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Nitrogenases are enzymes (EC that are produced by certain bacteria, such as cyanobacteria (blue-green algae). These enzymes are responsible for the reduction of nitrogen (N2) to ammonia (NH3). Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a key step in the process of nitrogen fixation. Nitrogen fixation is required for all forms of life, with nitrogen being essential for the biosynthesis of molecules (nucleotides, amino acids) that create plants, animals and other organisms.

Structure and mechanism[edit]

The equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (), the activation energy is very high ().[1] Nitrogenase acts as a catalyst, reducing this energy barrier such that the reaction can take place at ambient temperatures.

The nitrogenase complex consists of two proteins:

  • The homodimeric Fe protein, a reductase which has a high reducing power and is responsible for the supply of electrons.
  • The heterotetrameric MoFe protein, a nitrogenase which uses the electrons provided to reduce N2 to NH3.

Fe protein[edit]

The Fe protein is a dimer of identical subunits which contains one [Fe4S4] cluster and weighs approximately 60-64kDa.[2] The function of the Fe protein is to transfer electrons from a reducing agent, such as ferredoxin or flavodoxin to the MoFe protein. The transfer of electrons requires an input of chemical energy which comes from the binding and hydrolysis of ATP. The hydrolysis of ATP also causes a conformational change within the nitrogenase complex, bringing the Fe protein and MoFe protein closer together for easier electron transfer.

MoFe protein[edit]

The MoFe protein is a heterotetramer consisting of two α subunits and two β subunits, weighing approximately 240-250kDa.[2] The MoFe protein also contains two iron-sulfur clusters, known as P-clusters, located at the interface between the α and β subunits and two FeMo cofactors, within the α subunits.

  • The core (Fe8S7) of the P-cluster takes the form of two [Fe4S3] cubes linked by a central sulfur atom. Each P-cluster is covalently linked to the MoFe protein by six cysteine residues.
  • Each FeMo cofactor (Fe7MoS9C) consists of two non-identical clusters: [Fe4S3] and [MoFe3S3], which are linked by three sulfide ions. Each FeMo cofactor is covalently linked to the α subunit of the protein by one cysteine residue and one histidine residue.

Electrons from the Fe protein enter the MoFe protein at the P-clusters, which then transfer the electrons to the FeMo cofactors. Each FeMo cofactor then acts as a site for nitrogen fixation, with N2 binding in the central cavity of the cofactor. Here, Fe-N interactions causes weakening of the extremely strong triple bond linking the two nitrogen atoms, lowering the activation energy barrier required for reduction. Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH3), which is in turn bonded to glutamate to form glutamine. The nitrogenase reaction additionally produces molecular hydrogen as a side product.

Structure of the FeMo cofactor showing the sites of binding to nitrogenase (the amino acids cys and his).

Many mechanistic aspects of catalysis remain unknown. No crystallographic analysis has been reported on substrate bound to nitrogenase. One problem is that the enzyme is generally obtained as a mixture of states.. Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which binds to the enzyme and thereby prevents binding of dinitrogen. Dinitrogen prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and requires only one electron for reduction to ethylene.[3] Due to the oxidative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. Although the ability of some nitrogen fixers such as Azotobacteraceae to employ an oxygen-labile nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane, the effectiveness of such a mechanism has been questioned at oxygen concentrations above 70 µM (ambient concentration is 230 µM O2), as well as during additional nutrient limitations.[4]

The reaction that this enzyme performs is:


Nonspecific reactions[edit]

In addition to dinitrogen reduction, nitrogenases also reduce protons to dihydrogen, meaning nitrogenase is also a dehydrogenase. A list of other reactions carried out by nitrogenases is shown below:[6][7]

N=N+=O → N2 + H2O
N=N=N → N2 + NH3
CH4, NH3, H3C–CH3, H2C=CH2 (CH3NH2)
N≡C–R → RCH3 + NH3
C≡N–R → CH4, H3C–CH3, H2C=CH2, C3H8, C3H6, RNH2
O=C=SCO + H2S [8][9]
O=C=O → CO + H2O [8]
S=C=N → H2S + HCN [9]
O=C=N → H2O + HCN, CO + NH3 [9]

Furthermore, dihydrogen functions as a competitive inhibitor,[10] carbon monoxide functions as a non-competitive inhibitor,[6][7] and carbon disulfide functions as a rapid-equilibrium inhibitor[8] of nitrogenase.

Vanadium nitrogenases have also been shown to catalzye the conversion of CO into alkanes through a reaction comparable to Fischer-Tropsch synthesis.

Organisms that synthesize nitrogenase[edit]

There are two types of bacteria that synthesize nitrogenase and are required for nitrogen fixation. These are:

Similarity to other proteins[edit]

The three subunits of nitrogenase exhibit significant sequence similarity to three subunits of the light-independent version of protochlorophyllide reductase that performs the conversion of protochlorophyllide to chlorophyll. This protein is present in gymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution.[11]

Separately, two of the nitrogenase subunits (NifD and NifH) have homologues in methanogens that do not fix nitrogen e.g. Methanocaldococcus jannaschii.[12] Little is understood about the function of these "class IV" nif genes,[13] though they occur in many methanogens. In M. jannaschii they are known to interact with each other and are constitutively expressed.[12]

Measurement of nitrogenase activity[edit]

