MetaCyc Enzyme: Fe-Fe nitrogenase

Synonyms: iron-iron nitrogenase

Species: Azotobacter vinelandii

Subunit composition of Fe-Fe nitrogenase = [(AnfH)2][(AnfG)2(AnfK)2(AnfD)2]
         nitrogenase reductase monomer = (AnfH)2 (summary available)
                 nitrogenase reductase subunit = AnfH
         dinitrogenase component = (AnfG)2(AnfK)2(AnfD)2
                 dinitrogenase 3 subunit delta = AnfG (summary available)
                 dinitrogenase 3 subunit beta = AnfK (summary available)
                 dinitrogenase 3 subunit alpha = AnfD (summary available)


The reduction of the dinitrogen molecule to ammonia, a process known as nitrogen fixation, is catalyzed by the nitrogenase enzyme system in diazotrophs. The enzyme complex consists of two oxygen-sensitive metalloprotein components [Richards94]. The dinitrogenase reductase component is a Fe protein containing a single [4Fe4S] center that transfers electrons from an external electron donor (a ferredoxin or a flavodoxin, depending on the species) to the other component, dinitrogenase [OrmeJohnson92].

Three such systems have been described, and they differ in their protein structures and metal content in the active site. Type I contains molybedenum and iron (MoFe), type II contains vanadium and iron (VFe) and type III contains only iron (FeFe). Different genes and gene products make up the different types [Eady96]. All dinitrogenases contain two unique cofactors, an Fe8S7 complex known as the P-cluster, and the metal (Mo, V or Fe) containing cofactor. All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have one or more alternative nitrogenases.

About this Enzyme

An FeFe alternative nitrogenase was first isolated in Azotobacter vinelandii (which possesses all three types) and has also been characterized in other diazotrophs including the phototrophic purple bacterium Rhodobacter capsulatus. It is encoded by the anfHDGKOR operon under the control of the anfA transcriptional activator. The transcription of anfA is regulated by ammonium and traces of Mo [Kutsche96]. The anfO gene product is essential for Fe-nitrogenase dependent nitrogen reduction to ammonia and H2.

Component I of the Fe-nitrogenase is an α2β2δ2 hexameric protein. The Fe-nitrogenase complex has a higher rate of H2 evolving activity than the Mo enzyme [Schneider91]. Under optimal conditions for nitrogen reduction, the amount of H2 produced is almost two fold compared to the Mo nitrogenase [Sicking05].

The electron source for three nitrogenases of Azotobacter vinelandii is a flavodoxin encoded by the nifF gene, which shuttles electrons from pyruvate:flavin oxidoreductase (encoded by nifJ) [Tanaka77, Bennett88, Gangeswaran96]. However, when the genes encoding the FeFe nitrogenase of Azotobacter vinelandii were cloned and expressed in Escherichia coli, nitrogenase activity was not completely ablated by deletion of either nifF or nifJ. It is assumed that other genes in Escherichia coli may partially substitute for their function in electron transport [Chan00].

Interestingly, a gene encoding a [2Fe-2S] ferredoxin has been found in the nif gene cluster of Azotobacter vinelandii, that is very similar to the ferredoxin from Clostridium pasteurianum, which is known to act as the electron donor for the nitrogenase of that organism. It has been suggested that this ferredoxin may act as an alternative electron donor for the nitrogenase of Azotobacter vinelandii [Chatelet99].

