nitrogenase

Nitrogenase (PDB code=1n2c) (more details...)

Nitrogenase (EC 1.18.6.1) is the enzyme used by some organisms to fix atmospheric nitrogen gas (N₂). It is the only known family of enzymes which accomplishes this process. Dinitrogen is quite inert because of the strength of its N-N triple bond. To break one nitrogen atom away from another requires breaking all three of these chemical bonds.

Nitrogenase is a catalyst for the reaction:

N₂ + 6 H + energy → 2 NH₃

Whilst the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (ΔH⁰ = -45.2 kJ mol^-1 NH₃), the energy barrier to activation is generally insurmountable (E_A = 420 kJ mol^-1) without the assistance of catalysis^[1].

The enzyme therefore requires a great deal of chemical energy, released from the hydrolysis of ATP, and reducing agents, such as dithionite in vitro or ferredoxin in vivo. The enzyme is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein. Nitrogenase is supplied reducing power when it associates with the reduced, nucleotide-bound homodimeric Fe protein. The heterocomplex undergoes cycles of association and disassociation to transfer one electron, which is the limiting step in the process. ATP supplies the reducing power. Each electron transferred supplies enough energy to break one of dinitrogen's chemical bonds, though it has not yet been proven that exactly three cycles are sufficient to convert one molecule of N₂ to ammonia. Ultimately, nitrogenase bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH₃), which is in turn bonded to glutamate to form glutamine.

The exact mechanism of catalysis is unknown due to the difficulty in obtaining crystals of nitrogen bound to nitrogenase. This is because the resting state of MoFe protein does not bind nitrogen and also requires at least three electron transfers to perform catalysis. Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which acts competitively, blocking the active site from dinitrogen. Dinitrogen will prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and only requires only one electron for reduction.^[2]

All nitrogenases have an iron- and sulfur-containing cofactor that includes a heterometal complex in the active site (e.g. FeMoCo). In most, this heterometal has a central molybdenum atom, though in some species it is replaced by a vanadium atom^[3] or iron.

Due to the oxidiative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to avoid oxygen in vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. One known exception, a recently-discovered nitrogenase of Streptomyces thermoautotrophicus, is unaffected by the presence of oxygen [1]. The Azotobacteraceae are unique in their ability to employ an oxygen-labile nitrogenase under aerobic conditions. This ability has been attributed to a high metabolic rate allowing oxygen reduction at the membrane, but this idea has been shown to be unfounded and impossible at oxygen concentrations above 70 µM (ambient concentration is 230 µM O2), as well as during additional nutrient limitations.^[4]

The reaction that this enzyme performs is:

N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 P_i

Nonspecific Reactions

In addition to performing the reaction N≡N → 2 NH₃, nitrogenase is also capable of catalyzing the following reactions:^[5][6]

HC≡CH → H₂C=CH₂

N≡N–O → N₂ + H₂O

N≡N–N⁻ → N₂ + NH₃

C≡N⁻ → CH₄, NH₃, H₃C–CH₃, H₂C=CH₂ (CH₃NH₂)

N≡C–R → RCH₃ + NH₃

C≡N–R → CH₄, H₃C–CH₃, H₂C=CH₂, C₃H₈, C₃H₆, RNH₂

C=O=S → CO + H₂S ^[7]

O=C=O → CO + H₂O ^[7]

S=C=N^– → H₂S + HCN ^[8]

S=C=O → H₂S + CO ^[8]

O=C=N^– → H₂O + HCN, CO + NH₃ ^[8]

Furthermore, dihydrogen functions as a competitive inhibitor,^[9] carbon monoxide functions as a non-competitive inhibitor,^[5][6] and carbon disulfide functions as a rapid-equilibrium inhibitor^[7] of nitrogenase.

Organisms that synthesize nitrogenase

• Free-living diazotrophs, e.g.
  • Cyanobacteria (by means of differentiated heterocysts)
  • Azotobacteraceae
• Symbiotic diazotrophs, e.g.
  • Rhizobia
  • Frankia

Similarity to protochlorophyllide reductase

The light independent version of protochlorophyllide reductase that performs conversion from protochlorophyllide into chlorophyll also consists of three subunits that exhibit significant sequence similarity to the three subunits of nitrogenase. This protein is present in gymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution [2].

See also

• Nitrogen fixation

References