Nitrogenase
Encyclopedia
Nitrogenases are enzyme
s used by some organisms to fix atmospheric nitrogen
gas (N2). It is the only known family of enzymes that accomplish 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.
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 very high (EA = 420 kJ mol−1) without the assistance of catalysis.
In addition to reducing agents, such as dithionite
in vitro
, or ferredoxin
or flavodoxin
in vivo
, the enzymatic reduction of dinitrogen to ammonia therefore also requires an input of chemical energy, released from the hydrolysis
of ATP
, to overcome the activation energy barrier. The enzyme
is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein
. Electrons for the reduction of nitrogen are supplied to nitrogenase 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 rate-limiting step in nitrogen reduction. ATP supplies the energy to drive the transfer of electrons from the Fe protein to the MoFe protein. The reduction potential of each electron transferred to the MoFe protein is sufficient to break one of dinitrogen's chemical bonds, though it has not yet been shown that exactly three cycles are sufficient to convert one molecule of N2 to ammonia. Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia
(NH3), which is in turn bonded to glutamate to form glutamine
. The nitrogenase reaction additionally produces molecular hydrogen
as a side product.
The exact mechanism of catalysis
is unknown due to the difficulty in obtaining crystal
s 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 binds to the enzyme and thereby prevents binding of dinitrogen. Dinitrogen will prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and requires only one electron for reduction to ethylene
.
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 complex has a central molybdenum
atom, though in some species it is replaced by a vanadium
or iron
atom.
Due to the oxidative properties of oxygen
, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo
. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen http://www.jbc.org/cgi/reprint/272/42/26627.pdf. 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.
The reaction that this enzyme performs is:
Furthermore, dihydrogen functions as a competitive inhibitor
, carbon monoxide
functions as a non-competitive inhibitor
, and carbon disulfide
functions as a rapid-equilibrium inhibitor of nitrogenase.
Vanadium nitrogenase
s have also been shown to catalzye the conversion of CO into alkanes through a reaction comparable to Fischer-Tropsch synthesis.
Typically nitrogenases are coded by Nif gene
s.
that performs the conversion of protochlorophyllide
to 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 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=160407.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s used by some organisms to fix atmospheric nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
gas (N2). It is the only known family of enzymes that accomplish this process. Dinitrogen is quite inert because of the strength of its N-N triple bond
Triple bond
A triple bond in chemistry is a chemical bond between two chemical elements involving six bonding electrons instead of the usual two in a covalent single bond. The most common triple bond, that between two carbon atoms, can be found in alkynes. Other functional groups containing a triple bond are...
. To break one nitrogen atom away from another requires breaking all three of these chemical bonds.
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
Activation energy
In chemistry, activation energy is a term introduced in 1889 by the Swedish scientist Svante Arrhenius that is defined as the energy that must be overcome in order for a chemical reaction to occur. Activation energy may also be defined as the minimum energy required to start a chemical reaction...
to activation is very high (EA = 420 kJ mol−1) without the assistance of catalysis.
In addition to reducing agents, such as dithionite
Dithionite
The dithionite anion , is an oxoanion of sulfur formally derived from dithionous acid, H2S2O4.-Chemistry:Dithionous acid has not been detected either as a pure compound or in solution....
in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
, or ferredoxin
Ferredoxin
Ferredoxins are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions. The term "ferredoxin" was coined by D.C. Wharton of the DuPont Co...
or flavodoxin
Flavodoxin
Flavodoxins are electron-transfer proteins. Flavodoxin is a bacterial protein that includes flavin mononucleotide. The structure of flavodoxin is characterized by a five-stranded parallel beta sheet, surrounded by alpha helices at either side of the sheet. They have been isolated from prokaryotes,...
in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
, the enzymatic reduction of dinitrogen to ammonia therefore also requires an input of chemical energy, released from the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
, to overcome the activation energy barrier. The enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. Electrons for the reduction of nitrogen are supplied to nitrogenase when it associates with the reduced, nucleotide-bound homodimeric Fe protein. The heterocomplex undergoes cycles of association and disassociation to transfer one electron
Electron
The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...
, which is the rate-limiting step in nitrogen reduction. ATP supplies the energy to drive the transfer of electrons from the Fe protein to the MoFe protein. The reduction potential of each electron transferred to the MoFe protein is sufficient to break one of dinitrogen's chemical bonds, though it has not yet been shown that exactly three cycles are sufficient to convert one molecule of N2 to ammonia. Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia
Ammonia
Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...
(NH3), which is in turn bonded to glutamate to form glutamine
Glutamine
Glutamine is one of the 20 amino acids encoded by the standard genetic code. It is not recognized as an essential amino acid but may become conditionally essential in certain situations, including intensive athletic training or certain gastrointestinal disorders...
. The nitrogenase reaction additionally produces molecular hydrogen
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
as a side product.
The exact mechanism of catalysis
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
is unknown due to the difficulty in obtaining crystal
Crystal
A crystal or crystalline solid is a solid material whose constituent atoms, molecules, or ions are arranged in an orderly repeating pattern extending in all three spatial dimensions. The scientific study of crystals and crystal formation is known as crystallography...
s 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
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
. Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which binds to the enzyme and thereby prevents binding of dinitrogen. Dinitrogen will prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and requires only one electron for reduction to ethylene
Ethylene
Ethylene is a gaseous organic compound with the formula . It is the simplest alkene . Because it contains a carbon-carbon double bond, ethylene is classified as an unsaturated hydrocarbon. Ethylene is widely used in industry and is also a plant hormone...
.
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 complex has a central molybdenum
Molybdenum
Molybdenum , is a Group 6 chemical element with the symbol Mo and atomic number 42. The name is from Neo-Latin Molybdaenum, from Ancient Greek , meaning lead, itself proposed as a loanword from Anatolian Luvian and Lydian languages, since its ores were confused with lead ores...
atom, though in some species it is replaced by a vanadium
Vanadium
Vanadium is a chemical element with the symbol V and atomic number 23. It is a hard, silvery gray, ductile and malleable transition metal. The formation of an oxide layer stabilizes the metal against oxidation. The element is found only in chemically combined form in nature...
or iron
Iron
Iron is a chemical element with the symbol Fe and atomic number 26. It is a metal in the first transition series. It is the most common element forming the planet Earth as a whole, forming much of Earth's outer and inner core. It is the fourth most common element in the Earth's crust...
atom.
Due to the oxidative properties of oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen http://www.jbc.org/cgi/reprint/272/42/26627.pdf. Although the ability of some nitrogen fixers such as Azotobacteraceae
Azotobacteraceae
The family Azotobacteraceae contains aerobic diazotrophs with two Genera, Azomonas and Azotobacter, distinguished by the ability to form cysts. The family is also characterized by variable cell shape, the classic shape being ovoid while many are pleomorphic...
to employ an oxygen-labile nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
, 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.
The reaction that this enzyme performs is:
- N2 + 8 H+ + 8 + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi
Nonspecific Reactions
In addition to performing the reaction N≡N → 2 NH3, nitrogenase is also capable of catalyzing the following reactions:- HC≡CHAcetyleneAcetylene is the chemical compound with the formula C2H2. It is a hydrocarbon and the simplest alkyne. This colorless gas is widely used as a fuel and a chemical building block. It is unstable in pure form and thus is usually handled as a solution.As an alkyne, acetylene is unsaturated because...
→ H2C=CH2EthyleneEthylene is a gaseous organic compound with the formula . It is the simplest alkene . Because it contains a carbon-carbon double bond, ethylene is classified as an unsaturated hydrocarbon. Ethylene is widely used in industry and is also a plant hormone... - N≡N–ONitrous oxideNitrous oxide, commonly known as laughing gas or sweet air, is a chemical compound with the formula . It is an oxide of nitrogen. At room temperature, it is a colorless non-flammable gas, with a slightly sweet odor and taste. It is used in surgery and dentistry for its anesthetic and analgesic...
→ N2 + H2O - N=N=N–AzideAzide is the anion with the formula N3−. It is the conjugate base of hydrazoic acid. N3− is a linear anion that is isoelectronic with CO2 and N2O. Per valence bond theory, azide can be described by several resonance structures, an important one being N−=N+=N−...
→ N2 + NH3 - CyanideA cyanide is a chemical compound that contains the cyano group, -C≡N, which consists of a carbon atom triple-bonded to a nitrogen atom. Cyanides most commonly refer to salts of the anion CN−. Most cyanides are highly toxic....
→ CH4MethaneMethane is a chemical compound with the chemical formula . It is the simplest alkane, the principal component of natural gas, and probably the most abundant organic compound on earth. The relative abundance of methane makes it an attractive fuel...
, NH3, H3C–CH3EthaneEthane is a chemical compound with chemical formula C2H6. It is the only two-carbon alkane that is an aliphatic hydrocarbon. At standard temperature and pressure, ethane is a colorless, odorless gas....
, H2C=CH2 (CH3NH2) - N≡C–R → RCH3 + NH3
- C≡N–R → CH4, H3C–CH3, H2C=CH2, C3H8PropanePropane is a three-carbon alkane with the molecular formula , normally a gas, but compressible to a transportable liquid. A by-product of natural gas processing and petroleum refining, it is commonly used as a fuel for engines, oxy-gas torches, barbecues, portable stoves, and residential central...
, C3H6PropylenePropene, also known as propylene or methylethylene, is an unsaturated organic compound having the chemical formula C3H6. It has one double bond, and is the second simplest member of the alkene class of hydrocarbons, and it is also second in natural abundance.-Properties:At room temperature and...
, RNH2 - O=C=SCarbonyl sulfideCarbonyl sulfide is the chemical compound with the formula OCS. Commonly written as COS, it is a colourless flammable gas with an unpleasant odor. It is a linear molecule consisting of a carbonyl group double bonded to a sulfur atom...
→ COCarbon monoxideCarbon monoxide , also called carbonous oxide, is a colorless, odorless, and tasteless gas that is slightly lighter than air. It is highly toxic to humans and animals in higher quantities, although it is also produced in normal animal metabolism in low quantities, and is thought to have some normal...
+ H2SHydrogen sulfideHydrogen sulfide is the chemical compound with the formula . It is a colorless, very poisonous, flammable gas with the characteristic foul odor of expired eggs perceptible at concentrations as low as 0.00047 parts per million... - O=C=OCarbon dioxideCarbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
→ CO + H2O - S=C=N–ThiocyanateThiocyanate is the anion [SCN]−. It is the conjugate base of thiocyanic acid. Common derivatives include the colourless salts potassium thiocyanate and sodium thiocyanate. Organic compounds containing the functional group SCN are also called thiocyanates...
→ H2S + HCN - S=C=OCarbonyl sulfideCarbonyl sulfide is the chemical compound with the formula OCS. Commonly written as COS, it is a colourless flammable gas with an unpleasant odor. It is a linear molecule consisting of a carbonyl group double bonded to a sulfur atom...
→ H2S + CO - O=C=N–CyanateThe cyanate ion is an anion with the chemical formula written as [OCN]− or [NCO]−. In aqueous solution it acts as a base, forming isocyanic acid, HNCO. The cyanate ion is an ambidentate ligand, forming complexes with a metal ion in which either the nitrogen or oxygen atom may be the electron-pair...
→ H2O + HCN, CO + NH3
Furthermore, dihydrogen functions as a competitive inhibitor
Competitive inhibition
Competitive inhibition is a form of enzyme inhibition where binding of the inhibitor to the active site on the enzyme prevents binding of the substrate and vice versa.-Mechanism:...
