Nitrate reductase enzyme

A substituted pyrrole (Fig. 7c) was electropolymerised across the pores of an alumina membrane [83] and the resultant polymer film then acted as a permselective membrane across which anions could cross. This was then used to separate an analyte solution from an internal sensing solution containing nitrate reductase enzyme and methyl viologen. 

Nitrate reductase catalyzes the first step in conversion of nitrate to ammonia, the reduction of nitrate to nitrite (N02 ). The reaction multisubunit nitrate reductase enzyme, with Mr of about 800 kilodaltons, contains bound FAD, molybdenum, and a cytochrome called cytochrome 557 (which contains an Fe4S4 complex). Nitrate reductase carries out the overall reaction ... 

Genes affecting reaction to nutritional factors including phosphorus, iron, and chloride and the presence/absence of a constitutive nitrate reductase enzyme occur in soybean. Palmer et al. (2004) discuss these as well as leaf flavonol glucosides. 

The uptake of nitrate and subsequent conversion to reduced nitrogen in cells requires a change of five in the oxidation state and proceeds in a stepwise fashion. The initial reduction takes place via the nitrate/nitrite reductase enzyme present in phytoplankton and requires large amounts of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and of adenosine triphosphate (ATP) and thus of harvested light energy from photosystem II. Both the nitrogenase enzyme and the nitrate reductase enzyme require iron as a cofactor and are thus sensitive to iron availability. 

Nonlegumes respond to added Mo because of the requirements for Mo by their nitrate reductase enzymes. Molybdenum additions in the presence of high amounts of N in the soil have prevented depressed crop yields due to excess N. Grains such as com, wheat, and rice have shown improved utilization of N following Mo application, but again, not all cultivars have the same Mo requirements. Many crops in the Brassica family respond to added Mo. Some of the early studies describing whiptail in cauliflower also found that different Brassica crops required different amounts of Mo. 

DNRA is a process that reduces nitrate to ammonia (Figure 8.43). This process is carried out by dissimilative nitrate reductase enzymes. Dissimilative nitrate reductases are membrane-bound proteins, whose synthesis is repressed by molecular oxygen and thus are only synthesized under anoxic conditions. For this reason, DNRA is mediated by obligate anaerobes that use nitrate as alternate electron acceptor in the process of cellular respiration. 

Nitrate reductase enzymes which catalyse the reduction of nitrate to nitrite. All N.r. studied so far contain iron and molybdenum. In the sequence of electron transfer, molybdenum appears to be the ultimate acceptor, which then transfers electrons to nitrate during this process the molybdenum alternates between Mo(V) and Mo(VI). Dissimilatory N.r. from E. coii (also called respiratory N.r.) (EC 1.7.99.4) is a transmembrane protein, containing Mo, inorganic sulfur and nonheme iron, approximate M,... 

As mentioned above plants may utilise nitrate or ammonium ions as nitrogen source— the nitrate being converted to ammonia during assimilation. Associated with this variation in the otherwise monotonous plant diet of carbon dioxide, water and inorganic salts is one of the few examples of inducible enzymes which have been found in higher plants. The s5mthesis of nitrate reductase enzyme system is inhibited by ammonia in whose presence it is redundant and stimulated by nitrate for whose assimilation it is essential. 

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

Sulfate reducers can use a wide range of terminal electron acceptors, and sulfate can be replaced by nitrate as a respiratory substrate. Molybdenum-containing enzymes have been discovered in SRB (also see later discussion) and, in particular, D. desulfuricans, grown in the presence of nitrate, generates a complex enzymatic system containing the following molybdenum enzymes (a) aldehyde oxidoreduc-tase (AOR), which reduces adehydes to carboxylic acids (b) formate dehydrogenase (FDH), which oxidizes formate to CO2 and (c) nitrate reductase (the first isolated from a SRB), which completes the enzy-... 

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

D. desulfuricans is able to grow on nitrate, inducing two enzymes that responsible for the steps of conversion of nitrate to nitrite (nitrate reductase-NAP), which is an iron-sulfur Mo-containing enzyme, and that for conversion of nitrite to ammonia (nitrite reduc-tase-NIR), which is a heme-containing enzyme. Nitrate reductase from D. desulfuricans is the only characterized enzyme isolated from a sulfate reducer that has this function. The enzyme is a monomer of 74 kDa and contains two MGD bound to a molybdenum and one [4Fe-4S] center (228, 229) in a single polypeptide chain of 753 amino acids. FXAFS data on the native nitrate reductase show that besides the two pterins coordinated to the molybdenum, there is a cysteine and a nonsulfur ligand, probably a Mo-OH (G. N. George, personal communication). 

There are four different classes of nitrate reductases (234). The nitrate reductases from D. desulfuricans show a strong homology to the a-subunit of the class of periplasmic respiratory nitrate reductases, and also to some of the enzymes that are included on the class of cytoplasmic assimilatory nitrate reductases. Because of this fact, a proposal was made for a new class of monomeric NAP, which contains the minimal arrangement of metal centers to perform nitrate reduction one [4Fe-4S] cluster and a Mo bound to two MGD. 

Similar difficulties have been encountered in the case of complex enzymes such as fumarate reductase and nitrate reductase from E. coli, in which substituting certain Cys ligands led to the loss of several if not all the iron-sulfur centers (171, 172). However, in the case of nitrate reductase, which possesses one [3Fe-4S] and three [4Fe-4S] centers, it was possible to remove selectively one [4Fe-4S]... 

Some of the above mentioned studies also use two-layer ONIOM QM MM approaches to include the full protein in an MM description. Other examples of QM MM calculations of metal enzymes include heme oxygenase [89], nitrate reductase [90] and peptide deformylase [91]. Finally, we note that the ONIOM (I IF Amber) potential energy surface has been directly used in a molecular dynamics study (ONIOM/MD) of cytidine deaminase [92],... 

For nitrate reductase, evidence on the role of molybdenum in the catalytic mechanism of the enzyme from Neurospora was first presented in 1954 by Nicholas and Nason (21) and the position seems to have changed relatively little since then. The original conclusion (23) was that molybdenum functions as an electron carrier in the sequence ... 

So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain. 

The first interest in the electroreduction of N02 or NO catalyzed by metal complexes is to model the activity of nitrite reductase enzymes.327 There is also an extensive growth in studies related to the development of metal complex-based electrochemical sensors for NO determination in biological and environmental samples 328 329 Nitrate disproportionates to nitric oxide and nitrate in aqueous solution. 

The latter authors demonstrate in their latest paper101) that bluelight (under physiological conditions stimulating conidiation and LIAC) will depress nitrate reductase activity and increase the activity of the smaller subunit of the enzyme complex in Neurospora. It is therefore suggested to be a key enzyme for physiological bluelight action. 

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