Ferredoxin:thioredoxin reductase

 

Ferredoxin:thioredoxin reductase (FTR) transfers the light-generated redox signal received by the chloroplast [Fe2S2]2+,+ ferredoxin (Fdx) to thioredoxins (Trxs), as part of a redox regulatory system controlling the activity of a wide range of oxygenic photosynthesis enzymes in response to light. The active site consists of a [Fe4S4] cluster and an an adjacent redox-active disulphide. FTR converts two light-generated one-electron signals to one two-electron thiol signal which is then transmitted via dithiol/disulphide interchange reactions to specific enzymes which are critical to the regulation of the Calvin cycle.

Phylogenetic analyses of genomic sequences revealed that the catalytic subunit of FTR originated in microaerophilic bacteria where it may have functioned in regulating CO2 fixation. Also, FTR may have been acquired by later-evolving species via horizontal gene transfer. FTR-like enzymes, for example FDR, with structural and functional diversity have evolved to meet ecological needs.

 

Reference Protein and Structure

Sequence
Q55389 UniProt (1.8.7.2) IPR024707, IPR044166 (Sequence Homologues) (PDB Homologues)
Biological species
Synechocystis sp. PCC 6803 substr. Kazusa (Bacteria) Uniprot
PDB
1dj7 - CRYSTAL STRUCTURE OF FERREDOXIN THIOREDOXIN REDUCTASE (1.6 Å) PDBe PDBsum 1dj7
Catalytic CATH Domains
3.90.460.10 CATHdb (see all for 1dj7)
Cofactors
Tetra-mu3-sulfido-tetrairon (1)
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Enzyme Reaction (EC:1.8.7.2)

L-cystine residue
CHEBI:50058ChEBI
+
hydron
CHEBI:15378ChEBI
+
di-mu-sulfido-diiron(1+)
CHEBI:33738ChEBI
→
L-cysteine residue
CHEBI:29950ChEBI
+
di-mu-sulfido-diiron(2+)
CHEBI:33737ChEBI

Enzyme Mechanism

Introduction

One-electron reduction mechanism, formally viewed as two-electron reduction of the disulphide with concurrent one-electron oxidation of the cluster due to coordination of an additional cysteinate ligand. This anchors one of the active-site thiol ligands via cluster coordination while freeing the other thiol for nucleophilic attack of the Trx disulphide to form an FTR/Trx heterodisulphide intermediate. A subsequent one-electron reduction reduces the cluster to [Fe4S4]2+ and releases the electron-transfer thiol to reform the active-site disulphide with concomitant cleavage of the heterodisulphide and formation of the reduced dithiol form of Trx. Note, this mechanism is analogous to that followed by an FTR-like enzyme "FDR".

Catalytic Residues Roles

UniProt PDB* (1dj7)
His87 His86A His86 is proposed to play a functional role in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate. electrostatic stabiliser
Cys88 Cys87A Cleavage of heterosulphide occurs via reductive release of Cys87 and subsequent nucleophilic attack of the heterodisulphide by Cys87, resulting in cleavage of the heterosulphide and reformation of the active site disulphide. nucleofuge, metal ligand, nucleophile, activator, covalent catalysis, electrofuge
Cys58 Cys57A Interchange thiol responsible for attacking the Trx disulphide. metal ligand, nucleofuge, nucleophile
Cys86, Cys56, Cys77, Cys75 Cys85A, Cys55A, Cys76A, Cys74A Attached to [Fe4S4]3+/2+ cluster. metal ligand
*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

substitution (not covered by the Ingold mechanisms), coordination to a metal ion, cofactor used, electron transfer, bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex formation, proton transfer, native state of cofactor regenerated, decoordination from a metal ion, enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated, overall product formed

References

  1. Walters EM et al. (2005), J Am Chem Soc, 127, 9612-9624. Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism. DOI:10.1021/ja051909q. PMID:15984889.
  2. Kumar AK et al. (2015), Biochemistry, 54, 3122-3128. Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans. DOI:10.1021/acs.biochem.5b00137. PMID:25915695.
  3. Walters EM et al. (2009), Biochemistry, 48, 1016-1024. Role of histidine-86 in the catalytic mechanism of ferredoxin:thioredoxin reductase. DOI:10.1021/bi802074p. PMID:19132843.
  4. Walters EM et al. (2004), Photosynth Res, 79, 249-264. Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe-4S] Cluster Chemistry. DOI:10.1023/B:PRES.0000017195.05870.61. PMID:16328791.

