Formate dehydrogenase

 

Formate Dehydrogenase H (FDH-H) from Escherichi coli is a 79kDa polypeptide component of the anaerobic formate hydrogen lyase complex. The enzyme catalyses the oxidation of formate (produced from pyruvate during anaerobic growth) to carbon dioxide, in the absence of exogenous electron acceptors, with the concomitant release of two electrons and two protons.

 

Reference Protein and Structure

Sequence
P07658 UniProt (1.17.98.4) IPR006478 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1aa6 - REDUCED FORM OF FORMATE DEHYDROGENASE H FROM E. COLI (2.3 Å) PDBe PDBsum 1aa6
Catalytic CATH Domains
3.40.228.10 CATHdb 2.20.25.90 CATHdb (see all for 1aa6)
Cofactors
Molybdenum(6+) (1), Tetra-mu3-sulfido-tetrairon (1), Molybdopterin guanine dinucleotide (2)
Click To Show Structure

Enzyme Reaction (EC:1.17.1.9)

acceptor
CHEBI:15339ChEBI
+
hydron
CHEBI:15378ChEBI
+
formate
CHEBI:15740ChEBI
→
hydrogen donor
CHEBI:17499ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
Alternative enzyme names: FDH I, FDH II, N-FDH, NAD(+)-dependent formate dehydrogenase, NAD(+)-formate dehydrogenase, NAD(+)-linked formate dehydrogenase, Formate benzyl-viologen oxidoreductase, Formate dehydrogenase (NAD(+)), Formate hydrogenlyase, Formate-NAD(+) oxidoreductase, Formic acid dehydrogenase, Formic hydrogen-lyase, Hydrogenlyase,

Enzyme Mechanism

Introduction

Initially, formate binds to Mo 800 via one of its oxygens and displaces SeCys140. Formate is then oxidised to carbon dioxide. Two electrons are transferred to Mo800 reducing it from Mo(VI) to Mo(IV). The transfer of electrons from formate to Mo 800 may occur by direct two-electron transfer or by direct hydride transfer. The alpha proton of formate is transferred to SeCys140 and then to His141. A selenium-carboxylated intermediate may be formed before CO2 is released. Electrons from Mo(IV) are shuttled to the Fe4S4 cluster one at a time so an intermediate with Mo(V) and reduced Fe4S4 is produced. This is supported by evidence from electron paramagnetic resonance (EPR) experiments. Electron shuttling occurs by a ping pong mechanism through the partly delocalised ring of molybdopterin guanine dinucleotide (MGD) 802 and hydrogen bonds linking MGD, HOH 30, Lys44 and the Fe4S4 cluster. The Fe4S4 cluster is then oxidised by another electron acceptor. It is not known what functions as the final electron acceptor in vivo. Benzyl viologen (BV) has been used as the final electron acceptor in experiments. After Mo(V) is oxidised to Mo(IV) the proton on His141 can be released to the solvent.

Catalytic Residues Roles

UniProt PDB* (1aa6)
Arg333 Arg333A Stabilises the free selenol on SeCys140 after it is displaced from Mo by the substrate. electrostatic stabiliser
His141 His141A After the proton from formate is transferred to SeCys140 it is passed on to His141. Evidence from electron paramagnetic resonance also supports the protonation of His141 by the alpha proton of formate (PMID: 9036855). proton acceptor, electrostatic stabiliser
Lys44 Lys44A Facilitates electron transfer between HOH 30 and the Fe4S4 cluster. electrostatic stabiliser
Sec140 Sec140A Accepts the alpha proton from formate. A selenium-carboxylated intermediate may be formed on this residue during the oxidation of formate to CO2. nucleofuge, nucleophile, metal ligand, proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

coordination to a metal ion, overall product formed, overall reactant used, electron transfer, redox reaction, electron relay, native state of enzyme regenerated

References

  1. Boyington JC et al. (1997), Science, 275, 1305-1308. Crystal Structure of Formate Dehydrogenase H: Catalysis Involving Mo, Molybdopterin, Selenocysteine, and an Fe4S4 Cluster. DOI:10.1126/science.275.5304.1305. PMID:9036855.
  2. Leopoldini M et al. (2008), Chemistry, 14, 8674-8681. Reaction mechanism of molybdoenzyme formate dehydrogenase. DOI:10.1002/chem.200800906. PMID:18671310.
  3. Raaijmakers HC et al. (2006), J Biol Inorg Chem, 11, 849-854. Formate-reduced E. coli formate dehydrogenase H: the reinterpretation of the crystal structure suggests a new reaction mechanism. DOI:10.1007/s00775-006-0129-2. PMID:16830149.

