| Nitrogenase | |||||||||
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| Identifiers | |||||||||
| EC no. | 1.18.6.1 | ||||||||
| CAS no. | 9013-04-1 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
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| Nitrogenase-type Oxidoreductase (component 1 subunit alpha/beta) | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Symbol | Oxidored_nitro | ||||||
| Pfam | PF00148 | ||||||
| InterPro | IPR000510 | ||||||
| SCOP2 | 1mio /SCOPe /SUPFAM | ||||||
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| Nitrogenase iron protein NifH (component 2) | |||||||
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| Identifiers | |||||||
| Symbol | NifH | ||||||
| InterPro | IPR005977 | ||||||
| CATH | 1fp6 | ||||||
| SCOP2 | d1fp6a_ /SCOPe /SUPFAM | ||||||
| CDD | cd02040 | ||||||
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| Alternative nitrogenase (component 1) delta subunit | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Symbol | AnfG_VnfG | ||||||
| Pfam | PF03139 | ||||||
| InterPro | IPR004349 | ||||||
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Nitrogenases areenzymes (EC1.18.6.1EC1.19.6.1) that are produced by certainbacteria, such ascyanobacteria (blue-green bacteria) andrhizobacteria. These enzymes are responsible for thereduction ofnitrogen (N2) toammonia (NH3). Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a step in the process ofnitrogen fixation. Nitrogen fixation is required for all forms of life, with nitrogen being essential for thebiosynthesis ofmolecules (nucleotides,amino acids) that create plants, animals and other organisms. They are encoded by theNif genes orhomologs. They are related toprotochlorophyllide reductase.
Although the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negativeenthalpy of reaction (), theactivation energy is very high ().[1] Nitrogenase acts as acatalyst, 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.[2] The function of the Fe protein is to transfer electrons from areducing agent, such asferredoxin orflavodoxin to the nitrogenase protein. Ferredoxin or flavodoxin can be reduced by one of six mechanisms: 1. by apyruvate:ferredoxin oxidoreductase, 2. by a bi-directionalhydrogenase, 3. in aphotosynthetic reaction center, 4. by couplingelectron flow to dissipation of theproton motive force, 5. byelectron bifurcation, or 6. by aferredoxin:NADPH oxidoreductase.[3] The transfer of electrons requires an input of chemical energy which comes from the binding and hydrolysis ofATP. 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.[4]
The MoFe protein is a heterotetramer consisting of two α subunits and two β subunits, with a mass of approximately 240-250kDa.[2] The MoFe protein also contains twoiron–sulfur clusters, known as P-clusters, located at the interface between the α and β subunits and twoFeMo cofactors, within the α subunits. The oxidation state of Mo in these nitrogenases was formerly thought Mo(V), but more recent evidence is for Mo(III).[5] (Molybdenum in other enzymes is generally bound tomolybdopterin as fully oxidized Mo(VI)).
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.
The MoFe protein can be replaced by alternative nitrogenases in environments low in the Mo cofactor. Two types of such nitrogenases are known: thevanadium–iron (VFe;Vnf) type and the iron–iron (FeFe;Anf) type. Both form an assembly of two α subunits, two β subunits, and two δ (sometimes γ: 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.[6][7] The δ/γ subunit helps bind the cofactor in the FeFe nitrogenase.[8] 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.[9]
Most, if not all, natural organisms carrying genes for an alternative nitrogenase also carry genes for the regular MoFe nitrogenase.[9][10] The MoFe nitrogenase is the most efficient in that it wastes less ATP on reducing H+ into H2 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.[11]
The FeFe nitrogenase inAzotobacter vinelandii (a model organism for nitrogenase engineering) is organized in ananfHDGKOR operon. This operon still requires some of theNif genes to function. A minimal 10-gene operon that incorporates these additional essential genes has been constructed in the lab.[12]
Nitrogenase is an enzyme responsible for catalyzingnitrogen fixation, which is the reduction of nitrogen (N2) to ammonia (NH3) and a process vital to sustaining life on Earth.[13] There are three types of nitrogenase found in various nitrogen-fixing bacteria: molybdenum (Mo) nitrogenase,vanadium (V) nitrogenase, and iron-only (Fe) nitrogenase.[14] Molybdenum nitrogenase, which can be found indiazotrophs such aslegume-associatedrhizobia,[15][16] is the nitrogenase that has been studied the most extensively and thus is the most well characterized.[14] Vanadium nitrogenase and iron-only nitrogenase can both be found in select species of Azotobacter as an alternative nitrogenase.[15][17] Equations 1 and 2 show the balanced reactions of nitrogen fixation in molybdenum nitrogenase and vanadium nitrogenase respectively.
