Nitrite reductase
Encyclopedia
Nitrite reductase refers to any of several classes of enzyme
s that catalyze the reduction of nitrite
. There are two classes of NIR's. A multi haem enzyme reduces NO2 to a variety of products. Copper
containing enzymes carry out a single electron transfer to produce nitric oxide
.
s with two polypeptide chains. Different forms of this reductase catalyze the formation of nitric oxide
or nitrous oxide
. A version of this compound was originally called [Ferrocytochrome c-551:oxidoreductase
]. It was initially considered an oxidase. It catalyzes the reduction of NO2 to NO. This tetraheme enzyme has two 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.
. 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 between nitrogen
and oxygen
. 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. These acids are often arginine
and Histidine
. 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
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.
, Bordatella, Alcaligenes
, and Achromobacter
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 in their bonding structure. Each type 1 Cu is strongly bonded to a thiolate sulfur from a cysteine
and to two imidazole
nitrogens from different Histidines. This induces a distorted trigonal planar
molecular geometry
that is stabilized by two weaker interactions with a Methionine
sulfur and a peptide
oxygen.
The cysteine ligated to the type 1 Cu center is located directly next to a Histidine in the primary structure
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.
of the enzyme. The Cu is bound by nitrogens of two Histidines from one monomer
, 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 Asparagine
residue hydrogen bonds to one of the newly formed oxygen ligand
s. An incoming electron reduces the Cu from an oxidation state
(II) to state (I). This change in Cu 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 and leads to the cleavage of the N-O bond. The Cu is now a five coordination
compound bonded to nitric oxide
and water. Nitric oxide is released as Cu is oxidized to state (II) and returns to the resting configuration.
involved in reduction
of nitrate
to nitrite
. The nitrite is immediately reduced to ammonia
(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.
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 that catalyze the reduction of nitrite
Nitrite
The nitrite ion has the chemical formula NO2−. The anion is symmetric with equal N-O bond lengths and a O-N-O bond angle of ca. 120°. On protonation the unstable weak acid nitrous acid is produced. Nitrite can be oxidised or reduced, with product somewhat dependent on the oxidizing/reducing agent...
. There are two classes of NIR's. A multi haem enzyme reduces NO2 to a variety of products. Copper
Copper
Copper is a chemical element with the symbol Cu and atomic number 29. It is a ductile metal with very high thermal and electrical conductivity. Pure copper is soft and malleable; an exposed surface has a reddish-orange tarnish...
containing enzymes carry out a single electron transfer to produce nitric oxide
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
.
Iron based
There are several types of iron based enzymes. Cytochrome cd1, or pseudomonas cytochrome oxidase contains two c and two d type hemeHeme
A heme or haem is a prosthetic group that consists of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin. Not all porphyrins contain iron, but a substantial fraction of porphyrin-containing metalloproteins have heme as their prosthetic group; these are...
s with two polypeptide chains. Different forms of this reductase catalyze the formation of nitric oxide
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
or nitrous oxide
Nitrous oxide
Nitrous 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...
. A version of this compound was originally called [Ferrocytochrome c-551:oxidoreductase
Oxidoreductase
In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule to another...
]. It was initially considered an oxidase. It catalyzes the reduction of NO2 to NO. This tetraheme enzyme has two subunits
Protein subunit
In structural biology, a protein subunit or subunit protein is a single protein molecule that assembles with other protein molecules to form a protein complex: a multimeric or oligomeric protein. Many naturally occurring proteins and enzymes are multimeric...
, 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 stateOxidation state
In chemistry, the oxidation state is an indicator of the degree of oxidation of an atom in a chemical compound. The formal oxidation state is the hypothetical charge that an atom would have if all bonds to atoms of different elements were 100% ionic. Oxidation states are typically represented by...
. 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
Electron density
Electron density is the measure of the probability of an electron being present at a specific location.In molecules, regions of electron density are usually found around the atom, and its bonds...
into the nitrite antibonding orbital between 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...
and 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...
