View source for Nitrogenase

{{Short description|Class of enzymes}}
{{infobox enzyme
| Name       = Nitrogenase
| EC_number  = 1.18.6.1
| CAS_number = 9013-04-1
| GO_code    =
| image      = Nitrogenase.png
| width      = 
| caption    =  
}}
{{Infobox protein family
|Name=Nitrogenase-type Oxidoreductase (component 1 subunit alpha/beta)
|Pfam=PF00148
|Symbol=Oxidored_nitro 
|InterPro=IPR000510
|SCOP=1mio
}}
{{Infobox protein family
|Name=Nitrogenase iron protein NifH (component 2)
|Symbol=NifH
|CDD=cd02040
|InterPro=IPR005977
|SCOP=d1fp6a_
|CATH=1fp6
}}
{{Infobox protein family
|Name=Alternative nitrogenase (component 1) delta subunit
|Pfam=PF03139
|Symbol=AnfG_VnfG
|InterPro=IPR004349
}}
<!-- [[File:Nitrogenase.png|thumb|Nitrogenase]] -->
'''Nitrogenases''' are [[enzymes]] ({{EC number|1.18.6.1}}{{EC number|1.19.6.1}}) that are produced by certain [[bacteria]], such as [[cyanobacteria]] (blue-green bacteria) and [[rhizobacteria]]. These enzymes are responsible for the [[Organic redox reaction|reduction]] of [[nitrogen]] (N<sub>2</sub>) to [[ammonia]] (NH<sub>3</sub>). Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a 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. They are encoded by the [[Nif gene]]s or [[Homologous chromosome|homologs]]. They are related to [[protochlorophyllide reductase]].

==Classification and structure==

Although the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative [[enthalpy of reaction]] (<math> \Delta H^{0} = -45.2 \ \mathrm{kJ} \, \mathrm{mol^{-1}} \; \mathrm{NH_3} </math>), the [[activation energy]] is very high (<math> E_\mathrm{A} = 230-420 \ \mathrm{kJ} \, \mathrm{mol^{-1}} </math>).<ref>{{cite journal | last1 = Modak | first1 = J. M. | name-list-style = vanc | year = 2002 | title = Haber Process for Ammonia Synthesis | journal = Resonance | volume = 7 | issue = 9| pages = 69–77 | doi=10.1007/bf02836187| s2cid = 195305228 }}</ref> Nitrogenase acts as a [[catalyst]], reducing this energy barrier such that the reaction can take place at ambient temperatures.

A usual assembly consists of two components:

# The [[homodimeric]] Fe-only [[protein]], the [[reductase]] which has a high reducing power and is responsible for a supply of electrons.
# The [[heterotetrameric]] MoFe protein, a nitrogenase which uses the electrons provided to reduce N<sub>2</sub> to NH<sub>3</sub>. In some assemblies it is replaced by a homologous alternative.

[[File:FeMoco cluster.svg|thumb|170px|Structure of the [[FeMoco|FeMo cofactor]] showing the sites of binding to nitrogenase (the amino acids cys and his).|left]]

===Reductase===
The Fe protein, the dinitrogenase reductase or NifH, is a dimer of identical subunits which contains one [Fe<sub>4</sub>S<sub>4</sub>] cluster and has a mass of approximately 60-64kDa.<ref name="Burges1996">{{Cite journal |vauthors=Burges BK, Lowe DJ | title=Mechanism of Molybdenum Nitrogenase | journal=[[Chemical Reviews]] | volume=96 | issue=7 | year=1996 | pages=2983–3011 | doi=10.1021/cr950055x | pmid=11848849}}</ref> The function of the Fe protein is to transfer electrons from a [[reducing agent]], such as [[ferredoxin]] or [[flavodoxin]] to the nitrogenase protein. Ferredoxin or flavodoxin can be reduced by one of six mechanisms: 1. by a [[pyruvate:ferredoxin oxidoreductase]], 2. by a bi-directional [[hydrogenase]], 3. in a [[photosynthetic reaction center]], 4. by coupling [[electron transport chain|electron flow]] to dissipation of the [[proton motive force]], 5. by [[electron bifurcation]], or 6. by a [[ferredoxin:NADPH oxidoreductase]].<ref>{{cite journal | vauthors = Alleman AB, Peters JW | year = 2023 | title = Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria | journal = Appl Environ Microbiol | volume = 89 |issue= 5 | pages = e00378-23 | doi = 10.1128/aem.00378-23 | pmid =37154716 | pmc = 10231201| bibcode = 2023ApEnM..89E.378A }}</ref> The transfer of electrons requires an input of chemical energy which comes from the binding and hydrolysis of [[Adenosine triphosphate|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.<ref name="pmid11913144">{{cite journal | vauthors = Lawson DM, Smith BE | title = Molybdenum nitrogenases: a crystallographic and mechanistic view | journal = Metal Ions in Biological Systems | volume = 39 | pages = 75–119 | date = 2002 | pmid = 11913144 }}</ref>

===Nitrogenase===
The MoFe protein is a heterotetramer consisting of two &alpha; subunits and two &beta; subunits, with a mass of approximately 240-250kDa.<ref name="Burges1996"/> The MoFe protein also contains two [[iron–sulfur cluster]]s, known as P-clusters, located at the interface between the &alpha; and &beta; subunits and two [[FeMo cofactor]]s, within the &alpha; subunits. The oxidation state of Mo in these nitrogenases was formerly thought Mo(V), but more recent evidence is for Mo(III).<ref>{{cite journal| pmc=4510703 | pmid=26213424 | doi=10.1002/zaac.201400446 | volume=641 | issue=1 | title=Molybdenum L-Edge XAS Spectra of MoFe Nitrogenase | year=2015 | journal=Z Anorg Allg Chem | pages=65–71 | vauthors=Bjornsson R, Delgado-Jaime MU, Lima FA, Sippel D, Schlesier J, Weyhermüller T, Einsle O, Neese F, DeBeer S}}</ref> (Molybdenum in other enzymes is generally bound to [[molybdopterin]] as fully oxidized Mo(VI)).

* The core (Fe<sub>8</sub>S<sub>7</sub>) of the P-cluster takes the form of two [Fe<sub>4</sub>S<sub>3</sub>] cubes linked by a central sulfur atom. Each P-cluster is linked to the MoFe protein by six cysteine residues.
* Each FeMo cofactor (Fe<sub>7</sub>MoS<sub>9</sub>C) consists of two non-identical clusters: [Fe<sub>4</sub>S<sub>3</sub>] and [MoFe<sub>3</sub>S<sub>3</sub>], which are linked by three sulfide ions. Each FeMo cofactor is covalently linked to the &alpha; 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 N<sub>2</sub> binding in the central cavity of the cofactor.