| N2 + 8 H+ + 8 e− + 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16 Pi[13] | 1 |
| N2 + 18 H+ + 18 e− + 36 MgATP → 2 NH3 + 6 H2 + 36 MgADP + 36 Pi[8] | 2 |
| N2 + 20 H+ + 20 e− + 40 MgATP → 2 NH3 + 7 H2 + 40 MgADP + 40 Pi[8] | 3 |
Recent refinements to the kinetic framework of Mo-nitrogenase (1) suggest that the minimum energetic cost of N2 reduction is higher than previously assumed, corresponding to approximately 25 MgATP per N2. 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.[18]
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.[13] Component II is a Fe protein that contains the Fe-S cluster., which transfers electrons to Component I.[17] Component I contains 2 metal clusters: the P-cluster, and theFeMo-cofactor (FeMo-co, M-cluster). Mo is replaced by V or Fe in vanadium nitrogenase and iron-only nitrogenase respectively.[13][19] 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 N2 to NH3 takes place.
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.[13]Steady state, freeze quench, andstopped-flow kinetics measurements carried out in the 1970s and 1980s by Lowe, Thorneley, and others provided a kinetic basis for this process.[20][21] The Lowe-Thorneley (LT) kinetic model was developed from these experiments and documents the eight correlated proton and electron transfers required throughout the reaction.[13][20][21] Each intermediate stage is depicted as En where n = 0–8, corresponding to the number of equivalents transferred. The transfer of four equivalents are required before the productive addition of N2, although reaction of E3 with N2 is also possible.[20] 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.[22]
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.Electron paramagnetic resonance (EPR) characterization shows that this species has a spin of3/2.[23]
E1 – The one electron reduced intermediate has been trapped during turnover under N2.Mӧssbauer spectroscopy of the trapped intermediate indicates that the FeMo-co is integer spin greater than 1.[24]
E2 – This intermediate is proposed to contain the metal cluster in its resting oxidation state with the two added electrons stored in a bridginghydride 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 of3/2.[25]
E3 – This intermediate is proposed to be the singly reduced FeMo-co with one bridging hydride and one hydride.[13]
E4 – Termed the Janus intermediate after theRoman god of transitions, this intermediate is positioned after exactly half of the electron proton transfers and can either decay back to E0 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.[13] 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.[26] 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.[26]95Mo and57Fe ENDOR show that the hydrides bridge between two iron centers.[27] Cryoannealing of the trapped intermediate at -20 °C results in the successive loss of two hydrogen equivalents upon relaxation, proving that the isolated intermediate is consistent with the E4 state.[13] The decay of E4 to E2 + H2 and finally to E0 and 2H2 has confirmed the EPR signal associated with the E2 intermediate.[13]
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.[13]
While the mechanism for nitrogen fixation prior to the Janus E4 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.[13][28][29] 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.[13][28][29] 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 thenitrido in the distal pathway,[30] and thediazene andhydrazine in the alternating pathway.[13] 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.[13] Studies withMo generally point towards a distal pathway, while studies with Fe generally point towards an alternating pathway.[13][28][29][31][32]
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.[30][33] Specific support for the alternating pathway stems from a few studies. Iron only model clusters have been shown to catalytically reduce N2.[31][32] Smalltungsten clusters have also been shown to follow an alternating pathway for nitrogen fixation.[34] The vanadium nitrogenase releases hydrazine, an intermediate specific to the alternating mechanism.[13][35] 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.[13][28][29]
Binding of MgATP is one of the central events to occur in the mechanism employed by nitrogenase.Hydrolysis of the terminalphosphate group of MgATP provides the energy needed to transfer electrons from the Fe protein to the MoFe protein.[36] The binding interactions between the MgATP phosphate groups and theamino 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).[37] Three protein residues have been shown to have significant interactions with the phosphates.[20] In the absence of MgATP, asalt bridge exists between residue 15,lysine, and residue 125,aspartic acid.[37] Upon binding, this salt bridge is interrupted.Site-specific mutagenesis has demonstrated that when the lysine is substituted for aglutamine, the protein's affinity for MgATP is greatly reduced[38] and when the lysine is substituted for anarginine, MgATP cannot bind due to the salt bridge being too strong.[39] The necessity of specifically aspartic acid at site 125 has been shown through noting altered reactivity upon mutation of this residue toglutamic acid.[40] Residue 16, serine, has been shown to bind MgATP. Site-specific mutagenesis was used to demonstrate this fact.[40] This has led to a model in which the serine remains coordinated to the Mg2+ ion after phosphate hydrolysis in order to facilitate its association with a different phosphate of the now ADP molecule.[41] MgATP binding also induces significant conformational changes within the Fe protein.[20] Site-directed mutagenesis was employed to create mutants in which MgATP binds but does not induce a conformational change.[42] ComparingX-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.[42]
Many mechanistic aspects ofcatalysis 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 toethylene.[43] Due to the oxidative properties ofoxygen, most nitrogenases are irreversibly inhibited bydioxygen, which degradatively oxidizes the Fe-S cofactors.[citation needed] This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygenin vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration.[citation needed] Although the ability of some nitrogen fixers such asAzotobacteraceae to employ an oxygen-labile nitrogenase under aerobic conditions has been attributed to a highmetabolic rate, allowing oxygen reduction at thecell 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.[44] MoFe-nitrogenase inAzotobacter vinelandii is protected from oxidative damage by a small ferredoxin-like protein called Shethna protein II (FeSII). Underoxidative stress, the [Fe2-S2] 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.[45][46] A molecule found in the nitrogen-fixing nodules of leguminous plants,leghemoglobin, which can bind to dioxygen via aheme prosthetic group, plays a crucial role in buffering O2 at the active site of the nitrogenase, while concomitantly allowing for efficient respiration.[47]
In addition to dinitrogen reduction, nitrogenases also reduceprotons todihydrogen, meaning nitrogenase is also adehydrogenase. A list of other reactions carried out by nitrogenases is shown below:[48][49]
Furthermore,dihydrogen functions as acompetitive inhibitor,[52]carbon monoxide functions as anon-competitive inhibitor,[48][49] andcarbon disulfide functions as arapid-equilibrium inhibitor[50] of nitrogenase.
