Nitrous oxide reductases

Nitric oxide reductase (P) Nitrous oxide reductase (P) Ascorbate oxidase (P) Cytochrome oxidase (PM) Copper ATPase pumps (PM)... 

Nitrite reductase Nitrous oxide reductase Metallothionein ACE-1 (MAC)... 

Figure 3.13 Structure of the Cuz centre in nitrous oxide reductase. The central sulfide interacts with all four copper atoms. (From Rees, 2002. Copyright 2002 Annual Reviews.)...

Figure 14.13 Structure of the subunits of the homodimer nitrous oxide reductase (red and blue) and of the Cuz site. (From Chen et al., 2004. Reproduced with permission from John Wiley Sons., Inc.)...

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. 

Nitrosyidisulfonic acid, reaction mechanisms, 22 129, 130 Nitrous acid, 33 103 decomposition, rate constants, 22 157 as oxidizing agent, 22 133 reaction mechanisms, 22 143-156 electrophilic nitrosations, 22 144-152 with inorganic species, 22 148, 149 nitrite oxidation by metals, 22 152-154 oxidation by halogens, 22 154, 155 in solution, 22 143, 144 reduction by metals, 22 155, 156 Nitrous oxide reductase, 40 368 Nitroxyl, reaction mechanisms, 22 138 Nitrozation, pentaamminecobalt(III) complexes, 34 181... 

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... 

Bell, L. C., and Ferguson, S. J. (1991). Nitric and nitrous oxide reductases are active under aerobic conditions in cells of Thiosphaera pantotropha. Biochem. J. 273, 423-427. 

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. 

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. 

Snyder, S. W., and Hollocher, T. C. (1987). Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans. J. Biol. Chem. 262, 6515-6525. 

The expression and characterization of a recombinant subunit II of the archaebacterial terminal oxidase complex in Sulfolobus acidocaldarius was achieved. The binuclear CuA centre was shown to be correctly inserted. A protonation of one of the coordinating histidines was suggested from the pH-profile.109 The subunit is part of a supercomplex SoxM which also has been isolated in a catalytically competent form for the first time.110 Nitrous oxide reductase (NOR) was prepared from Hyphomicrobium denitrificans and charac-... 

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 5.1 Schematic representations of selected active sites of the copper proteins plastocyanin [56] (type 1, a) galactose oxidase [57] (type 2, b) oxy hemocyanin [58] (type 3, c) ascorbate oxidase [10] (type 4, or multicopper site, d) nitrous oxide reductase [59] (CuA site, e) cytochrome c oxidase [15]...

This type of active site is also known as a mixed-valence copper site. Similarly to the type 3 site, it contains a dinuclear copper core, but both copper ions have a formal oxidation state of +1.5 in the oxidized form. This site exhibits a characteristic seven-line pattern in the EPR spectra and is purple colored. Both copper ions have a tetrahedral geometry and are bridged by two sulfur atoms of two cysteinyl residues. Each copper ion is also coordinated by a nitrogen atom from a histidine residue. The function of this site is long-range electron transfer, and it can be found, for example, in cytochrome c oxidase [12-14], and nitrous oxide reductase (Figure 5.1 e). 

The Cuz active site consists of four copper ions, arranged in a distorted tetrahedron and coordinated by seven histidine residues and one hydroxide anion. This site was detected in nitrous oxide reductase [16, 17] (Figure 5.1g) and is involved in the reduction of N20 to N2. The copper ions in the tetranuclear cluster are bridged by an inorganic sulfur ion [18], which until recently was believed to be a hydroxide anion. Three copper ions are coordinated by two histidine residues, whereas the fourth is coordinated by only one, thus leaving a binding site for the substrate. 

The third class consists of proteins that are composed of one or more BCB domains fused to a sequence domain(s) characteristic of evolutionarily unrelated protein families. Such a mosaic domain organization has been found in the phytocyanin protein family, stellacyanins, uclacyanins, and the recently characterized dicyanins (Section V) in blood coagulation factor VIII (Section VIII) and in nitrous oxide reductase (Section IX). 

Fig. 11. The binuclear CuA (A) and tetranuclear CuZ (B) copper-binding sites of nitrous oxide reductase from Pseduomonas nautica (PDB Accession Code IQNl). The sulfur atom in the tetranuclear copper site is marked with an S.

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