==== Variations ====
The MoFe protein can be replaced by alternative nitrogenases in environments low in the Mo cofactor. Two types of such nitrogenases are known: the [[Vanadium nitrogenase|vanadium–iron]] (VFe; ''Vnf'') type and the iron–iron (FeFe; ''Anf'') type. Both form an assembly of two &alpha; subunits, two &beta; subunits, and two &delta; (sometimes &gamma;: VnfG/AnfG) subunits. The delta subunits are homologous to each other, and the alpha and beta subunits themselves are homologous to the ones found in MoFe nitrogenase. The gene clusters are also homologous, and these subunits are interchangeable to some degree. All nitrogenases use a similar Fe-S core cluster, and the variations come in the cofactor metal.<ref name=vnf>{{cite book |vauthors=Hales BJ|title=Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models and Commercial Processes |pages=255–279 |publisher=Springer Netherlands |isbn=978-1-4020-3611-8 |doi=10.1007/978-1-4020-3611-8_10 |chapter=Vanadium Nitrogenase|year=2004 }}</ref><ref name=anf>{{cite book |vauthors=Schneider K, Mueller A|title=Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models and Commercial Processes |pages=281–307 |publisher=Springer Netherlands |isbn=978-1-4020-3611-8 |doi=10.1007/978-1-4020-3611-8_11 |chapter=Iron-Only Nitrogenase: Exceptional Catalytic, Structural and Spectroscopic Features|year=2004 }}</ref> The &delta;/&gamma; subunit helps bind the cofactor in the FeFe nitrogenase.<ref name=Schmidt24>{{cite journal |last1=Schmidt |first1=Frederik V. |last2=Schulz |first2=Luca |last3=Zarzycki |first3=Jan |last4=Prinz |first4=Simone |last5=Oehlmann |first5=Niels N. |last6=Erb |first6=Tobias J. |last7=Rebelein |first7=Johannes G. |title=Structural insights into the iron nitrogenase complex |journal=Nature Structural & Molecular Biology |date=January 2024 |volume=31 |issue=1 |pages=150–158 |doi=10.1038/s41594-023-01124-2|pmid=38062208 |pmc=10803253 }}</ref> Based on the timing of its evolution, the subunit in VFe and FeFe nitrogenases is believed to have helped with the  prototypical alternative nitrogenase adapt to new metals.<ref name="Cuevas24">{{cite journal |last1=Cuevas-Zuviría |first1=Bruno |last2=Garcia |first2=Amanda K |last3=Rivier |first3=Alex J |last4=Rucker |first4=Holly R |last5=Carruthers |first5=Brooke M |last6=Kaçar |first6=Betül |title=Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation |journal=Molecular Biology and Evolution |date=2 April 2024 |volume=41 |issue=4 |doi=10.1093/molbev/msae067|pmid=38526235 |pmc=11018506 }}</ref>

Most, if not all, natural organisms carrying genes for an alternative nitrogenase also carry genes for the regular MoFe nitrogenase.<ref name="Cuevas24"/><ref>{{cite journal |last1=McRose |first1=Darcy L. |last2=Zhang |first2=Xinning |last3=Kraepiel |first3=Anne M. L. |last4=Morel |first4=François M. M. |title=Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments |journal=Frontiers in Microbiology |date=28 February 2017 |volume=8 |page=267 |doi=10.3389/fmicb.2017.00267|doi-access=free |pmid=28293220 |pmc=5328986 }}</ref> The MoFe nitrogenase is the most efficient in that it wastes less ATP on reducing H<sup>+</sup> into H<sub>2</sub> than the alternative nitrogenases (see [[#General mechanism]] below). When Mo is present, the expression of the alternative nitrogenases is repressed, so that only the more efficient enzyme is used.<ref>{{cite journal |last1=Mus |first1=F |last2=Alleman |first2=AB |last3=Pence |first3=N |last4=Seefeldt |first4=LC |last5=Peters |first5=JW |title=Exploring the alternatives of biological nitrogen fixation. |journal=Metallomics: Integrated Biometal Science |date=25 April 2018 |volume=10 |issue=4 |pages=523–538 |doi=10.1039/c8mt00038g |pmid=29629463|osti=1434118 }}</ref>

The FeFe nitrogenase in ''[[Azotobacter vinelandii]]'' (a model organism for nitrogenase engineering) is organized in an ''anfHDGKOR'' operon. This operon still requires some of the [[Nif gene]]s to function. A minimal 10-gene operon that incorporates these additional essential genes has been constructed in the lab.<ref>{{cite journal | vauthors = Yang J, Xie X, Wang X, Dixon R, Wang YP | title = Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 35 | pages = E3718-25 | date = September 2014 | pmid = 25139995 | doi = 10.1073/pnas.1411185111 | doi-access = free | pmc=4156695| bibcode = 2014PNAS..111E3718Y }}</ref>

{{clear}}

== Mechanism ==
[[File:Correct_Cartoon_Nitrogenase_with_Active_Sites_Highlighted.png|left|thumb|299x299px|Nitrogenase with catalytic sites highlighted. There are two sets of catalytic sites within each nitrogenase enzyme.]]
[[File:Cartoon_Nitrogenase_with_Active_Site_Magnified.png|left|thumb|299x299px|Nitrogenase with one set of metal clusters magnified. Electrons travel from the Fe-S cluster (yellow) to the P cluster (red), and end at the FeMo-co (orange).]]

=== General mechanism ===
[[File:Corrected_Nitrogenase_Active_Sites.jpg|thumb|278x278px|Catalytic sites within nitrogenase. Atoms are colored by element. Top: Fe-S Cluster Middle: P Cluster Bottom: FeMo-co/M-cluster]]
Nitrogenase is an enzyme responsible for catalyzing [[nitrogen fixation]], which is the reduction of nitrogen (N<sub>2</sub>) to ammonia (NH<sub>3</sub>) and a process vital to sustaining life on Earth.<ref name="Hoffman_2014">{{cite journal | vauthors = Hoffman BM, Lukoyanov D, Yang ZY, Dean DR, Seefeldt LC | title = Mechanism of nitrogen fixation by nitrogenase: the next stage | journal = Chemical Reviews | volume = 114 | issue = 8 | pages = 4041–62 | date = April 2014 | pmid = 24467365 | pmc = 4012840 | doi = 10.1021/cr400641x }}</ref> There are three types of nitrogenase found in various nitrogen-fixing bacteria:  molybdenum (Mo) nitrogenase, [[Vanadium nitrogenase|vanadium (V) nitrogenase]], and iron-only (Fe) nitrogenase.<ref name="Peters_2006">{{cite journal | vauthors = Peters JW, Szilagyi RK | title = Exploring new frontiers of nitrogenase structure and mechanism | journal = Current Opinion in Chemical Biology | volume = 10 | issue = 2 | pages = 101–8 | date = April 2006 | pmid = 16510305 | doi = 10.1016/j.cbpa.2006.02.019 | series = Bioinorganic chemistry / Biocatalysis and biotransformation }}</ref> Molybdenum nitrogenase, which can be found in [[diazotroph]]s such as [[legume]]-associated [[rhizobia]],<ref name="Rubio_2008">{{cite journal | vauthors = Rubio LM, Ludden PW | title = Biosynthesis of the iron-molybdenum cofactor of nitrogenase | journal = Annual Review of Microbiology | volume = 62 | issue = 1 | pages = 93–111 | date = 2008 | pmid = 18429691 | doi = 10.1146/annurev.micro.62.081307.162737 | doi-access = free }}</ref><ref>{{cite journal | last1 = Franche | first1 = Claudine | last2 = Lindström | first2 = Kristina | last3 = Elmerich | first3 = Claudine | name-list-style = vanc | date = December 2008 | title = Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants | journal = Plant and Soil |volume=321|issue=1–2|pages=35–59|doi=10.1007/s11104-008-9833-8| s2cid = 10892514 |issn=0032-079X}}</ref> is the nitrogenase that has been studied the most extensively and thus is the most well characterized.<ref name="Peters_2006" /> Vanadium nitrogenase and iron-only nitrogenase can both be found in select species of Azotobacter as an alternative nitrogenase.<ref name="Rubio_2008" /><ref name="Schneider_2004">{{cite book | title = Catalysts for Nitrogen Fixation | vauthors = Schneider K, Müller A |date= January 2004 | publisher = Springer Netherlands | isbn = 978-90-481-6675-6 | veditors = Smith BE, Richards RL, Newton WE | series = Nitrogen Fixation: Origins, Applications, and Research Progress|pages=281–307|doi=10.1007/978-1-4020-3611-8_11}}</ref> Equations 1 and 2 show the balanced reactions of nitrogen fixation in molybdenum nitrogenase and vanadium nitrogenase respectively.