Vanadium nitrogenases have also been shown to catalyze the conversion of CO intoalkanes through a reaction comparable toFischer-Tropsch synthesis.
There are two types of bacteria that synthesize nitrogenase and are required for nitrogen fixation. These are:
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,[53] as well as comparison ofAlphaFold2-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.[54] (The alternative nitrogenases break this pattern as they are found to co-occur with Group I and II.)[55] Group I cannot have diverged more than 2.5gigayears ago based on the timing of theGreat Oxidation Event.[53]
Ancestral sequence reconstruction has been used to reconstruct two group I nitrogenases, Anc2 representing the ancestor to all sampled nitrogenases inGammaproteobacteria and Anc1 representing a smaller group built aroundAzotobacter vinelandii,Agaribacterium haliotis, and environmental samples. They work more slowly than the modernA. vinelandii version but keeps a similar efficiency in ATP use (the ratio between formed H2 and reduced N2 is largely unchanged at around 2.1, with the exception of Anc1B which is more efficient.)[53]
TheNif genes include a maturaseNifEN responsible for assembling the precursor to the P-cluster called an O-cluster and transferring it ontoNifDK. It is also where the M-cluster is assembled with the help of nitrogenase component IINifH, before it is transferred toNifDK. Its structure is rather similar to the nitrogenase component INifDK, except that it usually carries a P-cluster and a L-cluster (the precursor to the M-cluster). WhenNifEN fromA. vinelandii is expressed inE. coli with the component IINifH, the resulting combination of proteins prove to work as a nitrogenasein vivo, boosting the growth of transformedE. coli in a nitrogen-deficient medium. TheYfhL ferredoxin naturally present inE. coli is able to work withNifH.[56] Among organisms carrying a Group IIINif,NifN is only found in archaea. The reason is unclear.[55]
One interpretation of the nitrogen-fixing ability ofNifEN and its similarity toNifDK is that there used to be a single nitrogenase with P- and L- clusters, beforegene duplication and increased amounts of available Mo after theGreat Oxidation Event allowed for the current situation to evolve (one copy becameNifEN with the gain of M-cluster, the other becameNifDK with the loss of P-cluster). Isotopic data suggests that Mo-based nitrogen fixation is no younger than 3.2gigayears 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.[57] A large 2022 study mostly supports the view aboutNifEN andNifDK being formed by a duplication.[55]
The three subunits of nitrogenase exhibit significant sequence similarity to three subunits of the light-independent version ofprotochlorophyllide reductase (DPOR, (ChlNB)2 + ChlL2) that performs the conversion ofprotochlorophyllide tochlorophyll. This protein is present ingymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution. The structural similarity puts it in the same superfamily as nitrogenase.[58] The enzyme forbacteriochlorophyll is similar and is calledchlorophyllide oxidoreductase (COR, (BchNB)2 + BchL2 or (BchYZ)2 + BchX2).[59]
Separately, two of the nitrogenase subunits (NifD and NifH) have homologues inmethanogens that do not fix nitrogen e.g.Methanocaldococcus jannaschii.[60] Little is understood about the function of these "class IV"nif genes as of 2004,[61] though they occur in many methanogens. InM. jannaschii they are known to interact with each other and are constitutively expressed.[60] The "group IV"Nif are closer toNif-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 itmonophyletic. There are two known kinds of functions: theCfbC type makescoenzyme F430 and theMar type can make methionine, ethylene, and methane.[55]
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 with15N 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.[62] Though first used in a laboratory setting to measure nitrogenase activity in extracts ofClostridium 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.[63]
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.
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.
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