Nitrite reductases reduction to ammonia

Oxidation of ammonia to nitrite, N02, and nitrate, N03, is called nitrification the reverse reaction is ammonification. Reduction from nitrite to nitrogen is called denitrification. All these reactions, and more, occur in enzyme systems, many of which include transition metals. A molybdenum enzyme, nitrate reductase, reduces nitrate to nitrite. Further reduction to ammonia seems to proceed by 2-electron steps, through an uncertain intermediate with a -fl oxidation state (possibly hyponitrite, N202 ) and hydroxylamine ... 

The E. coll enzyme can reduce nitrite and hydroxylamine to ammonia at the expense of NADPH (339). However, with the use of N-nitrite it was shown that hydroxylamine was not an intermediate in the reduction of nitrite. No cofactor requirements were shown for the E. coli enzyme, but similar to other flavin and metal requiring nitrite reductases it was inhibited by cyanide and mercurials. 

Nitrate and nitrite reductases Nitrate reduction to ammonia... 

The second category includes enzymes that typically catalyze proper oxygen atom transfer reactions to or from an available electron lone pair of a substrate, and can be further subdivided into two families. The first family includes sulfite oxidase and assimilatory nitrate reductase, the physiological functions of which are to reduce nitrate to nitrite in the first stage of its reduction to ammonia for use by the plant cell. The second family comprises bacterial enzymes such as dimethylsulfoxide... 

Nitrite reductases, 698 denitrifying, 727 dissimilatory, 726 reduction to ammonia, 725 respiration, 717 Nitrites reduction, 725 Nitro compounds arenes... 

For the assimilatory pathway it was proposed (Meyer and Schultze, 1894) that the reduction of nitrite to ammonia proceeded by three steps, each step involving the transfer of two electrons with the production of hyponitrite and hydroxylamine as intermediates. Although belief in this three step reductive pathway was maintained for 60 years, current evidence shows that nitrite is reduced to ammonia by the enzyme nitrite reductase according to this scheme. 

The reduction of nitrite to ammonia was described for bacteria with a fermentative rather than a respiratory metabolism [125]. However, growth of various bacteria by oxidation of non-fermentable substrates such as formate linked to the reduction of nitrite to ammonia, demonstrated that nitrite ammonification may also function as respiratory energy conserving process [126]. The enzymol-ogy and bioenergetics of respiratory nitrite ammonification have been recently reviewed [17]. In respiratory nitrite ammonification, nitrite is reduced to ammonia without the release of intermediate products, such as NO or N2O, in a six-electron step by a cytochrome c nitrite reductase, the so-caUed NrfA protein [Eq. (10)] [127-131]. 

In nitrite-ammonifying bacteria, especially in enterobacteria, there may exist two independently regulated dissimilatory ways of nitrite reduction to ammonia with two different physiological functions [125]. A cytoplasmic siroheme-dependent nitrite reductase activity (NADH nitrite oxidoreductase) [138,139] confers on... 

First, ferredoxin-nitrate reductase (EC 1.7.7.2) is a molybdenum-iron-sulfur protein that converts nitrate to nitrite while undergoing oxidation. Its structure is not yet well-defined. Then, ferridoxin-nitrite reductase (EC 1.7.7.1), an iron (as heme and at least one iron sulfur complex, vide infra) can continue the reduction to ammonia (NH3). 

The bis-hydroxylamine adduct [Fe (tpp)(NH20H)2] is stable at low temperatures, but decomposes to [Fe(tpp)(NO)] at room temperature. [Fe(porphyrin)(NO)] complexes can undergo one-and two-electron reduction the nature of the one-electron reduction product has been established by visible and resonance Raman spectroscopy. Reduction of [Fe(porphyrin)(NO)] complexes in the presence of phenols provides model systems for nitrite reductase conversion of coordinated nitrosyl to ammonia (assimilatory nitrite reduction), while further relevant information is available from the chemistry of [Fe (porphyrin)(N03)]. Iron porphyrin complexes with up to eight nitro substituents have been prepared and shown to catalyze oxidation of hydrocarbons by hydrogen peroxide and the hydroxylation of alkoxybenzenes. ... 

Based on crystallographic observations it was suggested that the HA intermediate is bound to the cytochrome reductase via the iron atom, Fe(II)—NH2OH, and undergoes subsequent reduction to produce the NH3 that then dissociates from the protein ". It is of interest that the specific activity of cytochrome c-nitrite reductase from S. deleyianum in the conversion of N02 to NH3 is only 2-fold greater than that recorded for the conversion of HA to ammonia by the same enzyme, an observation that strongly supports the involvement of HA as an intermediate in the catalytic reduction of nitrite to NH3 . 

Blom was the first to demonstrate, in 1928, the formation of HA by an unknown mixture of bacteria which utilized nitrate as their sole nitrogen source to produce ammonia , an observation substantiated by Lindsey and Rhines who generalized this reaction to a diverse set of microorganisms capable of producing NH3 by reduction of both nitrites and nitrates. The mechanism of the 6-electron reduction of nitrite to ammonia (i.e. conversion of the [N + 02] species to by bacterial cytochrome c nitrite reductase... 

Nitrite reductase and sulfite reductase are enzymes found in choroplasts and in prokaryotes that reduce nitrite to ammonia and sulfite to sulfide (Scott et al., 1978). Sulfite reductase also catalyzes reduction of nitrite at a lower rate. Both enzymes contain a siroheme prosthetic group linked to an iron-sulfur cluster. In siroheme, the porphyrinoid moiety is present in the more reduced chlorin form. Because NO lies between nitrite and ammonia in oxidation state, it is a potential intermediate. 

Nitrate reductases have been isolated from bacteria, plants and fungi and always contain molybdenum. Two types may be distinguished (a) the assimilatory nitrate reductases which catalyze the reduction of nitrate to nitrite, which ultimately is reduced to ammonia and used by... 

These include nitrite and trimethylammonium oxide. Nitrite undergoes a six-electron reduction to give ammonia in a reaction catalyzed by a nitrite reductase. Nitrite reductase activity with lactate and formate has been reported, although other donors may support the reduction of nitrite. It should be noted that at least three pathways in E. coli exist for the reduction of nitrite. 

While this formally may follow assimilatory (to NH3) or dissimilatory (to N2) pathways, it is becoming clear that a number of non-denitrifiers are also able to produce dinitrogen monoxide by reduction of nitrite.1513,1514 Furthermore, reduction of nitrite to ammonia by one pathway (the NADPH-sulfite reductase) does not appear to be an assimilatory process, but rather one in which catabolic reducing equivalents are removed. 

Green plants, algae, fungi, cyanobacteria and bacteria that assimilate nitrate also produce assimilatory nitrite reductases, which catalyze the six-electron reduction of nitrite to ammonia (equation 89). The formation of heme-nitrosyl intermediates has been detected in several cases,1515 while hydroxylamine is commonly thought to be an intermediate. Added hydroxylamine is rapidly reduced to ammonia. However, no intermediates are released, and ammonia is the only product... 

Similar mechanisms operate in the action of nitrate reductase and nitrite reductase. Both of these substances are produced from ammonia by oxidation. Plants and soil bacteria can reduce these compounds to provide ammonia for metabolism. The common agricultural fertilizer ammonium nitrate, NH4NO3, provides reduced nitrogen for plant growth directly, and by providing a substrate for nitrate reduction. NADH or NADPH is the electron donor for nitrate reductase, depending on the organism. 

That hydroxylamine might not be an obligatory intermediate, or occur as a free intermediate, in the reduction of nitrite to ammonia is suggested by the properties of nitrite reductases of Azotobacter chroococcum and Escherichia coli. The former is an adaptive enzyme, the formation of which requires nitrate or nitrite in the culture (31,2). It is FAD-depen-dent and presumably contains metals and p-mercuribenzoate inhibitable... 

The nitrite reductase of Azotobacter vinelandvi (A. agile) was extracted in soluble form by Nason and his colleagues (537). The preparation reduced nitrite and hydroxylamine in the presence of reduced nicotinamide-adenine dinucleotides and required flavin for maximal activity. FAD was shown to be specific for nitrite reduction, whereas both FAD and FMN were active for hydroxylamine reduction. The hydroxylamine reductase activity of the preparation was enhanced in the presence of Mn +. Ammonia was shown to be the product of nitrite reduction, but the product of hydroxylamine reduction was not identified. Another nitrite and hydroxylamine reductase, which had a Mn requirement, was also isolated and partially purified in Nason s laboratory from soybean... 

However, in both the yeast and the E. coli systems, the stoichiometries for NADPH S and SOj -rS - are 3 1 and 1 1, respectively. These results and the inability to detect 2-electron- and 4-electron-reduced intermediates in these systems have suggested that such intermediates, if present at all, must be firmly held on the surface of the enzyme. It has further been suggested that the presence of multiple flavins and hemes in the enzyme might be a device for achieving a rapid six-electron reduction of sulfite without the release of intermediates (414)- This situation is analogous to the four-electron reduction of 0, to 2H,0 by cytochrome oxidase and the six-electron reduction of nitrite to ammonia by various assimilatory nitrite reductases. However, unlike cytochrome oxidase,... 

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