{{NumBlk|:|N<sub>2</sub> + 8 H<sup>+</sup> + 8 e<sup>−</sup> + 16 MgATP → 2 NH<sub>3</sub> + H<sub>2</sub> + 16 MgADP + 16 P<sub>i</sub><ref name="Hoffman_2014" />            |{{EquationRef|1}}}}
{{NumBlk|:|N<sub>2</sub> + 18 H<sup>+</sup> + 18 e<sup>−</sup> + 36 MgATP → 2 NH<sub>3</sub> + 6 H<sub>2</sub> + 36 MgADP + 36 P<sub>i</sub><ref name=Schmidt24/>|{{EquationRef|2}}}}
{{NumBlk|:|N<sub>2</sub> + 20 H<sup>+</sup> + 20 e<sup>−</sup> + 40 MgATP → 2 NH<sub>3</sub> + 7 H<sub>2</sub> + 40 MgADP + 40 P<sub>i</sub><ref name=Schmidt24/>|{{EquationRef|3}}}}

Recent refinements to the kinetic framework of Mo-nitrogenase (1) suggest that the minimum energetic cost of N<sub>2</sub> reduction is higher than previously assumed, corresponding to approximately 25 MgATP per N<sub>2</sub>. This revision is based on the observation that electron transfer from the Fe protein to the FeMo cofactor is not always productive, as MgATP dependent conformational gating introduces a significant number of unproductive electron transfer cycles. This diminishes the overall efficiency of coupling between ATP hydrolysis and substrate reduction, consequently increasing the total ATP requirement for catalysis.<ref>{{Cite journal |last=Harris |first=Derek F. |last2=Dean |first2=Dennis R. |last3=Hoffman |first3=Brian M. |last4=Raugei |first4=Simone |last5=Seefeldt |first5=Lance C. |date=2025-10-15 |title=Toward a Unified Kinetic Model of Nitrogenase Catalysis |url=https://doi.org/10.1021/acscatal.5c05136 |journal=ACS Catalysis |pages=17893–17908 |doi=10.1021/acscatal.5c05136|url-access=subscription }}</ref> 

All nitrogenases are two-component systems made up of Component I (also known as dinitrogenase) and Component II (also known as dinitrogenase reductase). Component I is a MoFe protein in molybdenum nitrogenase, a VFe protein in vanadium nitrogenase, and an Fe protein in iron-only nitrogenase.<ref name="Hoffman_2014" /> Component II is a Fe protein that contains the Fe-S cluster., which transfers electrons to Component I.<ref name="Schneider_2004" /> Component I contains 2 metal clusters: the P-cluster, and the [[FeMoco|FeMo-cofactor]] (FeMo-co, M-cluster). Mo is replaced by V or Fe in vanadium nitrogenase and iron-only nitrogenase respectively.<ref name="Hoffman_2014" /><ref>{{Cite journal |last1=Schmidt |first1=Frederik V. |last2=Schulz |first2=Luca |last3=Zarzycki |first3=Jan |last4=Prinz |first4=Simone |last5=Oehlmann |first5=Niels N. |last6=Erb |first6=Tobias J. |last7=Rebelein |first7=Johannes G. |date=2023-12-07 |title=Structural insights into the iron nitrogenase complex |journal=Nature Structural & Molecular Biology |volume=31 |issue=1 |language=en |pages=150–158 |doi=10.1038/s41594-023-01124-2 |issn=1545-9985|doi-access=free |pmid=38062208 |pmc=10803253 }}</ref> During catalysis, 2 equivalents of MgATP are hydrolysed which helps to decrease the potential of the to the Fe-S cluster and drive reduction of the P-cluster, and finally to the FeMo-co, where reduction of N<sub>2</sub> to NH<sub>3</sub> takes place.

=== Lowe-Thorneley kinetic model ===
The reduction of nitrogen to two molecules of ammonia is carried out at the FeMo-co of Component I after the sequential addition of proton and electron equivalents from Component II.<ref name="Hoffman_2014" /> [[Steady state (chemistry)|Steady state]], freeze quench, and [[stopped-flow]] kinetics measurements carried out in the 70's and 80's by Lowe, Thorneley, and others provided a kinetic basis for this process.<ref name="Burgess_1996">{{cite journal |vauthors= Burgess BK, Lowe DJ |title= Mechanism of Molybdenum Nitrogenase |journal= Chemical Reviews |volume= 96 |issue=7 |pages= 2983–3012 |date= November 1996 |pmid= 11848849 |doi= 10.1021/cr950055x}}</ref><ref name="Wilson_2001">{{cite journal |vauthors= Wilson PE, Nyborg AC, Watt GD |title= Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform |journal= Biophysical Chemistry |volume= 91 |issue=3 |pages= 281–304 |date= July 2001 |pmid= 11551440 |doi= 10.1016/S0301-4622(01)00182-X }}</ref> The Lowe-Thorneley (LT) kinetic model was developed from these experiments and documents the eight correlated proton and electron transfers required throughout the reaction.<ref name="Hoffman_2014" /><ref name="Burgess_1996" /><ref name="Wilson_2001" /> Each intermediate stage is depicted as E<sub>n</sub> where n = 0–8, corresponding to the number of equivalents transferred. The transfer of four equivalents are required before the productive addition of N<sub>2</sub>, although reaction of E<sub>3</sub> with N<sub>2</sub> is also possible.<ref name="Burgess_1996" /> Notably, nitrogen reduction has been shown to require 8 equivalents of protons and electrons as opposed to the 6 equivalents predicted by the balanced chemical reaction.<ref>{{cite journal |vauthors= Simpson FB, Burris RH |title= A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase  |journal= Science |volume= 224 |issue= 4653 |pages= 1095–7 |date= June 1984 |pmid= 6585956 |doi= 10.1126/science.6585956 |bibcode= 1984Sci...224.1095S }}</ref>

=== Intermediates E<sub>0</sub> through E<sub>4</sub> ===
Spectroscopic characterization of these intermediates has allowed for greater understanding of nitrogen reduction by nitrogenase, however, the mechanism remains an active area of research and debate. Briefly listed below are spectroscopic experiments for the intermediates before the addition of nitrogen:

'''E<sub>0</sub>''' – This is the resting state of the enzyme before catalysis begins. [[Electron paramagnetic resonance]] (EPR) characterization shows that this species has a spin of <sup>3</sup>/<sub>2</sub>.<ref>{{cite journal | vauthors = Barney BM, Lee HI, Dos Santos PC, Hoffman BM, Dean DR, Seefeldt LC | title = Breaking the N2 triple bond: insights into the nitrogenase mechanism  | journal = Dalton Transactions | issue = 19 | pages = 2277–84 | date = May 2006 | pmid = 16688314 | doi = 10.1039/B517633F }}</ref>

'''E<sub>1</sub>''' – The one electron reduced intermediate has been trapped during turnover under N<sub>2</sub>. [[Mössbauer spectroscopy|Mӧssbauer]] spectroscopy of the trapped intermediate indicates that the FeMo-co is integer spin greater than 1.<ref>{{cite journal | last1 = Yoo | first1 = Sun Jae | last2 = Angove | first2 = Hayley C. | last3 = Papaefthymiou | first3 = Vasilios | last4 = Burgess | first4 = Barbara K. | last5 = Münck | first5 = Eckard | name-list-style = vanc | date = May 2000 | title = Mössbauer Study of the MoFe Protein of Nitrogenase from Azotobacter vinelandii Using Selective 57Fe Enrichment of the M-Centers | journal = Journal of the American Chemical Society | volume = 122 | issue = 20 | pages = 4926–4936 | doi = 10.1021/ja000254k | bibcode = 2000JAChS.122.4926Y }}</ref>
[[File:Lowe-Thorneley_Kinetic_Model.jpg|thumb|396x396px|Lowe-Thorneley kinetic model for reduction of nitrogen to ammonia by nitrogenase.]]
'''E<sub>2</sub>''' – This intermediate is proposed to contain the metal cluster in its resting oxidation state with the two added electrons stored in a bridging [[hydride]] and the additional proton bonded to a sulfur atom. Isolation of this intermediate in mutated enzymes shows that the FeMo-co is high spin and has a spin of <sup>3</sup>/<sub>2</sub>.<ref>{{cite journal | vauthors = Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM | title = Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state  | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 5 | pages = 1451–5 | date = January 2007 | pmid = 17251348 | pmc = 1785236 | doi = 10.1073/pnas.0610975104 | bibcode = 2007PNAS..104.1451L | doi-access = free }}</ref>

'''E<sub>3</sub>''' – This intermediate is proposed to be the singly reduced FeMo-co with one bridging hydride and one hydride.<ref name="Hoffman_2014" />

'''E<sub>4</sub>''' – Termed the Janus intermediate after the [[Janus|Roman god of transitions]], this intermediate is positioned after exactly half of the electron proton transfers and can either decay back to E<sub>0</sub> or proceed with nitrogen binding and finish the catalytic cycle. This intermediate is proposed to contain the FeMo-co in its resting oxidation state with two bridging hydrides and two sulfur bonded protons.<ref name="Hoffman_2014" /> This intermediate was first observed using freeze quench techniques with a mutated protein in which residue 70, a valine amino acid, is replaced with isoleucine.<ref name="Igarashi_2005">{{cite journal | vauthors = Igarashi RY, Laryukhin M, Dos Santos PC, Lee HI, Dean DR, Seefeldt LC, Hoffman BM | title = Trapping H- bound to the nitrogenase FeMo-cofactor active site during H2 evolution: characterization by ENDOR spectroscopy | journal = Journal of the American Chemical Society | volume = 127 | issue = 17 | pages = 6231–41 | date = May 2005 | pmid = 15853328 | doi = 10.1021/ja043596p | bibcode = 2005JAChS.127.6231I }}</ref> This modification prevents substrate access to the FeMo-co.  EPR characterization of this isolated intermediate shows a new species with a spin of ½. [[Electron nuclear double resonance]] (ENDOR) experiments have provided insight into the structure of this intermediate, revealing the presence of two bridging hydrides.<ref name="Igarashi_2005" />  <sup>95</sup>Mo and <sup>57</sup>Fe ENDOR show that the hydrides bridge between two iron centers.<ref>{{cite journal | vauthors = Doan PE, Telser J, Barney BM, Igarashi RY, Dean DR, Seefeldt LC, Hoffman BM | title = 57Fe ENDOR spectroscopy and 'electron inventory' analysis of the nitrogenase E4 intermediate suggest the metal-ion core of FeMo-cofactor cycles through only one redox couple | journal = Journal of the American Chemical Society | volume = 133 | issue = 43 | pages = 17329–40 | date = November 2011 | pmid = 21980917 | pmc = 3232045 | doi = 10.1021/ja205304t | bibcode = 2011JAChS.13317329D }}</ref> Cryoannealing of the trapped intermediate at -20&nbsp;°C results in the successive loss of two hydrogen equivalents upon relaxation, proving that the isolated intermediate is consistent with the E<sub>4</sub> state.<ref name="Hoffman_2014" /> The decay of E<sub>4</sub> to E<sub>2</sub> + H<sub>2</sub> and finally to E<sub>0</sub> and 2H<sub>2</sub> has confirmed the EPR signal associated with the E<sub>2</sub> intermediate.<ref name="Hoffman_2014" />

The above intermediates suggest that the metal cluster is cycled between its original oxidation state and a singly reduced state with additional electrons being stored in hydrides. It has alternatively been proposed that each step involves the formation of a hydride and that the metal cluster actually cycles between the original oxidation state and a singly oxidized state.<ref name="Hoffman_2014" />

=== Distal and alternating pathways for N<sub>2</sub> fixation ===
[[File:N2-fixation-mech.jpg|alt=|thumb|500x500px|Distal vs. alternating mechanistic pathways for nitrogen fixation in nitrogenase.]]
While the mechanism for nitrogen fixation prior to the Janus E<sub>4</sub> complex is generally agreed upon, there are currently two hypotheses for the exact pathway in the second half of the mechanism: the "distal" and the "alternating" pathway.<ref name="Hoffman_2014" /><ref name="Neese_2005">{{cite journal | vauthors = Neese F | title = The Yandulov/Schrock cycle and the nitrogenase reaction: pathways of nitrogen fixation studied by density functional theory  | journal = Angewandte Chemie | volume = 45 | issue = 2 | pages = 196–9 | date = December 2005 | pmid = 16342309 | doi = 10.1002/anie.200502667 }}</ref><ref name="Hinnemann_2008">{{cite journal|last1=Hinnemann|first1=Berit|last2=Nørskov|first2=Jens K. | name-list-style = vanc | title = Catalysis by Enzymes: The Biological Ammonia Synthesis | journal=Topics in Catalysis|volume=37|issue=1|pages=55–70 | date = 2008 | doi =10.1007/s11244-006-0002-0 |s2cid=93357657}}</ref> In the distal pathway, the terminal nitrogen is hydrogenated first, releases ammonia, then the nitrogen directly bound to the metal is hydrogenated. In the alternating pathway, one hydrogen is added to the terminal nitrogen, then one hydrogen is added to the nitrogen directly bound to the metal. This alternating pattern continues until ammonia is released.<ref name="Hoffman_2014" /><ref name="Neese_2005" /><ref name="Hinnemann_2008" /> Because each pathway favors a unique set of intermediates, attempts to determine which path is correct have generally focused on the isolation of said intermediates, such as the [[Metal nitrido complex|nitrido]] in the distal pathway,<ref name="Schrock_2005">{{cite journal | vauthors = Schrock RR | title = Catalytic reduction of dinitrogen to ammonia at a single molybdenum center | journal = Accounts of Chemical Research | volume = 38 | issue = 12 | pages = 955–62 | date = December 2005 | pmid = 16359167 | pmc = 2551323 | doi = 10.1021/ar0501121 }}</ref> and the [[diazene]] and [[hydrazine]] in the alternating pathway.<ref name="Hoffman_2014" /> Attempts to isolate the intermediates in nitrogenase itself have so far been unsuccessful, but the use of model complexes has allowed for the isolation of intermediates that support both sides depending on the metal center used.<ref name="Hoffman_2014" /> Studies with [[Molybdenum|Mo]] generally point towards a distal pathway, while studies with Fe generally point towards an alternating pathway.<ref name="Hoffman_2014" /><ref name="Neese_2005" /><ref name="Hinnemann_2008" /><ref name="Rodriguez_2011">{{cite journal | vauthors = Rodriguez MM, Bill E, Brennessel WW, Holland PL | title = N₂reduction and hydrogenation to ammonia by a molecular iron-potassium complex  | journal = Science | volume = 334 | issue = 6057 | pages = 780–3 | date = November 2011 | pmid = 22076372 | pmc = 3218428 | doi = 10.1126/science.1211906 | bibcode = 2011Sci...334..780R }}</ref><ref name="Anderson_2013">{{cite journal | vauthors = Anderson JS, Rittle J, Peters JC | title = Catalytic conversion of nitrogen to ammonia by an iron model complex  | journal = Nature | volume = 501 | issue = 7465 | pages = 84–7 | date = September 2013 | pmid = 24005414 | pmc = 3882122 | doi = 10.1038/nature12435 | bibcode = 2013Natur.501...84A }}</ref>