As with many assays for enzyme activity, it is possible to estimate nitrogenase activity by measuring the rate of conversion of the substrate (N2) to the product (NH3). Since NH3 is involved in other reactions in the cell, it is often desirable to label the substrate with 15N to provide accounting or "mass balance" of the added substrate. A more common assay, the acetylene reduction assay or ARA, estimates the activity of nitrogenase by taking advantage of the ability of the enzyme to reduce acetylene gas to ethylene gas. These gases are easily quantified using gas chromatography.[14] Though first used in a laboratory setting to measure nitrogenase activity in extracts of Clostridium pasteurianum cells, ARA has been applied to a wide range of test systems, including field studies where other techniques are difficult to deploy. For example, ARA was used successfully to demonstrate that bacteria associated with rice roots undergo seasonal and diurnal rhythms in nitrogenase activity, which were apparently controlled by the plant.[15]

Unfortunately, the conversion of data from nitrogenase assays to actual moles of N2 reduced (particularly in the case of ARA), is not always straightforward and may either underestimate or overestimate the true rate for a variety of reasons. For example, H2 competes with N2 but not acetylene for nitrogenase (leading to overestimates of nitrogenase by ARA). Bottle or chamber-based assays may produce negative impacts on microbial systems as a result of containment or disruption of the microenvironment through handling, leading to underestimation of nitrogenase. Despite these weaknesses, such assays are very useful in assessing relative rates or temporal patterns in nitrogenase activity.

See also[edit]


  1. ^ Modak, J. M. (2002). "Haber Process for Ammonia Synthesis". Resonance. 7: 69–77. doi:10.1007/bf02836187. 
  2. ^ a b Burges BK, Lowe DJ (1996). "Mechanism of Molybdenum Nitrogenase". Chemical Reviews. 96: 2983–3011. doi:10.1021/cr950055x. 
  3. ^ Seefeldt LC, Dance IG, Dean DR (Feb 2004). "Substrate interactions with nitrogenase: Fe versus Mo". Biochemistry. 43 (6): 1401–9. doi:10.1021/bi036038g. PMID 14769015. 
  4. ^ Oelze, J (2000). "Respiratory protection of nitrogenase in Azotobacter species: Is a widely-held hypothesis unequivocally supported by experimental evidence?". FEMS Microbiol Rev. 24 (4): 321–33. doi:10.1111/j.1574-6976.2000.tb00545.x. 
  5. ^ Igarashi RY, Seefeldt LC (2003). "Nitrogen fixation: the mechanism of the Mo-dependent nitrogenase". Critical Reviews in Biochemistry and Molecular Biology. 38 (4): 351–84. doi:10.1080/10409230391036766. PMID 14551236. 
  6. ^ a b Rivera-Ortiz JM, Burris RH (Aug 1975). "Interactions among substrates and inhibitors of nitrogenase". Journal of Bacteriology. 123 (2): 537–45. PMC 235759free to read. PMID 1150625. 
  7. ^ a b Schrauzer GN (Aug 1975). "Nonenzymatic simulation of nitrogenase reactions and the mechanism of biological nitrogen fixation". Angewandte Chemie. 14 (8): 514–22. doi:10.1002/anie.197505141. PMID 810048. 
  8. ^ a b c Seefeldt LC, Rasche ME, Ensign SA (Apr 1995). "Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase". Biochemistry. 34 (16): 5382–9. doi:10.1021/bi00016a009. PMID 7727396. 
  9. ^ a b c Rasche ME, Seefeldt LC (Jul 1997). "Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: kinetic characterization and EPR spectroscopic analysis". Biochemistry. 36 (28): 8574–85. doi:10.1021/bi970217e. PMID 9214303. 
  10. ^ Guth JH, Burris RH (Oct 1983). "Inhibition of nitrogenase-catalyzed NH3 formation by H2". Biochemistry. 22 (22): 5111–22. doi:10.1021/bi00291a010. PMID 6360203. 
  11. ^ Li J, Goldschmidt-Clermont M, Timko MP (Dec 1993). "Chloroplast-encoded chlB is required for light-independent protochlorophyllide reductase activity in Chlamydomonas reinhardtii". The Plant Cell. 5 (12): 1817–29. doi:10.1105/tpc.5.12.1817. PMC 160407free to read. PMID 8305874. 
  12. ^ a b Staples CR, Lahiri S, Raymond J, Von Herbulis L, Mukhophadhyay B, Blankenship RE (Oct 2007). "Expression and association of group IV nitrogenase NifD and NifH homologs in the non-nitrogen-fixing archaeon Methanocaldococcus jannaschii". Journal of Bacteriology. 189 (20): 7392–8. doi:10.1128/JB.00876-07. PMC 2168459free to read. PMID 17660283. 
  13. ^ Raymond J, Siefert JL, Staples CR, Blankenship RE (Mar 2004). "The natural history of nitrogen fixation". Molecular Biology and Evolution. 21 (3): 541–54. doi:10.1093/molbev/msh047. PMID 14694078. 
  14. ^ Dilworth MJ (Oct 1966). "Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum". Biochimica et Biophysica Acta. 127 (2): 285–94. doi:10.1016/0304-4165(66)90383-7. PMID 5964974. 
  15. ^ Sims GK, Dunigan EP (1984). "Diurnal and seasonal variations in nitrogenase activity (C2H2 reduction) of rice roots.". Soil Biology and Biochemistry. 16: 15–18. doi:10.1016/0038-0717(84)90118-4. 

Further reading[edit]