nitrite reductase
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Nitrite reductase refers to any of several classes of
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
s that catalyze the reduction of
nitrite The nitrite ion has the chemical formula . Nitrite (mostly sodium nitrite) is widely used throughout chemical and pharmaceutical industries. The nitrite anion is a pervasive intermediate in the nitrogen cycle in nature. The name nitrite also ...
. There are two classes of NIR's. A multi haem enzyme reduces NO2 to a variety of products.
Copper Copper is a chemical element with the symbol Cu (from la, cuprum) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pink ...
containing enzymes carry out a single electron transfer to produce
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its ...
.


Iron based

There are several types of iron based enzymes. Cytochrome cd1, or ''Pseudomonas'' cytochrome oxidase contains two c and two d type hemes with two
polypeptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...
chains. Different forms of this reductase catalyze the formation of
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its ...
or
nitrous oxide Nitrous oxide (dinitrogen oxide or dinitrogen monoxide), commonly known as laughing gas, nitrous, or nos, is a chemical compound, an oxide of nitrogen with the formula . At room temperature, it is a colourless non-flammable gas, and ha ...
. A version of this compound was originally called errocytochrome_c-551: errocytochrome_c-551:oxidoreductase">oxidoreductase.html"_;"title="errocytochrome_c-551:oxidoreductase">errocytochrome_c-551:oxidoreductase_It_was_initially_considered_an_oxidase.__It_catalyzes_the_reduction_of_NO2_to_NO.__This_tetraheme_enzyme_has_two_Protein_subunit.html" ;"title="oxidoreductase.html" ;"title="oxidoreductase.html" ;"title="errocytochrome c-551:oxidoreductase">errocytochrome c-551:oxidoreductase">oxidoreductase.html" ;"title="errocytochrome c-551:oxidoreductase">errocytochrome c-551:oxidoreductase It was initially considered an oxidase. It catalyzes the reduction of NO2 to NO. This tetraheme enzyme has two Protein subunit">subunits, each containing a c-type and a d-type heme. The reduced d hemes bind nitrite and convert it to product. Cytochrome c nitrite reductase (ccNIR) is a multiheme enzyme that converts nitrite to ammonia on each active site. The active site iron is bound to a protoporphyrin IX ring that is covalently linked to the enzyme's proteins.


Proposed mechanism

The ccNIR protein uses six electrons and seven hydrogens to reduce nitrite to ammonia. The active site of the enzyme contains an iron in a +2
oxidation state In chemistry, the oxidation state, or oxidation number, is the hypothetical charge of an atom if all of its bonds to different atoms were fully ionic. It describes the degree of oxidation (loss of electrons) of an atom in a chemical compound. C ...
. The oxidation level allows nitrite to bond more strongly than to the +3 state due to increased pi backbonding. This electronic effect transfers electron density into the nitrite
antibonding orbital In chemical bonding theory, an antibonding orbital is a type of molecular orbital that weakens the chemical bond between two atoms and helps to raise the energy of the molecule relative to the separated atoms. Such an orbital has one or more n ...
between
nitrogen Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
and
oxygen Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements ...
. The occupation of the LUMO decreases the strength of the N-O bond. A second electronic effect is the hydrogen bonding of both oxygens to nearby
amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
. These acids are often
arginine Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
and
Histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the ...
. The interactions lengthen the N-O bonds and facilitate cleavage of an oxygen from nitrogen. The Fe-NO bond is linear and has six shared valence electrons. This is not a stable state for an Fe-NO bond. However, a bent seven electron configuration is too stable to undergo further reaction without considerable energy input. To compensate for this barrier, two rapid, consecutive, single electron reductions form an eight electron complex. The electron transfer occurs before a shift in geometry from a linear to bent geometry. Two
protonation In chemistry, protonation (or hydronation) is the adding of a proton (or hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is removed from a Brønsted–Lowry acid ...
s of the nitrogen lead to an increased N-O bond distance. The resulting intermediate is a hydroxylamine. further protonation of the hydroxylamine leads to the breakage of the N-O bond to form water. The oxidation of iron from Fe(II) to Fe(III), coupled with a further protonation of nitrogen leads to the release of ammonia.


