Nitric Oxide-Reducing Nitrite Reductases

In contrast, many gram-negative bacteria contain a nitrate reductase (EC 1.7.99.4 and/or 1.9.6.1) that also reduces nitrate to nitrite although under anaerobic conditions. The dissimilatory nitrite reduction leading to denitrification encompasses then the reduction of nitrite to nitric oxide by dissimilatory nitrite reductases (NiR, EC 1.7.2.1) that, in combination with nitric oxide reductases (NOR) and nitrous oxide reductases (N2OR), transform nitrite into nitrogen ... 

Nitric oxide formed from nitrite by the catalysis of nitrite reductase is next reduced to nitrous oxide (N20). The reduction of nitric oxide is catalyzed by nitric oxide reductase (NO reductase) which is a cytochrome ebb (Carr and Ferguson,... 

The bacterial, iron-containing cd nitrite reductases constitute another family of enzymes catalyzing the one-electron reduction of nitrite to nitric oxide (74). These enzymes are homodimers of 60-kDa subunits, each containing one heme-c and one heme-rii. Extensive studies have established heme-c as the electron entry site, whereas heme-dj is the catalytic center where nitrite is reduced (95). Three-dimensional structures of two different cytochromes cd have been determined in oxidized and reduced states P. pantotrophus, Pp-NiR (96, 97) and P. aeruginosa, Pa-NiR (98, 99). In both enzymes, heme-c is covalently linked to the A-terminal a-helical domain and heme-di is bound noncovalently to the C-terminal B-propeller domain (Fig. 17). Intramolecular ET between c and di hemes is an essential step in the catalytic cycle this reaction has been studied by several groups using different methods (95, 100-106). The rate constants for Pa-NiR are on the order of 1 s (95, 100-102, 104), while intramolecular ET in Pp-NiR is significantly faster (rate constant of... 

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. 

Nitrite reductase (NAD(P)H) [EC 1.6.6.4] catalyzes the reaction of three NAD(P)H with nitrite to yield three NAD(P)+, NH4OH, and water. Cofactors for this enzyme include FAD, non-heme iron, and siroheme. (2) Nitrite reductase (cytochrome) [EC 1.7.2.1] is a copper-depen-dent system that catalyzes the reaction of nitric oxide with two ferricytochrome c and water to produce nitrite and two ferrocytochrome c. (3) Ferredoxin-nitrite reductase [EC 1.7.7.1], a heme- and iron-dependent enzyme, catalyzes the reaction of ammonia with three oxidized ferredoxin to produce nitrite and three reduced ferredoxin. (4) Nitrite reductase [EC 1.7.99.3] is a copper- and FAD-dependent enzyme that catalyzes the reaction of two nitric oxide with an acceptor substrate and two water to produce two nitrite and the reduced acceptor. 

Deletion of the regulatory nirQ gene results simultaneously in a loss of the ability to reduce both nitrite and NO in P. stutzeri (Braun and Zumft, 1992). Nitrite reductase is synthesized and is active in vitro but nitric oxide reductase is synthesized in an inactive form. Although the exact function of the nirQ gene product is unknown, the gene encodes a protein homologous with the NtrC family of transcriptional activators (Zumft, 1993). 

Yamanaka and co-workers (364-366) have crystallized a cytochrome oxidase from P. aeruginosa which oxidizes Pseudomonas ferrocytochrome c-551. It is also capable of nitrite reduction with a turnover number of 4000 moles nitrite reduced under anaerobic conditions to nitric oxide per minute at 37°. It is an adaptive enzyme, nitrate being essential for its biosynthesis. The enzyme has a molecular weight of 120,000, with two subunits of equivalent molecular weight, 2 heme c and 2 heme d groups per mole (Fig. 38) (366a). Nitrite reductase activity is 94% inhibited by 8 X 10 M KCN, but only by CO. The lack of CO inhibition appears to be related to the fact that the enzyme has a greater affinity for nitrite than for carbon monoxide. 

Figure 2 The dissimilatory denitrification pathway. NO3 is reduced to NO2 by a membrane-bound or periplasmic nitrate reductase(NaR). N02 is reduced to NO by either a cytochrome cdi or copper nitrite reductase (NiR). NO is reduced to N2O by nitric oxide reductase (NOR). N2O is reduced to N2 by nitrous oxide reductase (N2OR). Electron transport from uhiquinol (UQH2) at NaR and the cyt hcj complex is coupled to generation of a proton gradient...

Next, nitrite is reduced to nitric oxide by the catalysis of nitrite reductase. Two kinds of nitrite reductases are known in the denitrifying bacteria (N2 forming) cytochrome cdx-type enzyme (Yamanaka et al., 1960, 1961, 1963 Yamanaka and Okunuki, 1963a,b,c Yamanaka, 1964) and copper protein-type enzyme (Iwasaki and Matsubara, 1972). However, no case has been found in which one species of the denitrifying bacterium has both types of the enzymes simultaneously (Coyne et al., 1989). 

Xanthine oxidase can reduce nitrate to nitrite (Westerfield et al. 1959, Fridovich and Handler 1962). Xanthine oxidase and dissimilatory nitrate reductase share structural similarities. Both are molybdoenzymes and contain flavin adenine dinucleotide and Fe/S clusters (McCord 1985, Mitchell 1986, Payne et al. 1997). Zhang et al. (1998) reported that both purified bovine buttermilk xanthine oxidase and xanthine oxidase-containing inflamed human synovial tissue can generate NO by reducing nitrite in the presence of NADH. This nitrite reductase activity of xanthine oxidase may act as a supplement to the activity of nitric oxide synthase (NOS) to redistribute blood flow to ischaemic tissues when NOS activity is absent. 

The organization of the enzymes of denitrification in gram-negative bacteria, as determined by antibody labelling and electron microscopy studies, is shown in Figure 5. The first enzyme, nitrate reductase (NaR), resides in the cytoplasmic membrane with its active site accessed from the cytoplasmic side, necessitating transport of nitrate across both the periplasmic and cytoplasmic membranes. The product nitrite is transported back into the periplasmic space, where it is reduced by the nitrite reductase (NiR). Most NiR s appear to be soluble enzymes, although there have been reports of preparations in which the activity was associated with membrane fractions. The nitric oxide reductase (NoR) is also localized in the cytoplasmic membrane, and releases its product N2O back into the cytoplasmic space, where the soluble enzyme nitrous oxide reductase (NoS) converts it to N2. 

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