Pyruvate:ferredoxin oxidoreducatse

 

Pyruvate:ferrodoxin/flavodoxin reductases (PFORs) catalyse the oxidative decarboxylation of pyruvate to acetyl-CoA in anaerobic organisms. PFORs can occur in both obligately and facultatively anaerobic bacteria and also some eukaryotic microorganisms. PFORs are single-chain enzymes containing a thiamin pyrophosphate cofactor for the cleavage of carbon-carbon bonds next to a carbonyl group, and iron-sulphur clusters for electron transfer. Ferredoxin I and ferredoxin II, which are single 4Fe-4S cluster ferredoxins are the most effective electron carriers for PFORs in Desulfovibrio africanus.

 

Reference Protein and Structure

Sequence
P94692 UniProt (1.2.7.1) IPR011895 (Sequence Homologues) (PDB Homologues)
Biological species
Desulfovibrio africanus (Bacteria) Uniprot
PDB
2c3m - Crystal Structure Of Pyruvate-Ferredoxin Oxidoreductase From Desulfovibrio africanus (1.84 Å) PDBe PDBsum 2c3m
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 2c3m)
Cofactors
Tetra-mu3-sulfido-tetrairon (3), Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.2.7.1)

oxidized ferredoxin
CHEBI:17908ChEBI
+
pyruvate
CHEBI:15361ChEBI
+
coenzyme A(4-)
CHEBI:57287ChEBI
→
acetyl-CoA(4-)
CHEBI:57288ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
+
reduced ferredoxin
CHEBI:17513ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Pyruvate oxidoreductase, Pyruvate synthetase, Pyruvate:ferredoxin oxidoreductase, Pyruvic-ferredoxin oxidoreductase, 2-oxobutyrate synthase, Alpha-ketobutyrate-ferredoxin oxidoreductase, 2-ketobutyrate synthase, Alpha-ketobutyrate synthase, 2-oxobutyrate-ferredoxin oxidoreductase, 2-oxobutanoate:ferredoxin 2-oxidoreductase (CoA-propionylating), 2-oxobutanoate:ferredoxin 2-oxidoreductase (CoA-propanoylating),

Enzyme Mechanism

Introduction

The catalytic mechanism of PDC for the most part follows the principles of catalytic mechanisms of other TPP-dependent enzymes: carbonyl addition of pyruvate to the reactive C2 atom of the cofactor thiazolium ring yields the intermediate 2-(2-lactyl)-TDP (LTDP). The subsequent release of carbon dioxide produces resonating carbanion/enamine forms of 2-(1-hydroxyethyl)-TDP (HETDP, also known as hydroxyethylidene-TPP). The resonating form is considered to be a central and highly reactive intermediate state in TPP-dependent enzymes acting on pyruvate. However, unlike most other TPP-dependent enzymes in which the intermediate is oxidized, the carbanion/enamine in PDC is protonated at the C2α position, yielding C2alpha-hydroxylethylthiamine diphosphate (HETDP) before the final release of acetaldehyde completes the reaction.

Catalytic Residues Roles

UniProt PDB* (2c3m)
Glu64 Glu64(63)A Acts as the general acid/base that activates the TPP cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asn996, Thr31, Arg114 Asn996(995)A, Thr31(30)A, Arg114(113)A Help activate and stabilise the reactive intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
*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, assisted tautomerisation (not keto-enol), cofactor used, intermediate formation, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, hydride transfer, tautomerisation (not keto-enol), redox reaction, overall reactant used, native state of cofactor regenerated, decarboxylation, overall product formed, intermediate collapse, electron relay, homolysis, radical formation, colligation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Cavazza C et al. (2006), Structure, 14, 217-224. Flexibility of Thiamine Diphosphate Revealed by Kinetic Crystallographic Studies of the Reaction of Pyruvate-Ferredoxin Oxidoreductase with Pyruvate. DOI:10.1016/j.str.2005.10.013. PMID:16472741.
  2. Eram MS et al. (2013), Biomolecules, 3, 578-596. Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles. DOI:10.3390/biom3030578. PMID:24970182.
  3. Reed GH et al. (2012), Biochim Biophys Acta, 1824, 1291-1298. Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases. DOI:10.1016/j.bbapap.2011.11.010. PMID:22178227.
  4. Mansoorabadi SO et al. (2006), Biochemistry, 45, 7122-7131. EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase†. DOI:10.1021/bi0602516. PMID:16752902.
  5. Chabrière E et al. (2001), Science, 294, 2559-2563. Crystal Structure of the Free Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase. DOI:10.1126/science.1066198. PMID:11752578.
  6. Chabrière E et al. (1999), Nat Struct Biol, 6, 182-190. Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate. DOI:10.1038/5870. PMID:10048931.
  7. Pieulle L et al. (1995), Biochim Biophys Acta, 1250, 49-59. Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus. PMID:7612653.