Copper based

To date, there have been several types of Copper Nitrite Reductases discovered. These CuNIR are found in many different fungi and bacteria; for example, the bacterial genera '' Pseudomonas'', '' Bordetella'', '' Alcaligenes'', and ''
Achromobacter ''Achromobacter'' is a genus of bacteria, included in the family Alcaligenaceae in the order Burkholderiales. The cells are Gram-negative straight rods and are motile by using one to 20 peritrichous flagella. They are strictly aerobic and are ...
'' all contain CuNIR. What is common to all CuNIR is the presence of at least one type 1 copper center in the protein. These centers are similar to
Azurin Azurin is a small, periplasmic, bacterial blue copper protein found in ''Pseudomonas'', ''Bordetella'', or ''Alcaligenes'' bacteria. Azurin moderates single-electron transfer between enzymes associated with the cytochrome chain by undergoing oxid ...
in their bonding structure. Each type 1 Cu is strongly bonded to a thiolate sulfur from a
cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, some ...
, two
imidazole Imidazole (ImH) is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole, and has non ...
nitrogens from different Histidine residues, and a sulfur atom of an axial
Methionine Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical ...
ligand. This induces a distorted
tetrahedral molecular geometry In a tetrahedral molecular geometry, a central atom is located at the center with four substituents that are located at the corners of a tetrahedron. The bond angles are cos−1(−) = 109.4712206...° ≈ 109.5° when all four substituents a ...
. The cysteine ligated to the type 1 Cu center is located directly next to a Histidine in the
primary structure Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynth ...
of the amino acids. This Histidine is bound to the Type 2 Cu center responsible for binding and reducing nitrite. This Cys-His bridge plays an important role in facilitating rapid electron transfer from the type 1 center to the type 2.


Proposed mechanism

The type 2 copper center of a copper nitrite reductase is the
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate ( binding site) ...
of the enzyme. The Cu is bound by nitrogens of two Histidines from one
monomer In chemistry, a monomer ( ; '' mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization. Classification ...
, and bound by one Histidine from another monomer; the Cys-His bridge to the type 1 Cu. This gives the molecule a distorted tetrahedral geometry. In the resting state, the Cu is also binding a water molecule that is displaced by nitrite. As nitrite displaces water, Cu is bound by both oxygens in a bidentate fashion. A nearby
Aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
residue hydrogen bonds to one of the newly formed oxygen
ligand In coordination chemistry, a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's elect ...
s. An incoming electron reduces the Cu from
oxidation state In chemistry, the oxidation state, or oxidation number, is the hypothetical charge of an atom if all of its bonds to different atoms were fully ionic. It describes the degree of oxidation (loss of electrons) of an atom in a chemical compound. C ...
(II) to (I). This change facilitates a shift in nitrite binding so that the nitrogen is bound to Cu, and one oxygen has an extended bond length due to hydrogen bonding. A second hydrogen bond forms from Histidine or a nearby water molecule and leads to the cleavage of the N-O bond. The Cu is now five coordinate bonded to
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its ...
and water. Nitric oxide is released as Cu is oxidized to state (II) and returns to the resting configuration.


Assimilatory

Assimilatory nitrate reductase is an enzyme of the assimilative
metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run ...
involved in reduction of
nitrate Nitrate is a polyatomic ion with the chemical formula . Salts containing this ion are called nitrates. Nitrates are common components of fertilizers and explosives. Almost all inorganic nitrates are soluble in water. An example of an insolu ...
to
nitrite The nitrite ion has the chemical formula . Nitrite (mostly sodium nitrite) is widely used throughout chemical and pharmaceutical industries. The nitrite anion is a pervasive intermediate in the nitrogen cycle in nature. The name nitrite also ...
. The nitrite is immediately reduced to
ammonia Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous ...
(probably via hydroxylamine) by the activity of nitrite reductase. The term assimilatory refers to the fact that the product of the enzymatic activity remains in the organism. In this case, the product is ammonia which has an inhibitive effect on assimilatory nitrate reductase, thus ensuring that the organism produces the ammonia according to its requirements.


See also

*
Nitrite oxidoreductase Nitrite oxidoreductase (NOR or NXR) is an enzyme involved in nitrification. It is the last step in the process of aerobic ammonia oxidation, which is carried out by two groups of nitrifying bacteria: ammonia oxidizers such as '' Nitrosospira'', ''N ...
*
Ferredoxin—nitrite reductase In enzymology, a ferredoxin—nitrite reductase () is an enzyme that catalyzes the chemical reaction :NH3 + 2 H2O + 6 oxidized ferredoxin \rightleftharpoons nitrite + 6 reduced ferredoxin + 7 H+ The 3 substrates of this enzyme are NH3, H2O, a ...
(NiR) involved in the assimilation of nitrates by plants


References


Further reading

* * {{Portal bar, Biology, border=no EC 1.7 Copper enzymes