Step 1. Catalysis begins with the formate displacing the Mo-bound Sec140 group in the oxidised form of the enzyme.

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Catalytic Residues Roles

Residue Roles
Arg333A electrostatic stabiliser
Lys44A electrostatic stabiliser
His141A electrostatic stabiliser
Sec140A metal ligand
Sec140A nucleofuge

Chemical Components

coordination to a metal ion

Step 2. The formate proton is abstracted by Sec140. The CO2 molecule is released and two electrons are transferred to Mo, forming Mo(IV).

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Catalytic Residues Roles

Residue Roles
His141A electrostatic stabiliser
Arg333A electrostatic stabiliser
Sec140A proton acceptor

Chemical Components

overall product formed, overall reactant used, electron transfer, redox reaction

Step 3. An electron is shuttled from Mo(IV) to an electron acceptor (R) through an MGD ligand, a water molecule, then the Fe4S4 cluster. This results in a Mo(V) intermediate. The formate proton is transferred from Sec140 to His141. This leads to hydrogen bond formation between His141 and Sec140.

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Catalytic Residues Roles

Residue Roles
His141A electrostatic stabiliser, proton acceptor
Sec140A proton donor

Chemical Components

electron transfer, electron relay, electron transfer, redox reaction

Step 4. Once the Fe4S4 cluster is reoxidized, a second electron is transferred from Mo(V) to the Fe4S4 cluster. The oxidation of Mo(V) to Mo(VI) causes the hydrogen bond between Sec140 and His141 to break, releasing the proton of His141 to solvent. Sec140 re-cordinates to the Mo(VI) centre. After the Fe4S4 cluster is reoxidised for a second time, the enzyme is restored to its initial state.

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Catalytic Residues Roles

Residue Roles
Sec140A nucleophile

Chemical Components

native state of enzyme regenerated, electron relay, redox reaction
Download: Image, Marvin File

Step 1. Catalysis begins with the formate displacing the Mo-bound Sec140 group in the oxidised form of the enzyme.

Step 2. The formate proton is abstracted by Sec140. The CO2 molecule is released and two electrons are transferred to Mo, forming Mo(IV).

Step 3. An electron is shuttled from Mo(IV) to an electron acceptor (R) through an MGD ligand, a water molecule, then the Fe4S4 cluster. This results in a Mo(V) intermediate. The formate proton is transferred from Sec140 to His141. This leads to hydrogen bond formation between His141 and Sec140.

Step 4. Once the Fe4S4 cluster is reoxidized, a second electron is transferred from Mo(V) to the Fe4S4 cluster. The oxidation of Mo(V) to Mo(VI) causes the hydrogen bond between Sec140 and His141 to break, releasing the proton of His141 to solvent. Sec140 re-cordinates to the Mo(VI) centre. After the Fe4S4 cluster is reoxidised for a second time, the enzyme is restored to its initial state.

Products.

Introduction

The hydrogen of the SH ligand on the Mo(VI) centre is abstracted by the Sec140 residue. There is direct hydride transfer from formate to Mo(VI), releasing CO2 and forming a Mo-H intermediate. The proton is then removed by the sulphur anion, reducing Mo(VI) to Mo(IV). The native state of the enzyme is restored when two electrons are transferred to an electron acceptor through the MGD ligands and Fe4S4 cluster, and Sec140 is deprotonated.

