH contains a molybdenum cofactor, which is essential for catalytic activity. The molybdenum ion can exist in several oxidation states, but during the catalytic cycle of FDH, it is in the Mo(VI) state (Tishkov and Popov 2004). The catalytic mechanism of FDH can be divided into several steps: (i) Binding of formate: The formate substrate binds to the molybdenum cofactor in the active site of FDH, forming a complex that is stabilized by hydrogen bonding and electrostatic interactions. (ii) Deprotonation of formate: The formate substrate is deprotonated by a basic residue in the active site, forming a negatively charged intermediate. (iii) Transfer of electrons: The negatively charged intermediate is then oxidized by the molybdenum cofactor, which accepts two electrons and two protons from the formate substrate. The oxidation of formate to carbon dioxide is coupled with the reduction of the molybdenum cofactor from the Mo(VI) state to the Mo(IV) state. (iv) Transfer of electrons to the electron acceptor: The reduced molybdenum cofactor then transfers the two electrons to the electron acceptor, which is reduced in the process. (v) Release of products: The products of the reaction, carbon dioxide and the reduced electron acceptor, are released from the active site, and the enzyme is ready to catalyze another reaction (Li et al., 2021, Popinako capital A et al., 2022, Meneghello et al., 2023). Frohlich et al. proposed the working hypothesis of hydrogen transfer catalysis based on undissociated formic acid. Concurrent nucleophilic attack of water on the acid proton facilitates the hydride transfer to NAD⁺ (Frohlich et al., 2011). Cleavage of formic acid esters is a straightforward example of this hypothesis in action; in this case, the water molecule attacks the C1 atom of the alkyl or aryl residues in the substrate, resulting in the production of NADH⁺, CO₂, and the corresponding alcohol (Fig. 1). Formate dehydrogenase is a crucial cofactor regenerator among the several NAD (P)H-dependent oxidoreductases utilized in biocatalysts and biotransformation. However, the main issue is that most naturally occurring FDHs have poor catalytic activity and operational stability. A selenocysteine residue at the molybdenum core is essential to its catalytic activity, which is why it does not catalyze oxygen atom transfer events but successfully breaks a C—H bond (Schmidt et al., 2010, Srikanth et al., 2017). As efficient and reversible electrocatalysts for CO₂ reduction, metal-dependent formate dehydrogenase enzymes have recently come to the fore. Enzymes with Mo atoms have been discovered in almost every known living organism. However, their atomic and ionic radii and coordination chemistry are very similar (Karaguler et al., 2007, Alissandratos et al., 2013).

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Fig. 1

Explicitly demonstrates the putative catalytic mechanism for NAD⁺-mediated hydride transfer from formic acid and formic acid esters.

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1.10 Amino acid and its role in the catalytic site of FDH

FDH's catalytic region is rich in amino acids that contribute to the enzyme's activity. A thiolate-imidazolium ion pair is formed between a histidine residue and cysteine in the catalytic region of FDH, making cysteine an essential amino acid (Yang et al., 2020). This pair stabilize the formate intermediate generated during the reaction. Tryptophan is also significant since it is a proton acceptor in the process (Zhang et al., 2018). Tryptophan accepts the proton that is produced during the oxidation of formate to carbon dioxide and water. FDH's catalytic region also contains the amino acids serine, aspartate, and lysine, which all play important roles in substrate binding and electron transfer. The catalytic domains of FDH (residues 400–902) were analysed from a gene related to aldehyde dehydrogenase and are in charge of the oxidation of short-chain aldehydes to their corresponding acids. An intermediate linker connects two catalytic domains (residues 311–399). In the FDH mechanism, this intermediate linker domain shuttles a formyl from the N-terminal domain to the C-terminal domain, where it is covalently linked to the sulfhydryl group of the phosphor pantetheine arm. A number of different investigations has established FDH's catalytic domain (Karaguler et al., 2007).

1.11 Role of selenium-Cys in the catalytic site

In three different classes of bacterial enzymes-glycine reductase in a few clostridia, formate dehydrogenases in Escherichia coli, salmonella Clostridia, and a Methanococcus, and hydrogenases in Methanococcus and a few other anaerobic bacteria—selenocysteine is found in a very unique position (Eswayah et al., 2016). Nearly half of all proteins in nature are metalloproteases, and many of these proteins, including zinc finger proteins, blue copper proteins, azurine iron-sufur proteins, and others, utilise cysteine thiolates as ligands for their metal cofactors (Jiang and Lewis 2023). Several metalloenzymes, such as formate dehydrogenase and nitrate hydrogenase, use selenocysteine to coordinate metal ions. The C-se bond in selenocysteine is weak and can be quickly weakened under mild conditions, making it a useful precursor to other amino acids (Kumar et al., 2023). The catalytic activity of E. coli formate dehydrogenase H relies on a selenocysteine residue (SeCys-140 in the polypeptide chain), in addition to molybdenum, two molybdopterin guanine dinucleotide cofactors, and selenocysteine (Boyington et al., 1997, Khangulov et al., 1998). The crystal structure indicates that it consists of three subunits such as α, β, and γ. The catalytic subunit is made up of 982 amino acids, one of which is an inherent selenium cysteine (SeCys) residue found in their active sites (Maia et al., 2015). Changing the TGA codon to TGC results in a mutant variant of the enzyme [S] FDHH that contains Cys rather than SeCys. The enzyme's catalytic activity was drastically reduced due to the substitution of sulfur for serine (Ser) since the mutant version of the enzyme containing SeCys was catalytically inactive. The formate-hydrogen lyase complex selenoenzyme of Escherichia coli oxidizes formate to CO₂ and decreases equivalents that are passed to a hydrogenase. A free selenolate anion is formed when formate binds to molybdenum, which then displaces selenocysteine, releasing CO₂ and transferring two electrons to the molybdenum center (Hartmann et al., 2015, Fogeron et al., 2022).

