Nitrite reductase domain

Fig. 14. (a) Ribbon drawing of nitrite reductase donaain 1. (b and c) Schematic of nitrite reductase domain 1 topology. 

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. 

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,... 

Fe atoms. It had been anticipated that the c-type cytochrome center would have His/Met coordination, but His/His is observed. The former is the more usual coordination, especially at the high potential end E° > +200 mV) ofthe typical bacterial electron transfer chain to which the nitrite reductase is connected (Fig. 2) (7). The second curious feature is that the di heme iron is also six-coordinate thus, the enzyme does not offer a substrate-binding site at either heme. In addition to an expected axial histidine ligand there was an axial tyrosine (residue 25) ligand to the d heme (Fig. 4a). Each monomer is organized into two domains. 

The salient features of A. faecalis pseudoazurin are that (1) it has a Cu-Met bond length shorter than that of either plastocyanin or azurin (see Table III) (2) it has only one NH - S bond, as does plastocyanin and (3) its overall architecture resembles plastocyanin (see Fig. 4), with an extended carboxy terminus folded into two a helices [a preliminary sequence comparison suggested that the folding would resemble plastocyanin (Adman, 1985)]. It retains the exposed hydrophobic face found in azurin and plastocyanin. Just how it interacts with nitrite reductase is still a subject of investigation. It is intriguing that the carboxy-terminal portion folds up onto the face of the molecule where the unique portions of other blue proteins are the flap in azurin, and, as we see below in the multi-copper oxidase, entire domains. 

With the structure of ascorbate oxidase in hand, a new structurally based alignment of the sequences of ascorbate oxidase, laccase, and ceruloplasmin has been performed (Messerschmidt and Huber, 1990). In brief, while gene triplication for ceruloplasmin is still revelant, its sequence can be further subdivided into two domains per unit of triplicated sequence, or six domains in total. Each of these sequences bears some resemblance to each of the three domains of ascorbate oxidase, as does each of the two domains in laccase. The coppers of the trinuclear site of ceruloplasmin then are predicted to be bound between domains 1 and 6, with a type I site also lying in both domains 6 and 4 (see Huber, 1990). The relative orientation of each of these domains is not predicted by this alignment, but it turns out that the structure of nitrite reductase may shed some light on this (see Section V,C). 

Arrangement of subunits in nitrite reductase from Achromohacter cycloclastes. Domains are denoted D1 and D2, copper sites are shaded spheres, and Cu ligands are denoted by one-letter abbreviations C for cysteine sulfur and H for histidine imidazole nitrogen. From Fenderson et al. (1991). 

Some metalloflavoproteins contain heme groups. The previously mentioned flavocytochrome b2 of yeast is a 230-kDa tetramer, one domain of which carries riboflavin phosphate and another heme. A flavocytochrome from the photosynthetic sulfur bacterium Chromatium (cytochrome c-552)279 is a complex of a 21-kDa cytochrome c and a 46-kDa flavoprotein containing 8a-(S-cysteinyl)-FAD. The 670-kDa sulfite reductase of E. coli has an a8P4 subunit structure. The eight a chains bind four molecules of FAD and four of riboflavin phosphate, while the P chains bind three or four molecules of siroheme (Fig. 16-6) and also contain Fe4S4 clusters.280 281 Many nitrate and some nitrite reductases are flavoproteins which also contain Mo or... 

Two types of dissimilatory nitrite reductases catalyze step b of Eq. 18-30. Some bacteria use a copper-containing enzyme, which contains a type 1 (blue) copper bound to a (3 barrel domain of one subunit and a type 2 copper at the catalytic center. The type 1 copper is thought to receive electrons from the small copper-containing carrier pseudoazurin (Chapter... 

More prevalent is cytochrome cd2 nitrite reductase.340 343 346 The water-soluble periplasmic enzyme is a homodimer of 60-kDa subunits, each containing a c-type heme in a small N-terminal domain and heme dv a ferric dioxoisobacteriochlorin (Fig. 16-6). The... 

Campbell, W.H. Kinghom, J.R. (1990). Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends in Biochemistry 15, 315-19. 

The second class consists of multidomain blue copper proteins composed of exclusively two or more BCB domains and includes nitrite reductase (Section IV, E), multicopper blue oxidases such as laccase, ascorbate oxidase, ceruloplasmin, and hephaestin (Section VII), and some sequences found in extreme halophilic archaea (see Section V, E). 

The constrained nature of the copper center in BCB domains reduces its reorganization energy, which is considered an important feature for their function in long-range electron transfer processes. They are capable of tunneling electrons, usually over 10- to 12-A distances, intramolecu-larly within the same protein (in the case of multicopper oxidases and nitrite reductases) or intermolecularly between a donor and an acceptor protein (in the case of cupredoxins) in a thermodynamically favorable environment. 

Cutruzzola, F., Arese, M., Grasso, S., Bellelli, A., and Brunori, M., 1997, Tyrosine 10 in the c-haem domain is not involved in the catalytic mechanism of nitrite reductase from Pseudomonas aeruginosa, FEBS Lett. 412 3659369. 

Fig. 8. (a) Drawing of the trimer of nitrite reductase from Achromobacter cycloclastes. (b) Drawing of the interface between domain 1 (subunit A) and domain 2 of the adjacent symmetry-related molecule (subunit C) of nitrite reductase from A. cycloclastes. (c) Drawing of domain 1 and 3 of ascorbate oxidase. The type-1 copper is in domain 3 and the trinuclear copper center is between domain 1 and domain 3. The domains have an orientation similar to that of the corresponding domains of the nitrite reductase shown in b. The figure was produced by the RIBBON Program (S7). 

In the trimer of nitrite reductase a six-domain structure is realized, which is reminiscent of the six-domain structure of ceruloplasmin (112), which was deduced from the amino-acid sequence alignment with the other blue oxidases (101). However, the arrangement of the six gene segments in ceruloplasmin is not simply a triplication of an ancestral... 

Cytochrome cd nitrite reductase from Paracoceus pantotrophus has a different mechanism, with two identical subunits, each with domains containing a c-type cytochrome heme and a dj-type cytochrome heme. Electrons from external donors enter through the c heme the d heme is the site of nitrite reduction to NO and oxygen reduction to water. One of the puzzles of the mechanism is how the NO can escape from... 

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