Enzyme nitrous oxide reductase

Cu(I) state. It has been proposed that a site of similar structure is present in the enzyme nitrous oxide reductase (96, 97). 

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. 

The final step in the denitrification process is carried out by the soluble enzyme nitrous oxide reductase (NoS). This enzyme has been isolated from a number of sources, and is unusual in a number of ways. In most cases, it is a homodimer of ca. 74 kDa subunits with ca. 4 Cu/subunit, but the enzyme is bright purple or pink as isolated, depending on conditions, and becomes the typical blue color expected for copper proteins only after reduction with dithionite. A variety of spectroscopic studies strongly suggest that the enzyme contains at least one mixed-valent, thiolate-bridged Cu(l)—Cu(ll) unit that may well be similar to a binuclear copper center in cytochrome c oxidase (23). The reaction catalyzed by the enzyme is deceptively simple ... 

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... 

Coyle, C. L., Zumft, W. G., Kroneck, P. M. H., Kiimer, H., and Jakob, W. (1985). Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina. Purification and properties of a novel multicopper enzyme. Eur. J. Biochem 153, 459-467. 

Jones, A. M., Hollocher, T. C., and Knowles, R. (1992). Nitrous oxide reductase of Flexibacter canadensis A unique membrane-bound enzyme. FEMS Microbiol. Lett. 92, 205-210. 

A new representative of a multicopper cluster in a protein is Cuz in nitrous oxide reductase. As was discussed above this enzyme contains a binuclear CuA centre as in COX. While the latter in addition has CuB in the form of a copper-heme group, N20 reductase has Cuz which is the site of dinitrogen formation from the substrate N20. Recently a central inorganic sulfide has been found as a ligand to copper and multiple forms of Cuz were detected in the enzyme from Paracoccus pantotrophus.134 More recently a tetranuclear copper cluster with X-S bridges was proposed as structure for Cuz..135... 

Reduction of N20 to N2 by bacteria (Eq. 18-30, step d) is catalyzed by the copper-containing nitrous oxide reductase. The purple enzyme is a dimer of 66-kDa subunits, each containing four atoms of Cu.353 It has spectroscopic properties similar to those of cytochome c oxidase and a dinuclear copper-thiolate center similar to that of CuA in cytochrome c oxidase (p. 1030). 

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.

A comparative study of the metal centers in cytochrome c oxidase from several bacterial sources, including Thermus thermophilus and P. denitrificans, using EPR and MCD spectroscopy has established that in both cases cytochrome a is liganded by two histidine oxidases and the Cua center is identical to that in bovine cytochrome c oxidase (105, 106). The properties of the cytochrome Os/Cub dimer have not been established to be identical, although ferrocytochrome 03 is high-spin ferrous, as expected. Recent studies of the MCD properties of the Cua center in cytochrome c oxidase and a copper center in nitrous oxide reductase (107,108) show that the two centers are virtually identical. The evidence from the EPR hyperfine structure of the copper center in nitrous oxide reductase suggests that the center in this enzyme is a mixed-valence Cu(I)/Cu(II) dimer, which raises the interesting prospect that the Cua center in cytochrome c oxidase is also a dimeric copper species. 

Fujiwara and Fukumori, 1996). Nitric oxide reductase is also known, which lacks heme C and uses quinol as the electron donor (Suharti et al., 2001 de Vries et al., 2003). The cytochrome ebb-type enzyme has a molecular structure similar to the structure of the Cub binding portion in cytochrome c oxidase (Saraste and Castresana, 1994 Van der Oost et al., 1994 Zumft et al., 1994). Moreover, quinol NO reductase from Bacillus azotoformans is known to contain Cua. Nitrous oxide is further reduced to nitrogen gas (N2) by the catalysis of nitrous oxide reductase (N20 reductase) which is a multi-copper protein (Zumft and Matsubara, 1982). The structure of the copper-binding portion in the enzyme has been reported also to be similar to the structure of the Cua binding portion of cytochrome c oxidase (Chamock et al., 2000). 

Typical assessments of denitrification activity only assess the activity of the first three enzymes and do not consider the sensitivity of nitrous oxide reductase to toxic compounds. However, as hypothesized by others [14,15], it appears that nitrous oxide reductase is much more sensitive to TNT than the other three enzymes based on corresponding EC50 values of 400 mg kg-1 for the first three enzymes and 26 mg kg-1 for nitrous oxide reductase [10],... 

Nitrous oxide reductases are soluble, periplasmic Cu-containing enzymes encoded by nosZ that catalyze the final step in denitrification (Equation (10)). This reaction is difficult chemistry, since N2O is a poor ligand for transition metals, is kinetically inert, and none of the known metal/N20 complexes have been structurally characterized. No CU/N2O chemistry, other than that associated with N2OR, has been reported ... 

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). 

All purified nitrous oxide reductases to date are multicopper enzymes. N2O can serve as the terminal electron acceptor, catalyzing the overall 2e reduction in equation (16). Characterization of the copper sites in such a reductase has been described using resonance Raman spectroscopy. ... 

Nitrous oxide is reduced to nitrogen gas by the membrane-bound nitrous oxide reductase. The enzyme is inhibited by CO, CN, and azide indicating the involvement of a metal component (Thauer et al., 1977). No physiological donors have been identified and the enzyme is assayed using FADH2, reduced viologen dyes, or dichlorophenol indophenol as electron donors. 

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