1.12 Reaction mechanism of FDH

The anaerobic molybdoenzyme formate dehydrogenase oxidizes formate to carbon dioxide. At the density functional level in both vacuum and protein settings, two primary mechanisms have been examined to elucidate the process of catalysis in molybdoenzyme; these mechanisms differ in whether a SeCys residue directly coordinates to the molybdenum metal during catalysis (Leopoldini et al., 2008). The oxidation of formate is a major electron source for many pathways in anaerobic prokaryotes that use molecules other than dioxygen as final electron acceptors. The oxidation reaction releases two protons from small, neutral compounds like formic acid that have diffused across the cytoplasmic membrane into the periplasmic region. Kinetic and spectroscopic studies of E. coli FDH-H demonstrate that the oxidation of formate requires the breaking of the C—H bond to yield a CO₂ molecule, and that the proton of the substrate is transferred to an acceptor group near the molybdenum ion (Hartmann et al., 2016). The SeCys residue serves as the proton acceptor group in both suggestions, but the molybdenum formate interaction is accomplished quite differently in the two models. Raaijmakers proposed that the formate active site interaction takes place along the catalytic cycle after the Se⚌Mo link is broken with the Si ligand bound to molybdenum. Methane, methanol, and formate all support yeast and aerobic growth (Jormakka et al., 2003). Methanol, formaldehyde, formylglutathione, and formate are intermediates in the energy metabolism of these organisms, which ultimately results in carbon dioxide. Three moles of carbon dioxide and one mole of methane are produced from four formate moles when serving as the formate act substrate. After being oxidized to CO₂, the formate is converted to methane. Most likely, CO₂ is converted to formate as the first step. Then, the free formate could play a role in the production of methane (Friedebold and Bowien 1993).

1.13 Electron transfer reaction

Formate and hydrogen play pivotal roles in interspecies electron transfer in methanogenic environments. Hydrogen molecules are crucial in microbial metabolism because their midpoint redox potentials are similar to those of the redox couples H2/H⁺ and formate/CO₂, despite the fact that hydrogen and formate have different chemical and physical properties. When methane formation in syntrophic butyrate-degrading cultures from a reactor treatment could not be accounted for by interspecies hydrogen transfer alone, experiments showed that formate might be involved for electron transfer (Schuchmann et al., 2018). The most common C-type cytochrome C3 accepts electrons from periplasmic formate dehydrogenases to complete the reduction of quinine and is a physiological partner of periplasmic hydrogenases. Sulfate reduction in the cytoplasm requires electron transfer from the periplasmic dehydrogenases to membrane-bound complexes containing cytochrome C. When fumarate is added, menaquinone in the membrane of W. Succinogenase is reoxidized. Menaquinone's redox state is determined by formate dehydrogenase activities in the steady-state of electron transfer. Subunit and prosthetic group compositions are identical in the crystal structure of formate dehydrogenase-N from Escherichia coli (Heidelberg et al., 2004). The catalytic subunit carries four tetranuclear iron-sulfur clusters localized at an appropriate distance for rapid electron transfer from the molybdenum site to yield two electrons, CO₂ and a proton. While the electron transfer to the ion cluster, CO₂ and protons leaves the catalytic site via substrate crevice and also the E. coli encodes the another structurally related formate dehydrogenase isoenzymes such as formate dehydrogenase-O (FdhO/FdoGHI) that are known to acts as electron transfer sinks which required for oxidation of formate to CO₂ and H⁺ and plays a role in the electron transfer to nitrate reductase (Holm and Lo 2016, Kim et al., 2016).