Step 1. The interaction between the cluster and Cys87 promotes charge build-up at the unique Fe site in the resting enzyme, priming the active site for one-electron reduction from ferredoxin leading to cleavage of the disulphide and attachment of Cys87 to the cluster. Overall one-electron oxidation of the cluster to [Fe4S4]3+.

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Catalytic Residues Roles

Residue Roles
Cys87A activator, covalent catalysis, metal ligand
Cys85A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand
Cys57A nucleofuge
His86A electrostatic stabiliser
Cys87A electrofuge

Chemical Components

substitution (not covered by the Ingold mechanisms), coordination to a metal ion, cofactor used, electron transfer

Step 2. Cys57 performs a nucleophilic attack on the thioredoxin disulphide to form an FTR/Trx heterodisulphide intermediate.

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Catalytic Residues Roles

Residue Roles
Cys85A metal ligand
Cys87A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand
His86A electrostatic stabiliser
Cys57A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex formation

Step 3. Protonation of Trx thiol.

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Catalytic Residues Roles

Residue Roles
His86A electrostatic stabiliser
Cys85A metal ligand
Cys87A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand

Chemical Components

proton transfer

Step 4. One-electron reduction of the cluster to [Fe4S4]2+ and release of Cys87.

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Catalytic Residues Roles

Residue Roles
Cys87A nucleofuge
Cys85A metal ligand
Cys87A metal ligand
Cys57A metal ligand
Cys76A metal ligand
Cys74A metal ligand

Chemical Components

cofactor used, native state of cofactor regenerated, decoordination from a metal ion, electron transfer

Step 5. Cys87 reforms the active site disulphide with concomitant cleavage of the heterodisulphide intermediate.

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Catalytic Residues Roles

Residue Roles
Cys87A nucleophile
Cys55A electrofuge, electrophile
Cys85A metal ligand
Cys57A metal ligand
Cys76A metal ligand
Cys74A metal ligand

Chemical Components

enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated, ingold: bimolecular nucleophilic substitution

Step 6. Trx thiol protonation, forming the reduced dithiol product.

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Catalytic Residues Roles

Residue Roles
Cys85A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand

Chemical Components

proton transfer, overall product formed
Download: Image, Marvin File

Step 1. The interaction between the cluster and Cys87 promotes charge build-up at the unique Fe site in the resting enzyme, priming the active site for one-electron reduction from ferredoxin leading to cleavage of the disulphide and attachment of Cys87 to the cluster. Overall one-electron oxidation of the cluster to [Fe4S4]3+.

Step 2. Cys57 performs a nucleophilic attack on the thioredoxin disulphide to form an FTR/Trx heterodisulphide intermediate.

Step 3. Protonation of Trx thiol.

Step 4. One-electron reduction of the cluster to [Fe4S4]2+ and release of Cys87.

Step 5. Cys87 reforms the active site disulphide with concomitant cleavage of the heterodisulphide intermediate.

Step 6. Trx thiol protonation, forming the reduced dithiol product.

Products.

Introduction

In an alternative and equally viable mechanism, FTR is reduced by two electrons prior to interaction with Trx. The first single-electron-reduction forms a transient intermediate which is then further reduced to yield the two-electron-reduced species. The strong H-bonding interaction between formed by Cys87 frees Cys57 for nucleophilic attack of Trx. His86 is proposed to play a role in stabilising the two-electron intermediate via interaction with Cys87. This mechanism is very similar to that found in the more extensively studied nucleotide-dependent disulphide reductases.