Catalytic Residues Roles

UniProt PDB* (1aa6)
Arg333 Arg333A Arg333 interacts with the Sec140 ligand and forms a hydrogen bond to formate. hydrogen bond donor, electrostatic stabiliser
Sec140 Sec140A In this proposal Sec140 is not initially coordinated to the Mo(VI) centre. Sec140 deprotonates the sulphur ligand attached to Mo. proton acceptor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, hydride transfer, overall reactant used, overall product formed, bimolecular elimination, redox reaction, electron transfer, native state of enzyme regenerated, electron relay

References

  1. Tiberti M et al. (2012), Inorg Chem, 51, 8331-8339. Evidence for the Formation of a Mo–H Intermediate in the Catalytic Cycle of Formate Dehydrogenase. DOI:/10.1021/ic300863d.
  2. Hartmann T et al. (2015), Biochim Biophys Acta, 1854, 1090-1100. Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria. DOI:10.1016/j.bbapap.2014.12.006. PMID:25514355.

Step 1. The selenide ion of Sec140 abstracts a proton from the Mo-bound sulphur. Arg333 stabilises unprotonated Sec140 and forms a hydrogen bond with the oxygen of coordinated formate.

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Catalytic Residues Roles

Residue Roles
Arg333A electrostatic stabiliser, hydrogen bond donor
Sec140A proton acceptor

Chemical Components

proton transfer

Step 2. B-elimination of a hydride from formate to the Mo(VI) centre occurs and CO2 is released.

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, overall reactant used, overall product formed, ingold: bimolecular elimination

Step 3. Proton transfer to the Mo-bound sulphur ion occurs, reducing the Mo centre to Mo(IV).

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Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, redox reaction, electron transfer

Step 4. The active site is restored when two electrons are transferred from Mo(IV) to the electron acceptor (R) and Sec140 is deprotonated.

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Catalytic Residues Roles

Residue Roles

Chemical Components

redox reaction, native state of enzyme regenerated, electron relay
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Step 1. The selenide ion of Sec140 abstracts a proton from the Mo-bound sulphur. Arg333 stabilises unprotonated Sec140 and forms a hydrogen bond with the oxygen of coordinated formate.

Step 2. B-elimination of a hydride from formate to the Mo(VI) centre occurs and CO2 is released.

Step 3. Proton transfer to the Mo-bound sulphur ion occurs, reducing the Mo centre to Mo(IV).

Step 4. The active site is restored when two electrons are transferred from Mo(IV) to the electron acceptor (R) and Sec140 is deprotonated.

Products.

Introduction

A computational study of the mechanism of formate dehydrogenase proposed that the inactive state of the enzyme contains a partial bond between the Mo-bound sulphur ligand and Sec140. An activation step occurs before catalysis involving a 'sulphur shift'. Firstly, formate binds to the sulphur ligand and is stabilised by the positive charge of Arg333. The 'sulphur shift' then occurs as formate coordinates to the Mo centre and displaces Sec140 which binds to sulphur with a single bond. The sulphur-selenium bond breaks and the resulting selenide ion abstracts the proton from formate. His141 stabilises the free selenide with hydrogen bonds and lowers the activation energy for proton abstraction. CO2 then forms an additional bond to the sulphur. Product release occurs when the Mo-oxygen bond breaks, inducing the cleavage of the sulphur-carbon bond. Two electrons are transferred from the reduced Mo(IV) centre to an unknown electron acceptor, via Lys44 and the Fe4S4 cluster. The active site is regenerated when Sec140 is deprotonated and recoordinates to Mo(VI). However, this proposal does not include a Mo(V) intermediate during formate oxidation which has been identified by EPR studies.

