Copper protein-type nitrite reductase

NOH is changed to nitrous oxide under oxygen-deficient conditions (Hooper and Terry, 1979), though the reduction of nitrite (or nitrous acid) once fronted to the gas may also occur (Poth and Focht, 1985 Yoshida, 1988). Indeed, the bacterium has a copper protein-type nitrite reductase which catalyzes the reduction of nitrite to nitric oxide and/or nitrous oxide (Hooper, 1968 Ritchie and Nicholas, 1972, 1974 Miller and Wood, 1983 DiSpirito et al., 1985). However, as Beaumont et al. (2002) have recently found that nitrous oxide is produced even by N. europaea in which the DNA encoding nitrite reductase has been destroyed, the formation of nitrous oxide by the bacterium seems attributable to the decomposition of NOH. Camera and Stein (2007) have recently reported similar results about the formation of nitrous oxide by the nitrite reductase-deficient mutant of the bacterium. 

The copper-protein type nitrite reductases have been purified from several denitrifying bacteria. They have 2 1 copper atoms in the molecule one copper per subunit (30 10 kDa). The enzymes catalyze the reduction of nitrite to nitric oxide, but the physiological electron donors for the enzymes have not been clarified, though they are said to be cytochrome c in some cases and a copper-protein in some cases (see Otsuka and Yamanaka, 1988). 

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). 

The physiological electron-donor of nitrite reductase is the type 1 copper protein pseudoazurin, which transfers a single electron to the type 1 copper center of nitrite reductase, from where it is transferred to the type 2 copper... 

It is interesting to speculate why nitrite reductase has its type I coppers in domains 1, whereas in hCP the mononuclear copper binding sites are retained in the domains 2,4, and 6 where they are comparatively buried in the protein. One possible reason can be related to the difference in functions of the two proteins. NR has to interact with a relatively large pseudo-azurin macromolecule in order for electron transfer to take place,... 

The NO/NO+ and NO/NO- self-exchange rates are quite slow (42). Therefore, the kinetics of nitric oxide electron transfer reactions are strongly affected by transition metal complexes, particularly by those that are labile and redox active which can serve to promote these reactions. Although iron is the most important metal target for nitric oxide in mammalian biology, other metal centers might also react with NO. For example, both cobalt (in the form of cobalamin) (43,44) and copper (in the form of different types of copper proteins) (45) have been identified as potential NO targets. In addition, a substantial fraction of the bacterial nitrite reductases (which catalyze reduction of NO2 to NO) are copper enzymes (46). The interactions of NO with such metal centers continue to be rich for further exploration. 

While nitrite reductases in many bacteria are haem proteins, some are copper containing homotrimers, which bind three Type I and three Type II copper centres. The Type I copper... 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

The nature of the copper in these proteins is not totally clear. Dooley et al (1988) reported that the Achromobacter protein may have two kinds of type I sites in a total of three copper sites per dimeric protein, while the A. faecalis protein was reported to be a tetrameric protein with both type I and type II coppers (KakutanielaZ., 1981). Interestingly, the Achromobacter protein is green. Both of these nitrite reductases accept electrons from a cupredoxin. 

The crystal structure of nitrite reductase (NiR) from Achromobacter cycloclastes was recently reported by Godden et al. (46). The protein contains both a type I and a type II copper site. The type I center has a distorted tetrahedral structure typical of type I copper, whereas the... 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

There are four different types of nitrite reductases the copper-containing protein Copper Enzymes in Denitrification and cytochrome cd perform a one-electron rednetion of nitrite to nitric oxide, and are involved in denitrification " the siroheme-containing protein and the cytochrome c ititrite reductase (cNiR) both perform the complete, six-electron reduction, of nitrite to ammonia. The cNiR is present in the y, 5 or e-subclasses of proteobacteria, and is encoded by the nrf operon (nitrite reduction with /ormate), which has different gene composition in the different classes of bacteria, having in common only the gene for the catalytic subunit, ntfA. 