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Created 11-Apr-2011 by Weerasinghe D , SRI International
Revised 20-Oct-2014 by Caspi R , SRI International

Enzymatic reaction of: nitrogenase

EC Number:

4 a reduced flavodoxin + N2 + 16 ATP + 16 H2O <=> 4 an oxidized flavodoxin + 2 ammonium + 16 ADP + 16 phosphate + H2 + 14 H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

In Pathways: nitrogen fixation II (flavodoxin)

Cofactors or Prosthetic Groups: Mg2+

Subunit of Fe-Fe nitrogenase: nitrogenase reductase monomer

Synonyms: Nitrogenase Fe protein 1, Nitrogenase component II

Gene: anfH Accession Number: G-12779 (MetaCyc)

Subunit composition of nitrogenase reductase monomer = [AnfH]2
         nitrogenase reductase subunit = AnfH

Molecular Weight of Polypeptide: 31.5 kD (from nucleotide sequence)

Unification Links: Protein Model Portal:P00459 , SMR:P00459 , UniProt:P00459

Relationship Links: InterPro:IN-FAMILY:IPR000392 , InterPro:IN-FAMILY:IPR005977 , InterPro:IN-FAMILY:IPR027417 , Panther:IN-FAMILY:PTHR13696:SF32 , PDB:Structure:1DE0 , PDB:Structure:1FP6 , PDB:Structure:1G1M , PDB:Structure:1G5P , PDB:Structure:1G20 , PDB:Structure:1G21 , PDB:Structure:1M1Y , PDB:Structure:1M34 , PDB:Structure:1N2C , PDB:Structure:1NIP , PDB:Structure:1RW4 , PDB:Structure:1XCP , PDB:Structure:1XD8 , PDB:Structure:1XD9 , PDB:Structure:1XDB , PDB:Structure:2AFH , PDB:Structure:2AFI , PDB:Structure:2AFK , PDB:Structure:2C8V , PDB:Structure:2NIP , Pfam:IN-FAMILY:PF00142 , Prints:IN-FAMILY:PR00091 , Prosite:IN-FAMILY:PS00692 , Prosite:IN-FAMILY:PS00746 , Prosite:IN-FAMILY:PS51026

This subunit is a homodimer associated with the molybdenum-iron nitrogenase component 2 and binds 1 4Fe-4S cluster per dimer.

Citations: [Jacobson89]

Subunit of Fe-Fe nitrogenase: dinitrogenase component

Subunit of dinitrogenase component: dinitrogenase 3 subunit delta

Synonyms: AnfG, nitrogenase iron-iron protein delta chain

Gene: anfG Accession Number: G-12782 (MetaCyc)

Molecular Weight: 15.3 kD (from nucleotide sequence)

Unification Links: UniProt:P16268

Relationship Links: InterPro:IN-FAMILY:IPR004349 , InterPro:IN-FAMILY:IPR014278 , Pfam:IN-FAMILY:PF03139 , ProDom:IN-FAMILY:PD006096

This subunit forms a hexamer of two alpha two beta and two delta chains which is part of the nitrogenase complex that catalyzes the key enzymatic reactions in nitrogen fixation.

Citations: [Joerger89]

Subunit of dinitrogenase component: dinitrogenase 3 subunit beta

Synonyms: AnfK, nitrogenase iron-iron protein beta chain

Gene: anfK Accession Number: G-12781 (MetaCyc)

Molecular Weight: 51.0 kD (from nucleotide sequence)

Unification Links: Protein Model Portal:P16267 , UniProt:P16267

Relationship Links: InterPro:IN-FAMILY:IPR000318 , InterPro:IN-FAMILY:IPR000510 , InterPro:IN-FAMILY:IPR014280 , Pfam:IN-FAMILY:PF00148 , Prosite:IN-FAMILY:PS00090 , Prosite:IN-FAMILY:PS00699

This subunit forms a hexamer of two alpha two beta and two delta chains which is part of the nitrogenase complex that catalyzes the key enzymatic reactions in nitrogen fixation.