Catalytic Residues Roles

UniProt PDB* (1aa6)
Arg333 Arg333A The positive charge of Arg333 stabilises the formate anion in the initial binding at the Mo(VI) centre. electrostatic stabiliser
His141 His141A Forms a hydrogen bond with the Sec140 anion which lowers the activation energy for proton abstraction from formate.
Lys44 Lys44A Involved in electron shuttling during the re-oxidation of Mo(IV) to Mo(VI).
Sec140 Sec140A Sec140 is displaced from the active site following formate coordination and the 'sulphur shift'. The selenide ion deprotonates formate. nucleofuge, metal ligand, proton acceptor, proton donor, electrophile
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

decoordination from a metal ion, coordination to a metal ion, proton transfer, overall product formed, native state of enzyme regenerated, redox reaction

References

  1. Mota CS et al. (2011), J Biol Inorg Chem, 16, 1255-1268. The mechanism of formate oxidation by metal-dependent formate dehydrogenases. DOI:10.1007/s00775-011-0813-8. PMID:21773834.
  2. Hartmann T et al. (2015), Biochim Biophys Acta, 1854, 1090-1100. Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria. DOI:10.1016/j.bbapap.2014.12.006. PMID:25514355.
  3. Boyington JC et al. (1997), Science, 275, 1305-1308. Crystal Structure of Formate Dehydrogenase H: Catalysis Involving Mo, Molybdopterin, Selenocysteine, and an Fe4S4 Cluster. DOI:10.1126/science.275.5304.1305. PMID:9036855.

Step 1. In the inactive form of the enzyme there is a partial bond between the Mo sulphur ligand and the Sec140 residue. Catalysis beings when the oxygen atom of formate binds to the sulphur. The formate is stabilised by Arg333.

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Catalytic Residues Roles

Residue Roles
Sec140A metal ligand
Arg333A electrostatic stabiliser

Chemical Components

Step 2. A sulphur shift occurs, in which the sulphur replaces the position of the Sec140 ligand, causing it to dissociate and form a covalently bound Sec140 intermediate to sulhpur. Formate binds to Mo(VI) via a single bond.

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Catalytic Residues Roles

Residue Roles
Sec140A electrophile, nucleofuge

Chemical Components

decoordination from a metal ion, coordination to a metal ion

Step 3. The sulphur-selenium single bond breaks.

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Catalytic Residues Roles

Residue Roles
Sec140A nucleofuge

Chemical Components

Step 4. His141 stabilises the free selenide. The selenide ion abstracts the formate proton and the remaining CO2 molecule forms and additional bond to the sulphur ligand.

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Catalytic Residues Roles

Residue Roles
Sec140A proton acceptor

Chemical Components

proton transfer, overall product formed

Step 5. Product release occurs prior to the reoxidation of Mo(IV). The Mo-oxygen bond breaks, which induces the break of the sulphur-carbon bond.

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Catalytic Residues Roles

Residue Roles

Chemical Components

overall product formed

Step 6. Two electrons are transferred from Mo(IV) to an electron acceptor (R) via Lys44 and the Fe4S4 cluster. The proton on Sec140 is likely to be removed by solvent. After deprotonation, the Sec140 residue rebinds to the Mo(VI) centre and the active site is regenerated.

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Catalytic Residues Roles

Residue Roles
Sec140A metal ligand, proton donor

Chemical Components

native state of enzyme regenerated, redox reaction
Download: Image, Marvin File

Step 1. In the inactive form of the enzyme there is a partial bond between the Mo sulphur ligand and the Sec140 residue. Catalysis beings when the oxygen atom of formate binds to the sulphur. The formate is stabilised by Arg333.

Step 2. A sulphur shift occurs, in which the sulphur replaces the position of the Sec140 ligand, causing it to dissociate and form a covalently bound Sec140 intermediate to sulhpur. Formate binds to Mo(VI) via a single bond.

Step 3. The sulphur-selenium single bond breaks.

Step 4. His141 stabilises the free selenide. The selenide ion abstracts the formate proton and the remaining CO2 molecule forms and additional bond to the sulphur ligand.

Step 5. Product release occurs prior to the reoxidation of Mo(IV). The Mo-oxygen bond breaks, which induces the break of the sulphur-carbon bond.

Step 6. Two electrons are transferred from Mo(IV) to an electron acceptor (R) via Lys44 and the Fe4S4 cluster. The proton on Sec140 is likely to be removed by solvent. After deprotonation, the Sec140 residue rebinds to the Mo(VI) centre and the active site is regenerated.

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett