Nitrite reductase location

Figure 2.7 (a) A front view of the nitrite reductase dimer with the five haems in each monomer in white, a bound Ca2+ ion in grey and Lys-133, which coordinates the active site iron of haem 1, in yellow, (b) The haem arrangement. The overall orientation corresponds to (a), with the active site located at haem 1. Reprinted with permission from Einsle et al., 1999. Copyright (1999), Macmillan Magazines Limited. 

Alefounder, P. R., and Ferguson, S. J. (1980). The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitri-ficans. Biochem.J. 192, 231-240. 

Many species of bacteria also have an assimilatory nitrite reductase which is located in the cytoplasm. There is relatively little known about such enzymes but the electron donor is throught to be NADPH and the active site again has siroheme (Cole, 1988). The assimilatory nitrite reductases of both plants and bacteria use nitrite that is provided as the product of the assimilatory nitrate reductases. Nitrate is a very common natural N source for plant and bacterial growth. 

Structure (of nitrite reductase from Alcaligenes faecalis). The subunits of nitrite reductase contain two domains. Each domain consists of a /1-barrel structure, similar to that in the small blue proteins [272]. The type 1 copper center is embedded in one of these /1-barrels and is coordinated by the ligands His 95 (domain 1), Cys 136, His 145, and Met 150 (domain 2) [26]. The type 2 copper center is coordinated by a water molecule and three histidine ligands His 100 and His 135 (subunit x) as well as His 306 (subunit y). It is situated at the interface of two subunits [272], The water molecule is displaced from the type 2 copper ion upon nitrite binding (Fig. 34). Although both copper ions are coordinated by neighboring residues, they are located approximately 12.5 A apart, which prevents a direct electron transfer [272]. 

Dissimilatory nitrite reductase of denitrifying bacteria is usually a soluble enzyme and it has been difficult to ascribe a phosphorylative function associated with the conversion of nitrite to nitric oxide. However, the demonstration by Wood (1978) that the terminal reductase in nitrite respiration is located in the periplasm implies that electrons generated in the cytoplasm must traverse the cytoplasmic membrane to the periplasmic nitrite reduction site. This location would require proton pumping, thus facilitating phosphorylation by the chemiosmotic mechanism. 

Most (Losadaet /., 1963 Ritenour er a/., 1%7 Dalling /a/., 1972a) but not all (Grant er al., 1970 Lips and Avissar, 1972) studies indicate that nitrite reductase from green leaves of higher plants is associated with isolated chloroplasts. Additional confirmation of the chloroplastic location has been published (Magalhaes et at., 1974 Miflin, 1974). These studies show that illuminated isolated intact chloroplasts without supplemental cofactors or enzymes can stoichiometrically reduce nitrite to ammonia. The rates of nitrite reduction were equivalent to in situ assimilation rates. 

In 1%7, Miflin reported that a particulate fraction from pea or barley roots was able to reduce nitrite using reduced benzyl viologen as the electron donor. Miflin (1970) and Bourne and Miflin (1970) isolated a barley root particle that reduced nitrate to ammonia when provided with pyruvate and ATP. The fractionation techniques used separated the nitrosome from the mitochondrial and peroxisomal fractions. Dalling et al. (1972b) found that 15% of the total nitrite reductase activity of wheat roots was associated with an organelle that was tentatively identified as a proplastid. A proplastid location for nitrite reductase in tissue culture cells has been indicated by Washitani and Sato (1977a,b). 

Assimilatory nitrase reductases (ANR), which catalyze the reduction of nitrate to nitrite, subsequently converted to NH4 by nitrite reductase, are also members of this group [137,138]. The prototypic SO, an enzyme located in the mitochondrial intermembrane, catalyzes the biologically essential oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of sulfur-containing methionine and cysteine amino acids. 

In one experiment washed chloroplasts were isolated and assayed for nitrite reductase, DAHP synthase-Mn and chorismate mutase-1 activities (Table 3). Since enzymes may fractionate with organelles by non-specific (or specific) association with the organelle surface, latency determinations were made. With this approach, activity determinations are made before and after rupture of the washed chloroplasts. If activities are located within the organelle, they are expected to increase dramatically following organelle disruption. Thus, nitrite reductase (chloroplast marker enzyme) gave a latency value of 16, a value similar to those obtained for DAHP synthase-Mn and chorismate mutase-1. The identity of chorismate mutase as the CM-1 isozyme was confirmed by its sensitivity to inhibition by L-tyrosine. 

Regardless of whether the process of nitrate reduction is located in photosynthetic or nonphotosynthetic tissues, it still involves a cytoplasmically located nitrate reductase [reaction (6)] and a nitrite reductase complex [reaction (7)], which is located in plastids. Possible sources of reductant for these reactions have been discussed in several reviews (e.g., Lee, 1980 Abrol et al., 1983 Smirnoff and Stewart, 1985) and the conclusion reached that in heterotrophic (nonphotosynthetic) nitrate assimilation the NADH required by nitrate reductase might be derived from glycolysis, from the oxidative pentose phosphate pathway, or even from mitochondrial dehydrogenases (see I e, 1980), whereas the pentose phosphate pathway may be of singular significance in supplying NADPH for nitrite assimilation. As indicated for root tissue by Ernes et al. 

The next step— the reduction of nitrite to ammonia—again may take place throughout the plant, although most is known about the process as it occurs in green tissues. Nitrite reductase in photosynthetic plant parts is located within the chloroplasts and the reaction it catalyses results in the transfer of 6 electrons from the photosynthetic electron transport system via ferredoxin (eqn. 65) to each molecule of nitrite. 

Canvin and Woo (1979) reported that under certain conditions Antimycin A (mitochondrial ATP site II inhibitor) was more effective in enhancing nitrite accumulation (75% of anaerobic control) by leaf discs than either amytal or rotenone (ATP site I inhibitors). In plant mitochondria, the malate dehydrogenase located on the outside of the inner membrane is capable of utilizing external NADH. They infer that in leaves under dark aerobic conditions, the mitochondria effectively compete with nitrate reductase for cytoplasmic NADH. Confirmation of this competition was afforded by a reconstituted system consisting of mitochondria, nitrate reductase, nitrate and NADH or NAD, malate, and malate dehydrogenase (Reed and Hageman, 1977). Nitrite production under aerobic conditions was 10% that observed under anaerobiosis. 

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