Nitrate reductase reducing substrates

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-... 

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. 

Fig. 1. The nitrate assimilation pathway in higher plants. The pathway of nitrate assimilation in the tobacco leaf is illustrated. In some other species an additional cytosolic GS is found in the leaf. The pathway in plant roots is more poorly documented and more variable GS in roots is mostly cytosolic, and some enzymes such as GOGAT are found as isoforms utilising alternate reducing substrates. T, expected nitrate carrier NR, nitrate reductase NiR, nitrite reductase GS, glutamine synthetase GOGAT, glutamate synthase Fd, ferredoxin Gin, glutamine Glu, glutamate.

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. 

Nitrate Reductase Activity. There are similarities between induced nitrate reductase activity and induced iron stress response. In both, biochemical reactions are induced, and a substrate is reduced N03 to N02 by nitrate reductase and Fe3+ to Fe2+ by a reductant activated in response to iron stress. Chemical reactions induced by iron stress increased the use of iron, and simultaneously increased nitrate reductase activity in roots (Figure 5) and in tops of iron-efficient tomato. This induced nitrate reductase activity declined when iron was made available to the plants. 

A cycle for substrate reduction can be devised by beginning at the reduced Mo(IV) state and reversing the direction of OAT and CEPT steps, as illustrated for the nitrate reductase reaction in Fig. 9(b). Electrons are provided to reduce Mo(VI) to Mo(IV) via cytochrome b and FAD. 

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... 

Certain bacteria can utilize nitrate nitrogen as the sole nitrogen source for the synthesis of all nitrogen containing compounds of the cell (Payne, 1973). This nitrate assimilation can occur under both aerobic and anaerobic conditions. In other instances (Payne, 1973) nitrate serves as a terminal hydrogen acceptor under anaerobic conditions and this process is called nitrate respiration. In both cases the product of nitrate reduction is nitrite. The nitrate reductases from bacteria have been differentiated by Pichinoty and Piechaud (1968) into nitrate reductase A which is membrane bound and can reduce chlorate in addition to nitrate as a substrate and nitrate reductase B which is... 

Dissimilatory nitrate reductases (Pichinoty type A) in membrane fractions from bacteria have been shown capable of utilizing a variety of respiratory Intermediates and reduced pyridine nucleotides for nitrate reduction (Cole and Wimpeny, 1968 Knook et ai, 1973 Burke and Lascelles, 1975 Enoch and Lester, 1975). Reduction of nitrate by the membrane fractions, when respiratory substrates or pyridine nucleotides serve as reduc-tant, is generally inhibited by azide, cyanide and p-chloromercuribenzoate. Nitrate reduction, mediated by respiratory substrates, could be inhibited by n-heptylhydroxyquinoline-N oxide (HONO) or dicoumoral (Ruiz-Herrera and DeMoss, 1%9 Knook et al., 1973 Burke and Lascelles, 1975). However, in Micrococcus denitrificans (Lam and Nicholas, 1969) and in Bacillus stearothermophilus (Downey, 1%6) nitrate reduction is not inhibited by... 

When particulate preparations from bacteria grown anaerobically in the presence of nitrate are incubated with respiratory substrates or reduced pyridine nucleotides, it is observed that the cytochromes become reduced (Knook et ai, 1973 Lam and Nicholas, 1969 Ruiz-Herrera and DeMoss, 1969 Vila et al., 1977). Because the cytochrome b component is reoxidized when nitrate is added, it appears the Mo-protein (nitrate reductase) transfers electrons from cytochrome b to nitrate. These observations on dissimilatory nitrate reduction by bacteria are summarized in the following scheme. 

The denitrification process could be described as a modular organization in which every biochemical reaction is catalyzed by specific reductase enzymes (Cuervo-Lopez et al., 2009). Four enzymatic reactions take place in the cell as follows (l) nitrate is reduced to nitrite by nitrate reductase (Nar) (ii) a subsequent reduction of nitrite to nitric oxide is carried out by nitrite reductase (Mr) (iu) afterwards, nitric oxide is reduced to nitrous oxide by the enzyme nitric oxide reductase (Nor) (iz ) finally, nitrous oxide is reduced to N2 by the enzyme nitrous oxide reductase (Nos) (Lalucat et al., 2006) (Table 9). These reactions take place when environmental conditions become anaerobic (Berks et al., 1995 Hochstein Tomlinson, 1988). The enzymatic reactions, which are thermodynamically favored, are carried out in the cell membrane and periplasmic space. Small half saturation constant values (Km) have been reported for different nitrogen substrates for some denitrifying bacteria, indicating that denitrifying enzymes have a high affinity for their substrate. However, several factors have to be considered, as the presence of small quantities of molybdenum, cooper and hem to ensure the successful enzymatic activity, as they are known cofactors for denitrifying enzymes. 

Finally, and perhaps most importantly, for nitrate reductase and sulfite oxidase, we have to inquire about the number of electrons transferred from the enzyme to the substrate, or vice versa. This brings us to the question of the valence states between which molybdenum in the enzymes cycles in the turnover processes. For both of these enzymes, the overall reaction is a two-electron process, as is the xanthine oxidase reaction. For xanthine oxidase, the evidence (Olson et al, 1974) favors xanthine reducing Mo(VI) directly to... 

The principles of the coordinated control of sulfate and nitrate assimilation were formulated by Reuveny and Filner (1977) following their pioneering work on the effect of nitrate and sulfate stress on the levels of ATP-sulfiirylase and nitrate reductase in cultured tobacco cells. Coordinated control involves independent internal control of the sulfate and nitrate assimilation pathways by their own substrates and products (such as discussed for the enzymes of sulfate assimilation in the previous section) and stimulation of the pathways of sulfate and nitrate assimilation by products of the other pathway. Thus for the sulfate assimilation pathway this involves stimulation by a reduced form of nitrogen while for the nitrate assimilation pathway it involves stimulation by a reduced form of sulfur. 

The conditions under which these function and their regulation depend on the organism. For example, in Escherichia coli, oxygen represses the synthesis of the other reductases, and under anaerobic conditions the reductases for fumarate, DMSO, and TMAO are repressed by nitrate. This does not apply to Wolinella succinogenes in which sulfur represses the synthesis of the more positive electron acceptors nitrate and fumarate (Lorenzen et al. 1993). The DMSO reductase from Escherichia coli (Weiner et al. 1988) has a broad substrate versatility, and is able to reduce a range of sulfoxides and A-oxides. Anaerobic sulfate reduction is not discussed here in detail. 

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