Nitrous oxidase

FIGURE 4.6 A selective gallery of more complex organic cofactors which are found in metalloproteins. (a) The Moco cofactor (b) the FeMo-cofactor, (c) the P-cluster of nitrogenase, (d) the H-cluster of microbial hydrogenases, and (e) the Cuz cluster of microbial nitrous oxidases. 

P-cluster of nitrogenases, the three types of metal clusters found in microbial hydrogenases, the unusual common structural features of which include CO ligands, and the Cuz cluster of microbial nitrous oxidases. The biosynthesis of some of these cofactors is discussed later in this chapter. 

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

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. 

The nitrous acid deamination of 2-amino-2-deoxy-D-mannose (8), in the favored conformation, the amino group ofwhich is axially attached, leads, in contrast, uniquely to D-glucose,15 characterized, after oxidation by nitric acid, as D-glucaric acid (9). This result has also been verified by direct crystallization of the D-glucose and by assay with D-glucose oxidase.44"... 

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. 

The other copper-only binuclear centre to be considered is the CuA or purple copper complex. It is part of the terminal oxidase in mitochondrial respiration, cytochrome c oxidase (COX). Its EPR signature, a seven-line spectrum, has since long been known to be different from the classes type 1 to 3 and arises from two copper ions in a 1.5 valence (or mixed valence) state, first proposed from EPR-analysis of a similar center in nitrous oxide (N20) reductase. There is a close correspondence between the blue and purple states of copper since each of the two copper ions in CuA can be considered as being structurally related to the mononuclear blue site coordination. 

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-... 

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). 

Nitrite reductase (dissimilatory) Nitric oxide reductase Nitrous oxide reductase Nitrite reductase Hydroxylamine oxidase Nitrite oxioreductase Hydroxylamine reductase Hydroxylamine oxioreductase... 

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. 

As mentioned above, ammonia is oxidized to nitrous acid via hydroxylamine in N. europaea first ammonia is oxidized to hydroxylamine by the catalysis of ammonia monooxygenase, and hydroxylamine formed is oxidized to nitrous acid by the catalysis of hydroxylamine oxidoreductase. Molecular oxygen is not necessary to the reaction itself of NH2OH — HN02 (Yamanaka and Sakano, 1980) but it is required for the consumption of electrons liberated from the reaction, NH2OH + H20 —> HN02 + 4H+ + 4c. Electrons thus liberated are transferred first to cytochrome c-554, then to cytochrome c-552, and finally oxidized with molecular oxygen by the catalysis of cytochrome c oxidase. Based on the results described above, the electron transfer pathway in the oxidation of ammonia to nitrite or nitrous acid by N. europaea will be presented as shown in Fig. 3.3. 

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). 

Ritchie GAF, Nicholas DJD (1972) Identification of sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea. Biochem J 126 1189-1191 Ritchie GAF, Nicholas DJD (1974) The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea. Biochem J 138 471 180... 

One may mention the relative lack of information on the possible toxicity mechanisms of other groups of explosives. The administration of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX 30-300 mg kg-1 daily for 13 weeks) to rats caused hypotriglycidire-mia, convulsions, and death [4], In contrast, pentaerythritol tetranitrate (PETN 0.5%-1.0% in standard diet for 13 weeks) was nontoxic to rats [80], RDX was much less cytotoxic to V79 and TK-6 mammalian cell cultures than TNT [9], There also are very few data on their reactions with mammalian enzymes. Rabbit liver cytochrome P-450 2B4 (EC 1.14.14.1) converted RDX into 4-nitro-2,4-diazabutanal, two nitrite ions, ammonium, and formaldehyde, consuming one equivalent NADPH [81]. However, it is unclear whether this slow reaction (kcat < 0.01 s-1) may contribute to the toxicity of RDX. Xanthine oxidase transformed octohydro-l,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) at a lower rate, 10.5 nmol h 1 mg 1 protein under anaerobic conditions, into nitrite, formaldehyde, nitrous oxide, formic acid, and ammonium [82], Our preliminary observations show that RDX was much less reactive substrate for P-450R and E. cloacae NR than NTO or ANTA [53], Thus, the mechanisms underlying toxicity of RDX remain undisclosed. 

Finally, the ET reactivity of the binuclear Cua site present in cytochrome c oxidase and nitrous oxide reductase illustrates an additional interesting... 

Type 1 copper proteins are the class of proteins for which cupredoxins were originally named. Type 1 copper proteins include both proteins with known electron transfer function (e.g., plastocyanin and rusticyanin), and proteins whose biological functions have not been determined conclusively (e.g., stellacyanin and plantacyanin). Although these proteins with unknown function cannot be called cupredoxins by the strict functional definition, they have been classified as cupredoxins because they share the same overall structural fold and metal-binding sites as cupredoxins. In addition, many multidomain proteins, such as laccase, ascorbate oxidase, and ceruloplasmin, contain multiple metal centers, one of which is a type 1 copper. Those cupredoxin centers are also included here. Finally, both the Cua center in cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR), and the red copper center in nitrocyanin will be discussed in this chapter because their metal centers are structurally related to the type 1 copper center and the protein domain that contains both centers share the same overall structural motif as those of cupredoxins. The Cua center also functions as an electron transfer agent. Like ferredoxins, which contain either dinuclear or tetranuclear iron-sulfur centers, cupredoxins may include either the mononuclear or the dinuclear copper center in their metal-binding sites. 

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. 

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