Nitric oxide reductase, an enzyme, catalyzes the reduction 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 ...

(NO) to

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 has ...

(N₂O). The enzyme participates in

nitrogen metabolism The nitrogen cycle is the biogeochemical cycle by which nitrogen is converted into multiple chemical forms as it circulates among atmospheric, terrestrial, and marine ecosystems. The conversion of nitrogen can be carried out through both biologi ...

and in the microbial defense against nitric oxide toxicity. The catalyzed reaction may be dependent on different participating small molecules:

Cytochrome c The cytochrome complex, or cyt ''c'', is a small hemeprotein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins and plays a major role in cell apoptosis. Cytochrome c is hig ...

(EC: 1.7.2.5, Nitric oxide reductase (cytochrome c)),

NADPH Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NADP ...

(EC:1.7.1.14), or

Menaquinone Vitamin K2 or menaquinone (MK) () is one of three types of vitamin K, the other two being vitamin K1 (phylloquinone) and K3 (menadione). K2 is both a tissue and bacterial product (derived from vitamin K1 in both cases) and is usually found in an ...

(EC:1.7.5.2).

Nomenclature

Nitric oxide reductase was assigned

Enzyme Commission number The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name for the correspon ...

(EC) 1.7.2.5. Enzyme Commission numbers are the standard naming system used for enzymes. The EC identifies the class, subclass, sub-subclass, and serial number of the enzyme. Nitric oxide reductase is in Class 1, therefore it is an

oxidoreductase In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually ...

The nitrogen cycle with the role of DNRA

Nitric oxide reductase belongs to the family of

s, specifically those acting on other nitrogenous compounds as donors with other acceptors. The

systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivial ...

of this enzyme class is nitrous-oxide:acceptor oxidoreductase (NO-forming). Other names in common use include nitrogen oxide reductase, and nitrous-oxide:(acceptor) oxidoreductase (NO-forming).

Function

Organisms reduce nitrate (NO₃⁻) to nitrogen gas (N₂) through the process of

denitrification Denitrification is a microbially facilitated process where nitrate (NO3−) is reduced and ultimately produces molecular nitrogen (N2) through a series of intermediate gaseous nitrogen oxide products. Facultative anaerobic bacteria perform denitr ...

, see Figure 1. Two important intermediates of the reduction pathway are nitric oxide (NO) and nitrous oxide (N₂O). The reducing reaction that transforms NO into N₂O is catalyzed by nitric oxide reductase (NOR). NO is reduced to N₂O also to prevent cellular toxicity. N₂O, a potent greenhouse gas, is released.

Reaction

In enzymology, a nitric oxide reductase (NOR) catalyzes the

chemical reaction A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the positions of electrons in the forming and break ...

: :2 NO + 2 e⁻ + 2 H⁺

\rightleftharpoons

N₂O + H₂O The enzyme acts on 2

(

substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (locomotion), the surface over which an organism lo ...

). The enzyme converts NO, electrons and protons to products:

, and H₂O. Inputs: 2 molecules of NO, 2 electrons, 2 protons Outputs: 1 molecule of N₂O, 1 molecule of H₂O

Mechanism

NOR catalyzes the formation of nitrogen to nitrogen (N--N) bonding. The conformation changes of the active site and attached ligands (ie. Glu211) allows NO to be positioned in the crowded binuclear center and form N--N bonds. The precise mechanism of catalysis is still unknown, although hypotheses have been proposed. Cordas et al. 2013 proposes three options: the trans-mechanism, the cis-FeB and the cis-heme b3 mechanisms. Based on the structure of the enzyme, Shiro 2012 proposes the following mechanism: (1) NO molecules bind at the binuclear center, (2) electrons are transferred from the ferrous irons to the NO, (3) charged NO molecules have the potential to form N to N bonds, and (4) N to O bonds are potentially broken by water, allowing for the N₂O and H₂O to be released. According to Hino et al. 2010, the changing charge of the active site causes NO to bind, form N₂O and leave the enzyme. The NOR active site is positioned near two hydrogen bound glutamic acids (Glu). The Glu groups provide an electron-negative charge to the active site. The electro-negative charge reduces the reaction potential for heme b3 and allows NO to bind to the binuclear activation site. Glu residues also provide protons needed for removal of N₂O and production of H₂O.

Structure

Subunits

NOR is made up of two subunits, NorC (small) and NorB (large), with a binuclear iron centre. The binuclear iron center is the active site. It is composed of two b-type

heme Heme, or haem (pronounced / hi:m/ ), is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream. Heme is biosynthesized in both the bone marrow and the liver. In biochemical terms, heme is a coordination complex "consis ...

s and a non-heme iron (FeB). The

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 ele ...

s are connected through a μ-oxo bridge. Histidine (His) residues are attached to the heme b3 in the small subunit. The hydrophilic region of the larger subunit has His and methionine (Met) ligands. Structure is similar to cytochrome oxidases. The active site is conserved between cNOR and qNOR, although differences (ie. heme type) occur between cNOR and qNOR.

Folding

Enzymatic folding produced 13 alpha-helices (12 from NorB, 1 from NorC) located within and through the membrane. The folded metalloenzyme transverses the membrane.

Species distribution

Bacteria, archaea and fungi use NOR. qNOR is found in denitrifying bacteria and archaea, as well as pathogenic bacteria not involved in denitrification. Denitrifying fungi reduce NO using P-450nor soluble enzyme.

Types

Three types of NOR were identified from bacteria: cNOR, qNOR, and qCuNOR. cNOR was found in denitrifying bacteria: ''

Paracoccus denitrificans ''Paracoccus denitrificans'', is a coccoid Coccoid means shaped like or resembling a coccus, that is, spherical.The noun coccoid or coccoids may refer to: * a level of organization, characterized by unicellular, non-flagellated, non-amoeboid ...

'', ''Halomonas halodenitrificans'', '' Pseudomonas nautica'', ''

Pseudomonas stutzeri ''Pseudomonas stutzeri'' is a Gram-negative soil bacterium that is motile, has a single polar flagellum, and is classified as bacillus, or rod-shaped. While this bacterium was first isolated from human spinal fluid, it has since been found in ...

'', and ''

Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common encapsulated, gram-negative, aerobic– facultatively anaerobic, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, ''P. aer ...

''. cNOR was first isolated from ''P''. ''aeruginosa.'' qNOR was isolated from ''

Geobacillus stearothermophilus ''Geobacillus stearothermophilus'' (previously ''Bacillus stearothermophilus'') is a rod-shaped, Gram-positive bacterium and a member of the phylum Bacillota. The bacterium is a thermophile and is widely distributed in soil, hot springs, ocean ...

''.