Nitrogenases are enzymes (EC 1.18.6.1EC 1.19.6.1) that are produced by certain bacteria, such as cyanobacteria (blue-green bacteria) and rhizobacteria. 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 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 genes or 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 ( Δ H 0 = − 45.2 k J m o l − 1 N H 3 {\displaystyle \Delta H^{0}=-45.2\ \mathrm {kJ} \,\mathrm {mol^{-1}} \;\mathrm {NH_{3}} } ), the activation energy is very high ( E A = 230 − 420 k J m o l − 1 {\displaystyle E_{\mathrm {A} }=230-420\ \mathrm {kJ} \,\mathrm {mol^{-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 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 N2 to NH3. In some assemblies it is replaced by a homologous alternative.
Reductase 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. 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 flow to dissipation of the proton motive force, 5. by electron bifurcation, or 6. by a ferredoxin:NADPH oxidoreductase. 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.
Nitrogenase The MoFe protein is a heterotetramer consisting of two α subunits and two β subunits, with a mass of approximately 240-250kDa. 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 oxidation state of Mo in these nitrogenases was formerly thought Mo(V), but more recent evidence is for Mo(III). (Molybdenum in other enzymes is generally bound to molybdopterin as fully oxidized Mo(VI)).
The core (Fe8S7) of the P-cluster takes the form of two [Fe4S3] cubes linked by a central sulfur atom. Each P-cluster is 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.
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–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. The δ/γ subunit helps bind the cofactor in the FeFe nitrogenase. 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. Most, if not all, natural organisms carrying genes for an alternative nitrogenase also carry genes for the regular MoFe nitrogenase. 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. 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 genes to function. A minimal 10-gene operon that incorporates these additional essential genes has been constructed in the lab.
Mechanism
General mechanism
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