Nitrite reductases catalyze both of the reactions below the physiological electron donors are either c-type cytochromes or small blue-copper proteins (eqnations 1 and 2). h28 xhe Type 1 center acts as an electron-accepting site, which then transfers the electron to the Type 2 copper where snbstrate binding and rednction occur. 

The copper-containing nitrite reductase from A. cycloclastes may also have evolved from this ancestral oxidase. Nitrite reductase is a two-domain protein that functions as a trimeric molecule. During its evolution from the ancestral copper oxidase, a gene inversion must have occurred, so that domain 2 of the ancestral oxidase is now domain 1 of nitrite reductase. Domain 1 of the ancestral oxidase lost its type-1 copper but has become domain 2 in nitrite reductase after the gene inversion. 

On the basis of the electronic, resonance Raman, and EPR spectra, the cysteine-containing copper proteins have been divided into four groups axial type 1 (e.g. plastocyanin), rhombic type 1 (e.g. nitrite reductase and stellacyanin), type 1.5, and type 2 (mutant) copper proteins [81]. We have studied the spectra of members of each group with the CASPT2 method [33,34,36,38]. 

Copper enzymes participate in two important reactions catalyzed by denitrifying bacteria. Nitrite reductases from species of Achromohactei and Alcaligene are trimeric proteins made up of 37-kDa subunits, each of which contains one type 1 (blue) copper and one type 2 (nonblue) copper. The first copper serves as an electron acceptor from a small blue pseudoazurin. LS44a second copper,... 

A number of other Cu electron transfer proteins which contain type-1 Cu centres (azurin, cemloplasmin, laccase, nitrite reductase, msticyanin, and stellacyanin) are known. They aU have three coordination positions contributed by 2 His and one Cys, similar to the copper coordination chemistry in plastocyanin — yet they span... 

Type 1 copper centers are found in small blue proteins, in blue oxidases and in nitrite reductase. In the latter, the distorted ligand geometry causes a shift in the absorption band resulting in a green instead of a blue enzyme [26,27]. 

The proteins treated in this section will only contain type 2 copper centers. Additional type 2 copper centers may be found in blue oxidases, in which they form trinuclear centers with type 3 copper ions, and in nitrite reductase. In nitrite reductase, the type 2 copper center is coupled to a type 1 copper center via a cysteine residue. 

Structure (of nitrite reductase from Alcaligenes faecalis). The subunits of nitrite reductase contain two domains. Each domain consists of a /1-barrel structure, similar to that in the small blue proteins [272]. The type 1 copper center is embedded in one of these /1-barrels and is coordinated by the ligands His 95 (domain 1), Cys 136, His 145, and Met 150 (domain 2) [26]. The type 2 copper center is coordinated by a water molecule and three histidine ligands His 100 and His 135 (subunit x) as well as His 306 (subunit y). It is situated at the interface of two subunits [272], The water molecule is displaced from the type 2 copper ion upon nitrite binding (Fig. 34). Although both copper ions are coordinated by neighboring residues, they are located approximately 12.5 A apart, which prevents a direct electron transfer [272]. 

Most proteins with type 1 copper centers are blue, although the nitrite reductases from Achromobacter cycloclastes, Alcaligenes faecalis, and Pseudomonas aureofaciens, are green [26,27]. This is probably caused by a distortion of the type 1 copper center, although the interrelation of distortion and absorption properties of the copper centers have not yet been clarified [27]. 

Nitrite reductase catalyses the reduction of NOj to NO. The enzyme s native state is that of a homotrimer. The monomers have two domains (domain 1 and 2) which show clear sequence homology to type 1 copper proteins [272], Thus, a phylogenetic relationship can be assumed for not only type 1 copper proteins but also for the domains themselves. Domain 1 of nitrite reductase contains type 1 copper, whereas a change in the copper-binding center of domain 2 resulted in its switch from a type 1 to a type 2 copper center. A similar switch from a type 1 to a type 2 copper center may be observed in the development of blue oxidases. 

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