Specific support for the distal pathway has mainly stemmed from the work of Schrock and Chatt, who successfully isolated the nitrido complex using Mo as the metal center in a model complex.<ref name="Schrock_2005" /><ref>{{cite journal | vauthors = Chatt J, Dilworth JR, Richards RL | title = Recent advances in chemistry of nitrogen-fixation | journal = Chem. Rev. | date = 1978 | volume = 78 | issue = 6 | pages = 589–625 | doi=10.1021/cr60316a001}}</ref> Specific support for the alternating pathway stems from a few studies. Iron only model clusters have been shown to catalytically reduce N<sub>2</sub>.<ref name="Rodriguez_2011" /><ref name="Anderson_2013" /> Small [[tungsten]] clusters have also been shown to follow an alternating pathway for nitrogen fixation.<ref>{{cite journal | vauthors = Murakami J, Yamaguchi W | title = Reduction of N2 by supported tungsten clusters gives a model of the process by nitrogenase  | journal = Scientific Reports | volume = 2 | article-number = 407 | date = 2012-05-14 | pmid = 22586517 | pmc = 3350986 | doi = 10.1038/srep00407 | bibcode = 2012NatSR...2..407M }}</ref> The vanadium nitrogenase releases hydrazine, an intermediate specific to the alternating mechanism.<ref name="Hoffman_2014" /><ref>{{cite journal | vauthors = Dilworth MJ, Eady RR | title = Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum  | journal = The Biochemical Journal | volume = 277 | issue = 2 | pages = 465–8 | date = July 1991 | pmid = 1859374 | doi = 10.1042/bj2770465 | pmc=1151257}}</ref> However, the lack of characterized intermediates in the native enzyme itself means that neither pathway has been definitively proven. Furthermore, computational studies have been found to support both sides, depending on whether the reaction site is assumed to be at Mo (distal) or at Fe (alternating) in the MoFe cofactor.<ref name="Hoffman_2014" /><ref name="Neese_2005" /><ref name="Hinnemann_2008" />

=== Mechanism of MgATP binding ===
Binding of MgATP is one of the central events to occur in the mechanism employed by nitrogenase. [[Hydrolysis]] of the terminal [[phosphate]] group of MgATP provides the energy needed to transfer electrons from the Fe protein to the MoFe protein.<ref>{{cite journal |vauthors= Hageman RV, Burris RH |title= Nitrogenase and nitrogenase reductase associate and dissociate with each catalytic cycle |journal= Proceedings of the National Academy of Sciences of the United States of America |volume=75 |issue=6 |pages= 2699–702 |date= June 1978 |pmid= 275837 |pmc= 392630 |doi= 10.1073/pnas.75.6.2699|bibcode= 1978PNAS...75.2699H |doi-access= free }}</ref> The binding interactions between the MgATP phosphate groups and the [[amino acid]] residues of the Fe protein are well understood by comparing to similar enzymes, while the interactions with the rest of the molecule are more elusive due to the lack of a Fe protein crystal structure with MgATP bound (as of 1996).<ref name="Georgiadis_1992">{{cite journal | vauthors = Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ, Rees DC | title = Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii | journal = Science | volume = 257 | issue = 5077 | pages = 1653–9 | date = September 1992 | pmid = 1529353 | doi=10.1126/science.1529353| bibcode = 1992Sci...257.1653G }}</ref> Three protein residues have been shown to have significant interactions with the phosphates.<ref name="Burgess_1996" /> In the absence of MgATP, a [[Salt bridge (protein and supramolecular)|salt bridge]] exists between residue 15, [[lysine]], and residue 125, [[aspartic acid]].<ref name="Georgiadis_1992" /> Upon binding, this salt bridge is interrupted. [[Site-directed mutagenesis|Site-specific mutagenesis]] has demonstrated that when the lysine is substituted for a [[glutamine]], the protein's affinity for MgATP is greatly reduced<ref>{{cite journal | vauthors = Seefeldt LC, Morgan TV, Dean DR, Mortenson LE | title = Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction  | journal = The Journal of Biological Chemistry | volume = 267 | issue = 10 | pages = 6680–8 | date = April 1992 | doi = 10.1016/S0021-9258(19)50480-X | pmid = 1313018 | doi-access = free }}</ref> and when the lysine is substituted for an [[arginine]], MgATP cannot bind due to the salt bridge being too strong.<ref>{{cite journal | vauthors = Ryle MJ, Lanzilotta WN, Mortenson LE, Watt GD, Seefeldt LC | title = Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes  | journal = The Journal of Biological Chemistry | volume = 270 | issue = 22 | pages = 13112–7 | date = June 1995 | pmid = 7768906 | doi = 10.1074/jbc.270.22.13112 | doi-access = free }}</ref> The necessity of specifically aspartic acid at site 125 has been shown through noting altered reactivity upon mutation of this residue to [[glutamic acid]].<ref name="Wolle_1992">{{cite journal | vauthors = Wolle D, Dean DR, Howard JB | title = Nucleotide-iron-sulfur cluster signal transduction in the nitrogenase iron-protein: the role of Asp125  | journal = Science | volume = 258 | issue = 5084 | pages = 992–5 | date = November 1992 | pmid = 1359643 | doi = 10.1126/science.1359643 | bibcode = 1992Sci...258..992W }}</ref> Residue 16, serine, has been shown to bind MgATP. Site-specific mutagenesis was used to demonstrate this fact.<ref name="Wolle_1992" /> This has led to a model in which the serine remains coordinated to the Mg<sup>2+</sup> ion after phosphate hydrolysis in order to facilitate its association with a different phosphate of the now ADP molecule.<ref>{{Cite journal|year=1962|title=Nuclear Magnetic Resonance Spectra of Adenosine Di- and Triphosphate | journal = Journal of Biological Chemistry|volume=237|pages=176–181|doi=10.1016/S0021-9258(18)81382-5 |doi-access=free |last1=Cohn |first1=Mildred |last2=Hughes |first2=Thomas R. }}</ref> MgATP binding also induces significant conformational changes within the Fe protein.<ref name="Burgess_1996" /> Site-directed mutagenesis was employed to create mutants in which MgATP binds but does not induce a conformational change.<ref name="Chen_1994">{{cite journal | vauthors = Chen L, Gavini N, Tsuruta H, Eliezer D, Burgess BK, Doniach S, Hodgson KO | title = MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering  | journal = The Journal of Biological Chemistry | volume = 269 | issue = 5 | pages = 3290–4 | date = February 1994 | doi = 10.1016/S0021-9258(17)41861-8 | pmid = 8106367 | doi-access = free }}</ref> Comparing [[X-ray scattering techniques|X-ray scattering]] data in the mutants versus in the wild-type protein led to the conclusion that the entire protein contracts upon MgATP binding, with a decrease in radius of approximately 2.0 Å.<ref name="Chen_1994" />