Citations: [Joerger89]

Subunit of dinitrogenase component: dinitrogenase 3 subunit alpha

Synonyms: AnfD

Gene: anfD Accession Number: G-12780 (MetaCyc)

Molecular Weight: 55.2 kD (from nucleotide sequence)

Unification Links: DIP:DIP-6252N , Mint:MINT-1508543 , Protein Model Portal:P07328 , SMR:P07328 , UniProt:P07328

Relationship Links: InterPro:IN-FAMILY:IPR000318 , InterPro:IN-FAMILY:IPR000510 , InterPro:IN-FAMILY:IPR005972 , InterPro:IN-FAMILY:IPR010143 , PDB:Structure:1FP4 , PDB:Structure:1G20 , PDB:Structure:1G21 , PDB:Structure:1L5H , PDB:Structure:1M1N , PDB:Structure:1M1Y , PDB:Structure:1M34 , PDB:Structure:1N2C , PDB:Structure:2AFH , PDB:Structure:2AFI , PDB:Structure:2AFK , PDB:Structure:2MIN , PDB:Structure:3K1A , PDB:Structure:3MIN , PDB:Structure:3U7Q , PDB:Structure:4ND8 , Pfam:IN-FAMILY:PF00148 , Prosite:IN-FAMILY:PS00090 , Prosite:IN-FAMILY:PS00699

This subunit forms a hexamer of two alpha two beta and two delta chains which is part of the nitrogenase complex that catalyzes the key enzymatic reactions in nitrogen fixation.

Citations: [Joerger89]


Bennett88: Bennett LT, Jacobson MR, Dean DR (1988). "Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii." J Biol Chem 263(3);1364-9. PMID: 3121629

Chan00: Chan JM, Wu W, Dean DR, Seefeldt LC (2000). "Construction and characterization of a heterodimeric iron protein: defining roles for adenosine triphosphate in nitrogenase catalysis." Biochemistry 39(24);7221-8. PMID: 10852721

Chatelet99: Chatelet C, Meyer J (1999). "The [2Fe-2S] protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the [2Fe-2S] ferredoxin from Clostridium pasteurianum." J Biol Inorg Chem 4(3);311-7. PMID: 10439076

Eady96: Eady RR (1996). "Structureminus signFunction Relationships of Alternative Nitrogenases." Chem Rev 96(7);3013-3030. PMID: 11848850

Gangeswaran96: Gangeswaran R, Eady RR (1996). "Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase." Biochem J 317 ( Pt 1);103-8. PMID: 8694750

Jacobson89: Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR (1989). "Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii." J Bacteriol 171(2);1017-27. PMID: 2644218

Joerger89: Joerger RD, Jacobson MR, Premakumar R, Wolfinger ED, Bishop PE (1989). "Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii." J Bacteriol 171(2);1075-86. PMID: 2644222

Kutsche96: Kutsche M, Leimkuhler S, Angermuller S, Klipp W (1996). "Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum." J Bacteriol 178(7);2010-7. PMID: 8606177

OrmeJohnson92: Orme-Johnson WH (1992). "Nitrogenase structure: where to now?." Science 257(5077);1639-40. PMID: 1529351

Richards94: Richards AJ, Lowe DJ, Richards RL, Thomson AJ, Smith BE (1994). "Electron-paramagnetic-resonance and magnetic-circular-dichroism studies of the binding of cyanide and thiols to the thiols to the iron-molybdenum cofactor from Klebsiella pneumoniae nitrogenase." Biochem J 297 ( Pt 2);373-8. PMID: 8297344

Schneider91: Schneider K, Muller A, Schramm U, Klipp W (1991). "Demonstration of a molybdenum- and vanadium-independent nitrogenase in a nifHDK-deletion mutant of Rhodobacter capsulatus." Eur J Biochem 195(3);653-61. PMID: 1999188

Sicking05: Sicking C, Brusch M, Lindackers A, Riedel KU, Schubert B, Isakovic N, Krall C, Klipp W, Drepper T, Schneider K, Masepohl B (2005). "Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus." J Bacteriol 187(1);92-8. PMID: 15601692

Tanaka77: Tanaka M, Haniu M, Yasunobu KT, Yoch DC (1977). "Complete amino acid sequence of azotoflavin, a flavodoxin from Azotobacter vinelandii." Biochemistry 16(16);3525-37. PMID: 889809

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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