Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a step in the process of nitrogen fixation.

Although the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (

[1] 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 Fe protein, the dinitrogenase reductase or NifH, is a dimer of identical subunits which contains one [Fe4S4] cluster and has a mass of approximately 60-64kDa.

[5] (Molybdenum in other enzymes is generally bound to molybdopterin as fully oxidized Mo(VI)).

The MoFe protein can be replaced by alternative nitrogenases in environments low in the Mo cofactor.

[16][17] The Lowe-Thorneley (LT) kinetic model was developed from these experiments and documents the eight correlated proton and electron transfers required throughout the reaction.

[18] 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: E0 – This is the resting state of the enzyme before catalysis begins.

[20] E2 – 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.

This intermediate is proposed to contain the FeMo-co in its resting oxidation state with two bridging hydrides and two sulfur bonded protons.

[9] 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.

[27][28] Small tungsten clusters have also been shown to follow an alternating pathway for nitrogen fixation.

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.

[32] 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).

[16] Site-directed mutagenesis was employed to create mutants in which MgATP binds but does not induce a conformational change.

[citation needed] This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo.

[citation needed] 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.

[40] 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 O2 at the active site of the nitrogenase, while concomitantly allowing for efficient respiration.

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

This protein is present in gymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution.

[47] Separately, two of the nitrogenase subunits (NifD and NifH) have homologues in methanogens that do not fix nitrogen e.g. Methanocaldococcus jannaschii.

[50] 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.

[51] 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.

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.