=== Other mechanistic details ===
Many mechanistic aspects of [[catalysis]] remain unknown.  No crystallographic analysis has been reported on substrate bound to nitrogenase.

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]].<ref>{{cite journal | vauthors = Seefeldt LC, Dance IG, Dean DR | title = Substrate interactions with nitrogenase: Fe versus Mo | journal = Biochemistry | volume = 43 | issue = 6 | pages = 1401–9 | date = February 2004 | pmid = 14769015 | doi = 10.1021/bi036038g }}</ref> Due to the oxidative properties of [[oxygen]], most nitrogenases are irreversibly inhibited by [[dioxygen]], which degradatively oxidizes the Fe-S cofactors.{{citation needed|date=February 2017}} 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.{{citation needed|date=February 2017}} 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 O<sub>2</sub>), as well as during additional nutrient limitations.<ref>{{cite journal | vauthors = Oelze J | title = Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence? | journal = FEMS Microbiology Reviews | volume = 24 | issue = 4 | pages = 321–33 | date = October 2000 | pmid = 10978541 | doi = 10.1111/j.1574-6976.2000.tb00545.x | doi-access = free }}</ref> MoFe-nitrogenase in ''[[Azotobacter vinelandii]]'' is protected from oxidative damage by a small ferredoxin-like protein called Shethna protein II (FeSII). Under [[oxidative stress]], the [Fe<sub>2</sub>-S<sub>2</sub>] cluster in FeSII becomes oxidized, triggering the formation of a filamentous complex involving FeSII and the Fe and MoFe subunits of nitrogenase. This complex locks the nitrogenase in an inactive, yet conformationally protected, state. This reversible “switch-off” mechanism, driven by a redox-sensitive conformational change in FeSII, is a key strategy the organism uses to shield nitrogenase from oxygen damage.<ref>{{Cite journal |last=Franke |first=Philipp |last2=Freiberger |first2=Simon |last3=Zhang |first3=Lin |last4=Einsle |first4=Oliver |date=2025-01-23 |title=Conformational protection of molybdenum nitrogenase by Shethna protein II |url=https://www.nature.com/articles/s41586-024-08355-3 |journal=Nature |language=en |volume=637 |issue=8047 |pages=998–1004 |doi=10.1038/s41586-024-08355-3 |issn=0028-0836 |pmc=11754109 |pmid=39779845}}</ref><ref>{{Cite journal |last=Schlesier |first=Julia |last2=Rohde |first2=Michael |last3=Gerhardt |first3=Stefan |last4=Einsle |first4=Oliver |date=2016-01-13 |title=A Conformational Switch Triggers Nitrogenase Protection from Oxygen Damage by Shethna Protein II (FeSII) |url=https://pubs.acs.org/doi/10.1021/jacs.5b10341 |journal=Journal of the American Chemical Society |language=en |volume=138 |issue=1 |pages=239–247 |doi=10.1021/jacs.5b10341 |issn=0002-7863|url-access=subscription }}</ref> A molecule found in the nitrogen-fixing nodules of leguminous plants, [[leghemoglobin]], which can bind to dioxygen via a [[heme]] prosthetic group, plays a crucial role in buffering O<sub>2</sub> at the active site of the nitrogenase, while concomitantly allowing for efficient respiration.<ref>{{Cite journal |last1=Ott |first1=Thomas |last2=van Dongen |first2=Joost T. |last3=Gu¨nther |first3=Catrin |last4=Krusell |first4=Lene |last5=Desbrosses |first5=Guilhem |last6=Vigeolas |first6=Helene |last7=Bock |first7=Vivien |last8=Czechowski |first8=Tomasz |last9=Geigenberger |first9=Peter |last10=Udvardi |first10=Michael K. |date=March 29, 2005 |title=Symbiotic Leghemoglobins Are Crucial for Nitrogen Fixation in Legume Root Nodules but Not for General Plant Growth and Development |journal=Current Biology |volume=15 |issue=6 |pages=531–535 |doi=10.1016/j.cub.2005.01.042 |pmid=15797021 |bibcode=2005CBio...15..531O |issn=0960-9822 |quote=Physiological analysis of nodules from LbRNAi plants revealed the crucial contribution of leghemoglobins to establishing low free-oxygen concentrations but high energy status in nodules, conditions that are necessary for effective SNF.}}</ref>

==Nonspecific reactions==
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:<ref name=Burris1>{{cite journal | vauthors = Rivera-Ortiz JM, Burris RH | title = Interactions among substrates and inhibitors of nitrogenase | journal = Journal of Bacteriology | volume = 123 | issue = 2 | pages = 537–45 | date = August 1975 | pmid = 1150625 | pmc = 235759 | doi =  10.1128/JB.123.2.537-545.1975}}</ref><ref name=Schrauzer>{{cite journal | vauthors = Schrauzer GN | title = Nonenzymatic simulation of nitrogenase reactions and the mechanism of biological nitrogen fixation | journal = Angewandte Chemie | volume = 14 | issue = 8 | pages = 514–22 | date = August 1975 | pmid = 810048 | doi = 10.1002/anie.197505141 }}</ref>
:[[acetylene|HC≡CH]] → [[ethylene|H<sub>2</sub>C=CH<sub>2</sub>]]
:[[nitrous oxide|N<sup>&ndash;</sup>=N<sup>+</sup>=O]] → N<sub>2</sub> + H<sub>2</sub>O
:[[azide|N=N=N<sup>&ndash;</sup>]] → N<sub>2</sub> + NH<sub>3</sub>
:[[cyanide|{{chem|C≡N|-}}]] → [[methane|CH<sub>4</sub>]], NH<sub>3</sub>, [[ethane|H<sub>3</sub>C–CH<sub>3</sub>]], H<sub>2</sub>C=CH<sub>2</sub> (CH<sub>3</sub>NH<sub>2</sub>)
:N≡C–R → RCH<sub>3</sub> + NH<sub>3</sub>
:C≡N–R → CH<sub>4</sub>, H<sub>3</sub>C–CH<sub>3</sub>, H<sub>2</sub>C=CH<sub>2</sub>, [[propane|C<sub>3</sub>H<sub>8</sub>]], [[propylene|C<sub>3</sub>H<sub>6</sub>]], RNH<sub>2</sub>
:[[Carbonyl sulfide|O=C=S]] → [[carbon monoxide|CO]] + [[Hydrogen sulfide|H<sub>2</sub>S]]<ref name=Seefeldt1>{{cite journal | vauthors = Seefeldt LC, Rasche ME, Ensign SA | title = Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase | journal = Biochemistry | volume = 34 | issue = 16 | pages = 5382–9 | date = April 1995 | pmid = 7727396 | doi = 10.1021/bi00016a009 }}</ref><ref name="Seefeldt2" />
:[[carbon dioxide|O=C=O]] → CO + H<sub>2</sub>O <ref name="Seefeldt1" />
:[[thiocyanate|S=C=N<sup>&ndash;</sup>]] → H<sub>2</sub>S + HCN <ref name=Seefeldt2>{{cite journal | vauthors = Rasche ME, Seefeldt LC | title = Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: kinetic characterization and EPR spectroscopic analysis | journal = Biochemistry | volume = 36 | issue = 28 | pages = 8574–85 | date = July 1997 | pmid = 9214303 | doi = 10.1021/bi970217e }}</ref>
:[[Cyanate|O=C=N<sup>&ndash;</sup>]] → H<sub>2</sub>O + [[Hydrogen cyanide|HCN]], CO + NH<sub>3</sub><ref name="Seefeldt2" />