Step 1. Glu64 deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor.

The cofactor adopts the V configuration that brings the 4 imino group of the aminopyrimidine ring close to the C2 carbon of the thiazolium ring [PMID:16472741]. Hydrogen bonding of the carboxylate group from Glu64 to N1' of the aminopyrimidine ring increases the basic nature of the 4' position by generating the 4'-imino tautomer [PMID:11752578]. Binding of the substrate triggers the proton transfers [PMID10048931]. The mechanism shown here is supported by crystallographic studies [PMID:16472741].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu64(63)A hydrogen bond acceptor
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), cofactor used, intermediate formation

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction that results in the cofactor undergoing double bond rearrangement that results in the deprotonation of Glu64.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Thr31(30)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond donor
Arg114(113)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, assisted tautomerisation (not keto-enol), intermediate formation

Step 3. The covalently bound pyruvate undergoes decarboxylation, resulting in double bond rearrangement, a single electron being transferred to ferredoxin via three iron-sulfur clusters and the formation of a one-electron bond between the cofactor and the formyl group.

The carbon dioxide reaction product remains tightly bound in the active site, consistent with the reversibility of the reaction [PMID:16472741]. The C2alpha-C2 bond between the acetyl and thiazole moieties is a one-electron unusually long bond PMID:11752578,PMID:16472741] and thus incompatible with enamine formation as postulated for a standard pyruvate decarboxylation cycle [PMID:16472741]. The generated intermediate shown here is supported by crystallographic studies [PMID:16472741].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Thr31(30)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond acceptor
Arg114(113)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, hydride transfer, ingold: bimolecular nucleophilic addition, tautomerisation (not keto-enol), redox reaction, overall reactant used, cofactor used, native state of cofactor regenerated, decarboxylation, overall product formed, intermediate collapse, intermediate formation, electron relay

Step 4. The C-C one electron bond between the cofator and the formyl group fragments.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond acceptor
Arg114(113)A hydrogen bond donor

Chemical Components

homolysis, intermediate collapse, intermediate formation

Step 5. Water deprotonates CoA, which initiates a single electron transfer to ferredoxin through three iron-sulfur clusters.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond acceptor
Arg114(113)A hydrogen bond donor

Chemical Components

proton transfer, radical formation, redox reaction, overall reactant used, electron relay, intermediate formation, overall product formed

Step 6. The CoA thiyl and the acetyl radicals undergo colligation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond acceptor
Arg114(113)A hydrogen bond donor

Chemical Components

colligation, intermediate terminated, overall product formed

Step 7. The cofactor is regenerated through the nitrogen donating its lone pair into the ring to satisfy the electron deficient carbon.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn996(995)A hydrogen bond donor, electrostatic stabiliser
Glu64(63)A hydrogen bond acceptor

Chemical Components

tautomerisation (not keto-enol), intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Glu64 deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor.

The cofactor adopts the V configuration that brings the 4 imino group of the aminopyrimidine ring close to the C2 carbon of the thiazolium ring [PMID:16472741]. Hydrogen bonding of the carboxylate group from Glu64 to N1' of the aminopyrimidine ring increases the basic nature of the 4' position by generating the 4'-imino tautomer [PMID:11752578]. Binding of the substrate triggers the proton transfers [PMID10048931]. The mechanism shown here is supported by crystallographic studies [PMID:16472741].

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction that results in the cofactor undergoing double bond rearrangement that results in the deprotonation of Glu64.

Step 3. The covalently bound pyruvate undergoes decarboxylation, resulting in double bond rearrangement, a single electron being transferred to ferredoxin via three iron-sulfur clusters and the formation of a one-electron bond between the cofactor and the formyl group.

The carbon dioxide reaction product remains tightly bound in the active site, consistent with the reversibility of the reaction [PMID:16472741]. The C2alpha-C2 bond between the acetyl and thiazole moieties is a one-electron unusually long bond PMID:11752578,PMID:16472741] and thus incompatible with enamine formation as postulated for a standard pyruvate decarboxylation cycle [PMID:16472741]. The generated intermediate shown here is supported by crystallographic studies [PMID:16472741].

Step 4. The C-C one electron bond between the cofator and the formyl group fragments.

Step 5. Water deprotonates CoA, which initiates a single electron transfer to ferredoxin through three iron-sulfur clusters.

Step 6. The CoA thiyl and the acetyl radicals undergo colligation.

Step 7. The cofactor is regenerated through the nitrogen donating its lone pair into the ring to satisfy the electron deficient carbon.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Craig Porter