. The occupation of the LUMO
HOMO/LUMO
HOMO and LUMO are acronyms for highest occupied molecular orbital and lowest unoccupied molecular orbital, respectively. The energy difference between the HOMO and LUMO is termed the HOMO-LUMO gap...
decreases the strength of the N-O bond. A second electronic effect is the hydrogen bonding of both oxygens to nearby amino acids. These acids are often arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
and Histidine
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
. 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
Protonation
In chemistry, protonation is the addition of a proton to an atom, molecule, or ion. Some classic examples include*the protonation of water by sulfuric acid:*the protonation of isobutene in the formation of a carbocation:2C=CH2 + HBF4 → 3C+ + BF4−*the protonation of ammonia in the...
s of the nitrogen lead to an increased N-O bond distance. The resulting intermediate is a hydroxylamine
Hydroxylamine
Hydroxylamine is an inorganic compound with the formula NH2OH. The pure material is a white, unstable crystalline, hygroscopic compound. However, hydroxylamine is almost always provided and used as an aqueous solution. It is used to prepare oximes, an important functional group. It is also an...
. 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 plants and bacteria; for example, the bacterial genera PseudomonasPseudomonas
Pseudomonas is a genus of gammaproteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species.Recently, 16S rRNA sequence analysis has redefined the taxonomy of many bacterial species. As a result, the genus Pseudomonas includes strains formerly classified in the...
, Bordatella, Alcaligenes
Alcaligenes
Alcaligenes is a genus of Gram-negative, aerobic, rod-shaped bacteria. The species are motile with one or more peritrichous flagella.Alcaligenes species have been used for the industrial production of non-standard amino acids; A. eutrophus also produces the biopolymer polyhydroxybutyrate .-External...
, and Achromobacter
Achromobacter
The Achromobacter are a genus of bacteria, included in the order Burkholderiales. The cells are straight rods and are motile by using 1 – 20 peritrichous flagella. They are strictly aerobic and are found in water and soils....
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 in their bonding structure. Each type 1 Cu is strongly bonded to a thiolate sulfur from a cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
and to two imidazole
Imidazole
Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...
nitrogens from different Histidines. This induces a distorted trigonal planar
Trigonal planar
In chemistry, trigonal planar is a molecular geometry model with one atom at the center and three atoms at the corners of a triangle, called peripheral atoms, all in one plane. In an ideal trigonal planar species, all three ligands are identical and all bond angles are 120°. Such species belong to...
molecular geometry
Molecular geometry
Molecular geometry or molecular structure is the three-dimensional arrangement of the atoms that constitute a molecule. It determines several properties of a substance including its reactivity, polarity, phase of matter, color, magnetism, and biological activity.- Molecular geometry determination...
that is stabilized by two weaker interactions with a Methionine
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
sulfur and a peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
oxygen.
The cysteine ligated to the type 1 Cu center is located directly next to a Histidine in the primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...
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 siteActive site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of the enzyme. The Cu is bound by nitrogens of two Histidines from one monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
, 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 Asparagine
Asparagine
Asparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
residue hydrogen bonds to one of the newly formed oxygen ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
s. An incoming electron reduces the Cu from an oxidation state
Oxidation state
In chemistry, the oxidation state is an indicator of the degree of oxidation of an atom in a chemical compound. The formal oxidation state is the hypothetical charge that an atom would have if all bonds to atoms of different elements were 100% ionic. Oxidation states are typically represented by...
(II) to state (I). This change in Cu 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 and leads to the cleavage of the N-O bond. The Cu is now a five coordination
Coordination number
In chemistry and crystallography, the coordination number of a central atom in a molecule or crystal is the number of its nearest neighbours. This number is determined somewhat differently for molecules and for crystals....
compound bonded to nitric oxide
Nitric oxide
Nitric oxide, also known as nitrogen monoxide, is a diatomic molecule with chemical formula NO. It is a free radical and is an important intermediate in the chemical industry...
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 metabolismMetabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
involved in reduction
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
of nitrate
Nitrate
The nitrate ion is a polyatomic ion with the molecular formula NO and a molecular mass of 62.0049 g/mol. It is the conjugate base of nitric acid, consisting of one central nitrogen atom surrounded by three identically-bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a...
to nitrite
Nitrite
The nitrite ion has the chemical formula NO2−. The anion is symmetric with equal N-O bond lengths and a O-N-O bond angle of ca. 120°. On protonation the unstable weak acid nitrous acid is produced. Nitrite can be oxidised or reduced, with product somewhat dependent on the oxidizing/reducing agent...
. The nitrite is immediately reduced to 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...
(probably via hydroxylamine
Hydroxylamine
Hydroxylamine is an inorganic compound with the formula NH2OH. The pure material is a white, unstable crystalline, hygroscopic compound. However, hydroxylamine is almost always provided and used as an aqueous solution. It is used to prepare oximes, an important functional group. It is also an...
) 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.