Furthermore, [[dihydrogen]] functions as a [[competitive inhibition|competitive inhibitor]],<ref name=Burris2>{{cite journal | vauthors = Guth JH, Burris RH | title = Inhibition of nitrogenase-catalyzed NH3 formation by H2 | journal = Biochemistry | volume = 22 | issue = 22 | pages = 5111–22 | date = October 1983 | pmid = 6360203 | doi = 10.1021/bi00291a010 }}</ref> [[carbon monoxide]] functions as a [[Non-competitive inhibition|non-competitive inhibitor]],<ref name="Burris1" /><ref name="Schrauzer" /> and [[carbon disulfide]] functions as a [[Enzyme inhibitor#Reversible inhibitors|rapid-equilibrium inhibitor]]<ref name="Seefeldt1" /> of nitrogenase.

[[Vanadium nitrogenase]]s have also been shown to catalyze the conversion of CO into [[alkanes]] through a reaction comparable to [[Fischer–Tropsch process|Fischer-Tropsch synthesis]].

==Organisms that synthesize nitrogenase==

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

* Free-living bacteria (non-[[symbiotic]]), examples include:
** [[Cyanobacteria]] (blue-green algae)
** [[Green sulfur bacteria]]
** ''[[Azotobacter]]''
* Mutualistic bacteria (symbiotic), examples include:
** ''[[Rhizobium]]'', associated with [[legumes]]
** ''[[Azospirillum]]'', associated with [[grass]]es
** ''[[Frankia]]'', associated with [[actinorhizal plant]]s

== Evolution ==

=== Internal evolution ===
Nitrogenases are divided into three groups, clades, or classes, named using roman numerals I through III. The alternative nitrogenases are nested in class III. The same grouping is recovered from sequence comparison,<ref name=Garcia23>{{cite journal |last1=Garcia |first1=Amanda K |last2=Harris |first2=Derek F |last3=Rivier |first3=Alex J |last4=Carruthers |first4=Brooke M |last5=Pinochet-Barros |first5=Azul |last6=Seefeldt |first6=Lance C |last7=Kaçar |first7=Betül |title=Nitrogenase resurrection and the evolution of a singular enzymatic mechanism |journal=eLife |date=17 February 2023 |volume=12 |doi=10.7554/eLife.85003|doi-access=free |pmid=36799917 |bibcode=2023eLife..1285003G |pmc=9977276 }}</ref> as well as comparison of [[AlphaFold]]2-predicted structures. Nif-I is primarily found in aerobic or at least facultatively anaerobic diazotrophs with large Nif gene families, while the two other types are almost exclusively found in anaerobic diazotrophs with smaller gene networks.<ref>{{cite journal |last1=Cuevas-Zuviría |first1=Bruno |last2=Detemple |first2=Franka |last3=Amritkar |first3=Kaustubh |last4=Garcia |first4=Amanda K. |last5=Seefeldt |first5=Lance C. |last6=Einsle |first6=Oliver |last7=Kaçar |first7=Betül |title=Nitrogenase structural evolution across Earth's history |journal=eLife |date=9 April 2025 |volume=14 |doi=10.7554/eLife.105613.1 |doi-access=free |language=en}}</ref> (The alternative nitrogenases break this pattern as they are found to co-occur with Group I and II.)<ref name=Pi22/> Group I cannot have diverged more than 2.5 [[gigayear]]s ago based on the timing of the [[Great Oxidation Event]].<ref name=Garcia23/>

[[Ancestral sequence reconstruction]] has been used to reconstruct two group I nitrogenases, Anc2 representing the ancestor to all sampled nitrogenases in [[Gammaproteobacteria]] and Anc1 representing a smaller group built around ''Azotobacter vinelandii'', ''Agaribacterium haliotis'', and environmental samples. They work more slowly than the modern ''A. vinelandii'' version but keeps a similar efficiency in ATP use (the ratio between formed H<sub>2</sub> and reduced N<sub>2</sub> is largely unchanged at around 2.1, with the exception of Anc1B which is more efficient.)<ref name=Garcia23/>

===Similarity to other proteins===
The [[Nif gene]]s include a maturase ''NifEN'' responsible for assembling the precursor to the P-cluster called an O-cluster and transferring it onto ''NifDK''. It is also where the M-cluster is assembled with the help of nitrogenase component II ''NifH'', before it is transferred to ''NifDK''. Its structure is rather similar to the nitrogenase component I ''NifDK'', except that it usually carries a P-cluster and a L-cluster (the precursor to the M-cluster). When ''NifEN'' from ''A. vinelandii'' is expressed in ''E. coli'' with the component II ''NifH'', the resulting combination of proteins prove to work as a nitrogenase ''in vivo'', boosting the growth of transformed ''E. coli'' in a nitrogen-deficient medium. The ''YfhL'' ferredoxin naturally present in ''E. coli'' is able to work with ''NifH''.<ref>{{cite journal |last1=Liu |first1=Yiling A. |last2=Lee |first2=Chi Chung |last3=Górecki |first3=Kamil |last4=Stiebritz |first4=Martin T. |last5=Duffin |first5=Calder |last6=Solomon |first6=Joseph B. |last7=Ribbe |first7=Markus W. |last8=Hu |first8=Yilin |title=Heterologous synthesis of a simplified nitrogenase analog in Escherichia coli |journal=Science Advances |date=2 May 2025 |volume=11 |issue=18 |article-number=eadw6785 |doi=10.1126/sciadv.adw6785|pmid=40315313 |bibcode=2025SciA...11.6785L |doi-access=free |pmc=12047441 }}</ref> Among organisms carrying a Group III ''Nif'', ''NifN'' is only found in archaea. The reason is unclear.<ref name="Pi22"/>

