Nitrogenase


Nitrogenases[] are enzymes that are produced by certain bacteria, such as cyanobacteria and rhizobacteria. These enzymes are responsible for the reduction of nitrogen to ammonia. 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 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, the activation energy is very high. 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:
  1. The homodimeric Fe-only protein, the reductase which has a high reducing power and is responsible for a supply of electrons.
  2. 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 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, but more recent evidence is for Mo..
  • The core of the P-cluster takes the form of two cubes linked by a central sulfur atom. Each P-cluster is linked to the MoFe protein by six cysteine residues.
  • Each FeMo cofactor consists of two non-identical clusters: and , 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 type and the iron–iron type. Both form an assembly of two α subunits, two β subunits, and two δ 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. 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 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

Nitrogenase is an enzyme responsible for catalyzing nitrogen fixation, which is the reduction of nitrogen to ammonia and a process vital to sustaining life on Earth. There are three types of nitrogenase found in various nitrogen-fixing bacteria: molybdenum nitrogenase, vanadium nitrogenase, and iron-only nitrogenase. Molybdenum nitrogenase, which can be found in diazotrophs such as legume-associated rhizobia, is the nitrogenase that has been studied the most extensively and thus is the most well characterized. Vanadium nitrogenase and iron-only nitrogenase can both be found in select species of Azotobacter as an alternative nitrogenase. Equations 1 and 2 show the balanced reactions of nitrogen fixation in molybdenum nitrogenase and vanadium nitrogenase respectively.
Recent refinements to the kinetic framework of Mo-nitrogenase 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.
All nitrogenases are two-component systems made up of Component I and Component II. Component I is a MoFe protein in molybdenum nitrogenase, a VFe protein in vanadium nitrogenase, and an Fe protein in iron-only nitrogenase. Component II is a Fe protein that contains the Fe-S cluster., which transfers electrons to Component I. Component I contains 2 metal clusters: the P-cluster, and the FeMo-cofactor. Mo is replaced by V or Fe in vanadium nitrogenase and iron-only nitrogenase respectively. 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.

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. Steady state, freeze quench, and stopped-flow kinetics measurements carried out in the 1970s and 1980s by Lowe, Thorneley, and others provided a kinetic basis for this process. The Lowe-Thorneley kinetic model was developed from these experiments and documents the eight correlated proton and electron transfers required throughout the reaction. 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. 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.

Intermediates E0 through E4

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 characterization shows that this species has a spin of 3/2.
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.
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. Isolation of this intermediate in mutated enzymes shows that the FeMo-co is high spin and has a spin of 3/2.
E3 – This intermediate is proposed to be the singly reduced FeMo-co with one bridging hydride and one hydride.
E4 – Termed the Janus intermediate after the Roman 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. 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. 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 experiments have provided insight into the structure of this intermediate, revealing the presence of two bridging hydrides. 95Mo and 57Fe ENDOR show that the hydrides bridge between two iron centers. 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. The decay of E4 to E2 + H2 and finally to E0 and 2H2 has confirmed the EPR signal associated with the E2 intermediate.
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.