1.14 The sulfur shift: An activation mechanism for formate dehydrogenase

The sulfur shift is an activation mechanism for formate dehydrogenase (FDH) that involves a unique conformational change in the enzyme's active site. This mechanism is used by certain variants of FDH, such as the molybdopterin-containing FDH from the bacterium Rhodobacter capsulatus. In the sulfur shift mechanism, a conserved cysteine residue in the enzyme's active site undergoes a reversible oxidation to a persulfide (R-S-S-H) state (Mardina et al., 2016, Dong and Ryde 2018). This persulfide intermediate then reacts with the formate substrate, forming a covalent thioformate intermediate (R-S-CO₂). The covalent thioformate intermediate is then rearranged in a process known as the sulfur shift, which involves the transfer of the thioformate group to a neighboring sulfur atom in a molybdopterin cofactor. This results in forming a new molybdopterin-sulfur linkage and releasing carbon dioxide and a thiol. The sulfur shift mechanism has been proposed to increase the rate of formate oxidation by facilitating the transfer of the formate group to the molybdopterin cofactor. This mechanism is thought to be particularly effective in FDH variants that lack a conserved arginine residue in the active site, which is thought to play a similar role in other FDH variants. The density functional theory calculation revealed that mainly three combinations of three simulations primitive charge are involved in sulfur shift mechanism; 1) the unbinding of Cys140 in Nap and SeCys140 in FDH from the shell to the second shell of the molybdenum ion and interact very closely with each other forming a quasi-covalent bond, 2) charge of the coordination position of the sulfide to the place previously occupied by the residue and the coordination of the substrates to the MO ion, 3) this process opens free coordination position at the metal site that is promptly occupied by the substrate that is, nitrate in Nap and formate in FDH (Fig. 2). The sulfur shift mechanism also found in other mechanistic proposals; Hofmann showed that nitrate reaction with a persulfate molybdenum complex fragment is favored over the oxidation of molybdenum bound sulfur atom (Cerqueira et al., 2013).

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Fig. 2

Represent the Sulfur shift mechanism of formate dehydrogenase.

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1.15 Comparative analysis of structure of FDH

The first and native structure NADP –dependent FDH identified in proteobacterium Burkholderia stabilis. It shows lower affinity and accepts both phosphorylated cofactor and NAD⁺. Recently Fogal et al. characterized new structural information on FDH from Granulicella mallensis (GraFDH). It contains the common feature of bacterial formate dehydrogenase with two monomers, each having a cofactor binding or catalytic domain. In addition, feature of secondary structure and tertiary structure analysis revealed that the GraFDH is the closest homolog of Pseudomonas sp. 101 (Singh et al., 2018). It shares several enzymatic features including contact area due to long loop protruding from each 1L and 4L hydrogen bond. Similar to Pseudomonas sp.101 GraFDH also shows the complex with NADP⁺ and NaN3, however the sequence of GraFDH is shorter than Pseudomonas and Moraxella enzyme those have 10 to 11 amino acids longer (Fogal et al., 2015). The crystal structure of Candida boidinii FDH (CbFDH) composed of two domains named as NAD binding or catalytic domain consist of 15 α-helices and 13 β-strands like all the member of dehydrogenase family. The catalytic domain of the CbFDH shows the Rossmann fold structure with three layer of polypeptide chain. Both the domains are connected by two long helices (H6 and H15) where the active site residues are located (Tishkov and Popov 2004).

In case of the structure of formate dehydrogenase H of E coli contains more than two cofactors such as selenocysteine, molybdenum and two molybdopterin dinucleotide (MGD) cofactors. In addition, Fe4S4 cluster is present in the active site responsible for catalyzing the oxidation of formate to carbon dioxide. The crystal structure of FDHH from E. coli shows four αβ domains (Fig. 3) with small antiparallel β-sheets. The MGD domain II and III closely resemble the classical dinucleotide binding fold. Recently new interpretation of the FDHH of E. coli was evaluated with new reaction mechanism. The FDH of methyltrophic bacteria Pseudomonas sp. has two globular domains (domain I and II). The first domain is formed by the residues 147–333 responsible for the NAD⁺ binding, and the second domain acts as catalytic domain formed by the residues 1–147 and 333–347. The FDH structure of Desulfovibrio gigas is the first reported tungsten containing enzyme. It consists of three subunits (α, β and γ), the larger subunit is similar to the FDH of the E. coli and have the essential conserved residues such as SeCys158, His159 and Arg407 at the active site (Selvaraj et al., 2014). The α and β subunits is similar to FDH of Desulfovibrio vulgaris Hildenborough, however instead of tungsten those have molybdenum with MGD, SeCys cofactors and 4Fe-4S cluster. Molybdenum containing FDH plays a key role in different metabolic pathways and electron transfer due to the presence of FeS cluster. The sequence similarity of the above-mentioned structure shows in Fig. 4 and the structural comparison represented in Fig. 5.

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Fig. 3

Shows the structural representation of FDHH of E. coli.

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Fig. 4

Structural comparison of structure of CbFDH (green; PDBID: 2FSS), PseFDH (Cyan; PDB ID: 2GO1) and GraFDH (Megenta; PDB ID: 4XYB).

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Fig. 5

Sequence alignment of formate dehydrogenase enzymes of from bacteria CbFDH, GraFDH, PseFDH, FDH of E. coli, BstFDH. (*) symbol denoted the conserved amino acids and (:) symbol denotes the semi-conservative substation.

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