Catalytic Residues Roles

UniProt PDB* (1dj7)
His87 His86A His86 is proposed to play a functional role in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate. electrostatic stabiliser, proton donor
Cys88 Cys87A Cleavage of heterosulphide occurs via nucleophilic attack of the heterodisulphide by Cys87, resulting in cleavage of the heterosulphide and reformation of the active site disulphide. hydrogen bond donor, metal ligand, proton acceptor, proton donor, covalent catalysis, electrofuge, electrophile
Cys58 Cys57A Interchange thiol responsible for attacking the Trx disulphide. nucleophile, proton acceptor, proton donor, electrofuge, electrophile
Cys86, Cys56, Cys77, Cys75 Cys85A, Cys55A, Cys76A, Cys74A Attached to [Fe4S4]3+/2+ cluster. metal ligand
*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

electron transfer, cofactor used, coordination to a metal ion, substitution (not covered by the Ingold mechanisms), decoordination from a metal ion, native state of cofactor regenerated, proton transfer, enzyme-substrate complex formation, intermediate formation, bimolecular nucleophilic substitution, intermediate terminated, enzyme-substrate complex cleavage, native state of enzyme regenerated, overall product formed

References

  1. Walters EM et al. (2005), J Am Chem Soc, 127, 9612-9624. Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism. DOI:10.1021/ja051909q. PMID:15984889.
  2. Walters EM et al. (2009), Biochemistry, 48, 1016-1024. Role of histidine-86 in the catalytic mechanism of ferredoxin:thioredoxin reductase. DOI:10.1021/bi802074p. PMID:19132843.

Step 1. The interaction between the cluster and Cys87 promotes charge build-up at the unique Fe site in the resting enzyme, priming the active site for one-electron reduction from ferredoxin leading to cleavage of the disulphide and attachment of Cys87 to the cluster. Overall one-electron oxidation of the cluster to [Fe4S4]3+.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys74A metal ligand
Cys76A metal ligand
Cys55A metal ligand
Cys85A metal ligand
Cys87A metal ligand
His86A electrostatic stabiliser
Cys57A proton acceptor
Cys87A electrofuge, covalent catalysis

Chemical Components

electron transfer, cofactor used, coordination to a metal ion, substitution (not covered by the Ingold mechanisms)

Step 2. Further one-electron reduction and release of Cys87.

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Catalytic Residues Roles

Residue Roles
Cys87A covalent catalysis
His86A proton donor, electrostatic stabiliser
Cys87A proton acceptor

Chemical Components

decoordination from a metal ion, cofactor used, electron transfer, native state of cofactor regenerated, proton transfer

Step 3. Cys57 performs a nucleophilic attack on the thioredoxin disulphide to form an FTR/Trx heterodisulphide intermediate. This is facilitated by a hydrogen bond between Cys87 and Cys55.

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Catalytic Residues Roles

Residue Roles
Cys55A hydrogen bond acceptor
Cys87A hydrogen bond donor
His86A electrostatic stabiliser
Cys85A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand
Cys57A nucleophile, electrofuge, electrophile, proton donor

Chemical Components

enzyme-substrate complex formation, intermediate formation, ingold: bimolecular nucleophilic substitution, proton transfer

Step 4. Cys87 reforms the active site disulphide with concomitant cleavage of the heterodisulphide intermediate.

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Catalytic Residues Roles

Residue Roles
Cys87A hydrogen bond donor
Cys55A hydrogen bond acceptor
Cys85A metal ligand
Cys55A metal ligand
Cys76A metal ligand
Cys74A metal ligand
Cys57A nucleophile
Cys87A electrophile, proton donor
His86A electrostatic stabiliser

Chemical Components

intermediate terminated, enzyme-substrate complex cleavage, native state of enzyme regenerated, proton transfer, overall product formed
Download: Image, Marvin File

Step 1. The interaction between the cluster and Cys87 promotes charge build-up at the unique Fe site in the resting enzyme, priming the active site for one-electron reduction from ferredoxin leading to cleavage of the disulphide and attachment of Cys87 to the cluster. Overall one-electron oxidation of the cluster to [Fe4S4]3+.

Step 2. Further one-electron reduction and release of Cys87.

Step 3. Cys57 performs a nucleophilic attack on the thioredoxin disulphide to form an FTR/Trx heterodisulphide intermediate. This is facilitated by a hydrogen bond between Cys87 and Cys55.

Step 4. Cys87 reforms the active site disulphide with concomitant cleavage of the heterodisulphide intermediate.

Products.

Contributors

Noa Marson, Antonio Ribeiro