Nitrate reductases enzymatic activity

Concerning nitrate uptake and assimilation (Albuzio et al., 1986) in barley seedlings incubated with HS from a grassland soil and their fractions—nitrate reductase (NR), glutamate dehydrogenase (GDH),and glutamine synthetase (GS) activities— were analyzed together with the rate of nitrate uptake. The enzymatic activities turned out to be stimulated by the treatment of unfractioned humus extract with increases compared to controls of 65%, 35% and 45% respectively. 

In all photoautotrophs, reduction of NOj" to NH4 is achieved in two distinct enzymatic steps (Campbell, 2001). First, assimilatory nitrate reductase (NR) catalyzes the two electron reduction from NOj" to NO2. NR is a large soluble cytoplasmic enzyme with FAD (flavin adinine dinucleotide), an iron-containing cytochrome and molybdopterin prosthetic groups, and requires NADH and/or NADPH as an electron donor (Guerrero et al, 1981). Functional NR is in the form of a homodimer and therefore requires two atoms of iron per enzyme. Following transport into the chloroplast, NO2 undergoes a 6 e reduction to NH4 via assimilatory nitrite reductase (NiR). NiR, a soluble chloroplastic enzyme, contains five iron atoms per active enzyme molecule, and requires photosynthetically reduced ferredoxin as an electron donor (Guerrero et al., 1981). 

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. 

Nitrate reductases (NaR) with an iron-sulfur center are used for nitrate conversion. Generally, nitrate is enzymatically reduced and NaR is in the oxidized form, which can be electrochemically reduced. However, the direct electron transfer between an enzyme and an electrode is strongly limited due to the fact that (1) the distance between the electrode surface and the redox active site of the enzyme, which is normally inside the globular protein, is large and (2) the orientation of donor to acceptor sites depends on the method of the immobilization of the enzyme at the electrode.Thus, low molar mass redox mediators including qui-nones, metal complexes, ferricyanide, derivatives of ferrocene, and organic redox dyes " have been used to facilitate the electron transfer between electrode and enzyme (Fig. 11.5). 

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