One interpretation of the nitrogen-fixing ability of ''NifEN'' and its similarity to ''NifDK'' is that there used to be a single nitrogenase with P- and L- clusters, before [[gene duplication]] and increased amounts of available Mo after the [[Great Oxidation Event]] allowed for the current situation to evolve (one copy became ''NifEN'' with the gain of M-cluster, the other became ''NifDK'' with the loss of P-cluster). Isotopic data suggests that Mo-based nitrogen fixation is no younger than 3.2 [[gigayear]]s old. There is also a similarity between the α and β subunits in nitrogenase, its maturase, and related proteins; the two halves might have been a single gene of unknown function to begin with and became duplicated before nitrogenase diverged from BChNB.<ref>{{cite journal |last1=Lee |first1=Chi Chung |last2=Górecki |first2=Kamil |last3=Stang |first3=Martin |last4=Ribbe |first4=Markus W. |last5=Hu |first5=Yilin |title=Cofactor maturase NifEN: A prototype ancient nitrogenase? |journal=Science Advances |date=14 June 2024 |volume=10 |issue=24 |article-number=eado6169 |doi=10.1126/sciadv.ado6169|pmid=38865457 |pmc=11168457 |bibcode=2024SciA...10O6169L }}</ref> A large 2022 study mostly supports the view about ''NifEN'' and ''NifDK'' being formed by a duplication.<ref name=Pi22/>

The three subunits of nitrogenase exhibit significant sequence similarity to three subunits of the light-independent version of [[protochlorophyllide reductase]] (DPOR, (ChlNB)<sub>2</sub> + ChlL<sub>2</sub>) that performs the conversion of [[protochlorophyllide]] to [[chlorophyll]]. This protein is present in [[gymnosperm]]s, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution. The structual similarity puts it in the same superfamily as nitrogenase.<ref>{{cite journal | vauthors = Li J, Goldschmidt-Clermont M, Timko MP | title = Chloroplast-encoded chlB is required for light-independent protochlorophyllide reductase activity in Chlamydomonas reinhardtii | journal = The Plant Cell | volume = 5 | issue = 12 | pages = 1817–29 | date = December 1993 | pmid = 8305874 | pmc = 160407 | doi = 10.1105/tpc.5.12.1817 }}</ref> The enzyme for [[bacteriochlorophyll]] is similar and is called [[chlorophyllide oxidoreductase]] (COR, (BchNB)<sub>2</sub> + BchL<sub>2</sub> or (BchYZ)<sub>2</sub> + BchX<sub>2</sub>).<ref>{{cite journal |last1=Wätzlich |first1=D |last2=Bröcker |first2=MJ |last3=Uliczka |first3=F |last4=Ribbe |first4=M |last5=Virus |first5=S |last6=Jahn |first6=D |last7=Moser |first7=J |title=Chimeric nitrogenase-like enzymes of (bacterio)chlorophyll biosynthesis. |journal=The Journal of Biological Chemistry |date=5 June 2009 |volume=284 |issue=23 |pages=15530–40 |doi=10.1074/jbc.M901331200 |doi-access=free |pmid=19336405 |pmc=2708849}}</ref>

Separately, two of the nitrogenase subunits (NifD and NifH) have homologues in [[methanogen]]s that do not fix nitrogen e.g. ''[[Methanocaldococcus jannaschii]]''.<ref name=jannaschii>{{cite journal | vauthors = Staples CR, Lahiri S, Raymond J, Von Herbulis L, Mukhophadhyay B, Blankenship RE | title = Expression and association of group IV nitrogenase NifD and NifH homologs in the non-nitrogen-fixing archaeon Methanocaldococcus jannaschii | journal = Journal of Bacteriology | volume = 189 | issue = 20 | pages = 7392–8 | date = October 2007 | pmid = 17660283 | pmc = 2168459 | doi = 10.1128/JB.00876-07 }}</ref> Little is understood about the function of these "class IV" ''nif'' genes as of 2004,<ref>{{cite journal | vauthors = Raymond J, Siefert JL, Staples CR, Blankenship RE|author4-link=Robert E. Blankenship | title = The natural history of nitrogen fixation | journal = Molecular Biology and Evolution | volume = 21 | issue = 3 | pages = 541–54 | date = March 2004 | pmid = 14694078 | doi = 10.1093/molbev/msh047 | doi-access = free }}</ref> though they occur in many methanogens. In ''M. jannaschii'' they are known to interact with each other and are constitutively expressed.<ref name=jannaschii /> The "group IV" ''Nif'' are closer to ''Nif''-I/II/III (the real nitrogenases) than DPOR and COR are. They do not have the DK vs EN duplication. The group has previously been considered paraphyletic, but a more recent analysis finds it [[monophyletic]]. There are two known kinds of functions: the ''CfbC'' type makes [[coenzyme F430]] and the ''Mar'' type can make methionine, ethylene, and methane.<ref name=Pi22>{{cite journal |last1=Pi |first1=Hong-Wei |last2=Lin |first2=Jinn-Jy |last3=Chen |first3=Chi-An |last4=Wang |first4=Po-Hsiang |last5=Chiang |first5=Yin-Ru |last6=Huang |first6=Chieh-Chen |last7=Young |first7=Chiu-Chung |last8=Li |first8=Wen-Hsiung |title=Origin and Evolution of Nitrogen Fixation in Prokaryotes |journal=Molecular Biology and Evolution |date=1 September 2022 |volume=39 |issue=9 |doi=10.1093/molbev/msac181|pmid=35993177 |pmc=9447857 }}</ref>

==Measurement of nitrogenase activity ==
As with many assays for enzyme activity, it is possible to estimate nitrogenase activity by measuring the rate of conversion of the substrate (N<sub>2</sub>) to the product (NH<sub>3</sub>).  Since NH<sub>3</sub> is involved in other reactions in the cell, it is often desirable to label the substrate with <sup>15</sup>N 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.<ref>{{cite journal | vauthors = Dilworth MJ | title = Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 127 | issue = 2 | pages = 285–94 | date = October 1966 | pmid = 5964974 | doi = 10.1016/0304-4165(66)90383-7 }}</ref>  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.<ref>{{cite journal | vauthors = Sims GK, Dunigan EP | year = 1984 | title = Diurnal and seasonal variations in nitrogenase activity (C<sub>2</sub>H<sub>2</sub> reduction) of rice roots.  | journal = Soil Biology and Biochemistry | volume = 16 | pages = 15–18 | doi=10.1016/0038-0717(84)90118-4}}</ref>

Unfortunately, the conversion of data from nitrogenase assays to actual moles of N<sub>2</sub> 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, H<sub>2</sub> competes with N<sub>2</sub> 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 ==
* [[Nitrogen fixation]]
* [[Abiological nitrogen fixation]]

== References ==
{{Reflist|33em}}

==Further reading==
{{refbegin}}
* {{cite journal | vauthors = Zumft WG, Mortenson LE | title = The nitrogen-fixing complex of bacteria | journal = Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics | volume = 416 | issue = 1 | pages = 1–52 | date = March 1975 | pmid = 164247 | doi = 10.1016/0304-4173(75)90012-9 }}
{{refend}}

==External links==
*{{Commons category-inline}}

{{Other oxidoreductases}}
{{Enzymes}}
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[[Category:EC 1.18.6]]
[[Category:Iron–sulfur proteins]]
[[Category:Nitrogen cycle]]
[[Category:Molybdenum enzymes]]