, carbon monoxide
Carbon monoxide
Carbon monoxide , also called carbonous oxide, is a colorless, odorless, and tasteless gas that is slightly lighter than air. It is highly toxic to humans and animals in higher quantities, although it is also produced in normal animal metabolism in low quantities, and is thought to have some normal...
functions as a non-competitive inhibitor
Non-competitive inhibition
Non-competitive inhibition is a type of enzyme inhibition where the inhibitor reduces the activity of the enzyme, by binding not to the active site on the enzyme, but to a different site...
, and carbon disulfide
Carbon disulfide
Carbon disulfide is a colorless volatile liquid with the formula CS2. The compound is used frequently as a building block in organic chemistry as well as an industrial and chemical non-polar solvent...
functions as a rapid-equilibrium inhibitor of nitrogenase.
Vanadium nitrogenase
Vanadium nitrogenase
Vanadium nitrogenase is a key enzyme for nitrogen fixation found in nitrogen-fixing bacteria, and is used as an alternative to molybdenum nitrogenase when molybdenum is unavailable. An important component of the nitrogen cycle, vanadium nitrogenase converts nitrogen gas to ammonia, thereby making...
s have also been shown to catalzye the conversion of CO into alkanes through a reaction comparable to Fischer-Tropsch synthesis.
Organisms that synthesize nitrogenase
- Free-living diazotrophDiazotrophDiazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia.A diazotroph is an organism that is able to grow without external sources of fixed nitrogen. Examples of organisms that do this are rhizobia and Frankia and Azospirillum. All diazotrophs...
s, e.g.- Cyanobacteria (by means of differentiated heterocystHeterocystHeterocysts are specialized nitrogen-fixing cells formed by some filamentous cyanobacteria, such as Nostoc punctiforme, Cylindrospermum stagnale and Anabaena sphaerica, during nitrogen starvation. They fix nitrogen from dinitrogen in the air using the enzyme nitrogenase, in order to provide the...
s) - AzotobacteraceaeAzotobacteraceaeThe family Azotobacteraceae contains aerobic diazotrophs with two Genera, Azomonas and Azotobacter, distinguished by the ability to form cysts. The family is also characterized by variable cell shape, the classic shape being ovoid while many are pleomorphic...
- Cyanobacteria (by means of differentiated heterocyst
- Symbiotic diazotrophs, e.g.
- RhizobiaRhizobiaRhizobia are soil bacteria that fix nitrogen after becoming established inside root nodules of legumes . Rhizobia require a plant host; they cannot independently fix nitrogen...
- FrankiaFrankiaFrankia is a genus of nitrogen fixing, filamentous bacteria that live in symbiosis with actinorhizal plants, similar to the Rhizobia bacteria that are found in the root nodules of legumes in the Fabaceae family. Bacteria of this genus also form root nodules.The genus Frankia was originally named by...
- Rhizobia
Typically nitrogenases are coded by Nif gene
Nif gene
The nif gene is the gene responsible for the coding of proteins related and associated with the fixation of atmospheric nitrogen into a form of nitrogen available to plants...
s.
Similarity to protochlorophyllide reductase
The light-independent version of protochlorophyllide reductaseProtochlorophyllide reductase
In enzymology, a protochlorophyllide reductase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are chlorophyllide a and NADP+, whereas its 3 products are protochlorophyllide, NADPH, and H+....
that performs the conversion of protochlorophyllide
Protochlorophyllide
Protochlorophyllide, or monovinyl protochlorophyllide, is an immediate precursor of chlorophyll a that lacks the phytol side-chain of chlorophyll. Unlike chlorophyll, protochlorophyllide is highly fluorescent; mutants that accumulate it glow red if irradiated with blue light...
to chlorophyll
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
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 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=160407.