Nitrite and Nitrous Oxide Reductases

Mechanisms of Biological Electron Transfer 884 3. Nitrite and Nitrous Oxide Reductases... 

Nitrite reductases and nitrous oxide reductases are relatively newly found copper-containing proteins involved in bacterial denitrification. N2O reductase may bear a relationship to cytochrome oxidase and, indeed, parallels it somewhat in function, being the terminal electron acceptor in its pathway. 

Jiingst, A., Braun, C., and Zumft, W. G. (1991a). Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification. Mol. Gen. Genet. 225, 241-248. 

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 ... 

Anaerobic Growth. In the absence of molecular oxygen, electron flow is dependent on the availability of N-oxides that may serve as alternative electron acceptors 8,10,44, 45, 46). At the shift from oxygen limitation to anaerobiosis, the makeup of the respiratory network drastically changes. The expression of nitrate, nitrite, nitric oxide, and nitrous oxide reductases as well as of the blue copper-containing electron carrier pseudoazurin is established, and in a concerted action these terminal oxidoreductases couple the removal of electrons from the respiratory network to the reduction of the corresponding... 

One interesting feature of cupredoxins is that members of its family display a variety of intense and beautiful colors, from blue (e.g., plastocyanin and azurin), to green (e.g., plantacyanin and some nitrite reductases), to red (e.g., nitrosocyanin), to yellow (e.g., some model cupredoxin proteins and compounds), and to purple (e.g., Cua center from cytochrome c oxidase and nitrous oxide reductase). This rainbow of colors makes cupredoxins both fun to work with and challenging to study. The structure and function of each of the cupredoxin and structurally related centers will be described below and the origin of the color will be explained. 

Each step of the denitrification pathway is catalyzed by a distinct enzyme, nitrogen oxide reductase (nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase), that transfers electrons from the chain to the particular intermediate. Thermodynamically, in the absence of oxygen, nitrogen oxides are the most preferred electron acceptors by facultative bacterial groups. The role of nitrogen oxides in regulating organic matter decomposition has been discussed in earlier chapters (see Chapter 5). 

Nitrite Reductase. Bacterial denitrification in anaerobic microorganisms involves a four-step process, with the overall reduction of nitrate (N03 ) to dinitrogen (N2). Each reaction in the reduction process is catalyzed by one or more different metalloenzymes, which have various transition metals found in diverse ligand environments nitrate reductase (NOR, molybdenum), nitrite reductase (NiR, iron or copper), nitric oxide reductase (NOR, heme and nonheme iron), and nitrous oxide reductase (N2OR, copper) (9). 

Although the pathway of Eq. (1) is now based on much evidence (Section 111) and is unambiguous in the case of at least one bacterium [Pseudomonas stutzeri strain Zobell (f. sp. P. perfectomarina)], there have been alternative hypothesis. One hypothesis, advanced by the Hollocher group (Garber and Hollocher, 1981 St. John and Hollocher, 1977), considered NO as a likely intermediate, but one that remained at least partly enzyme-bound and was not entirely free to diffuse. This view was based on the outcome of certain kinetic and isotope experiments which can be summarized as follows. When denitrifying bacteria were challenged simultaneously with [ N]nitrite and ordinary NO, the cells reduced both compounds concomitantly to N2 (or to N2O in the presence of acetylene which is a specific inhibitor (Balderston et al., 1976 Yoshinari and Knowles, 1976) of nitrous oxide reductase). In the process, little NO was generally detected in the gas phase pool of NO and there was relatively little isotopically mixed N2O formed. That is, most of the N and N reduced to NjO appeared as N2O... 

Wharton, D. C., and Weintraub, S. T. (1980). Identification of nitric oxide and nitrous oxide as products of nitrate reduction by Pseudomonas cytochrome oxidase (nitrite reductase). Biochem. Biophys. Res. Commun. 97, 236-242. 

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...

Figure 12 A diagram of the nitrogen cycle with catalyzing enzymes and metal requirements of each step. NIT, nitrogenase AMO, ammonium mono-oxygenase HAO, hydroxylamine oxidoreductase NAR, membrane-bound respiratory nitrate reductase NAP, periplasmic respiratory nitrate reductase NR, assimila-tory nitrate reductase NIR, respiratory nitrite reductase NiR, assimilatory nitrite reductase NOR, nitric oxide reductase N2OR, nitrous oxide reductase.

Biochemically, in denitrification electrons are transported via cytochromes to nitrogen oxide reductases. These reductases are indicated in the scheme below as follows (1) nitrate reductase, (2) nitrite reductase, (3) nitric oxide reductase, and (4) nitrous oxide reductase. 

Denitrification enzyme activity Catabolic nitrate redaction involves several enzymes including dissimilatory nitrate rednctase (nitrate to nitrite), nitrite rednctase (nitrite to nitric oxide), nitric oxide reductase (nitric oxide to nitrons oxide), and nitrons oxide reductase (nitrous oxide to nitrogen gas). The combined effect of all these enzymes is operationally dehned as denitrification enzyme activity (DEA), which is now rontinely measnred under... 

In the biochemical context, it has been well noted that the chemistry of NO (and of its conjugated acid, FINO (i8)) is quite distinct from that of NO (22). Its occurrence in catalytic cycles of NO synthase, nitrite reductase, and nitric oxide reductase (NOR) has been postulated (23). For example, in the multi-iron containing enzyme NOR two NO molecules are converted reductively to nitrous oxide, N2O, with nitroxyl (NO ), and hyponitrite (N202 ) (24) as putative intermediates (23). 

Heme di (107), which was isolated by Timkovich et al (70) and Chang et al (71) occurs as one of two cofactors in the reductase cytochrome cd. Cytochrome cd participates in the reduction of nitrite to nitrous oxide (N2O) in chemoautotrophic bacteria, such as Pseudomonas aeruginosa, Paracoccus denitrificans, and Thiobacillus denitrificans (13). From recent investigations it seems very likely that cytochrome cdi mediates the nitrite reduction to nitric oxide (NO) and that a second enzyme produces N2O from NO (13). A structure was... 

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. 

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. 

Nitric oxide and iron nitrosyl complexes have been observed in the reduction of nitrite by bacterial nitrite reductases, which contain iron chlorin or iron isobac-terichlorin [151]. A specific nitric oxide reductase also exists to convert NO to nitrous oxide [9]. Iron complexes of chlorins, isobacteriochlorins, and porphyrins, as well as ruthenium and osmium polypyridines, and cobalt and nickel... 

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