Nitrous reductase function

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. 

As the focus of this review is on copper-dioxygen chemistry, we shall briefly summarize major aspects of the active site chemistry of those proteins involved in 02 processing. The active site structure and chemistry of hemocyanin (He, 02 carrier) and tyrosinase (Tyr, monooxygenase) will be emphasized, since the chemical studies described herein are most relevant to their function. The major classes of these proteins and their origins, primary functions, and leading references are provided in Table 1. Other classes of copper proteins not included here are blue electron carriers [13], copper-thiolate proteins (metallothioneines) [17], and NO reductases (e.g., nitrite [NIR] [18] or nitrous oxide [19]). 

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). 

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. 

A number of different enzymes can carry out the reduction of nitrite to either ammonium or nitric oxide and/or nitrous oxide. The latter types are involved with the denitrification process (Payne, 1973) and will not be considered here. Among the enzymes that catalyze the six-electron reduction of nitrite to ammonia, several different types are recognized. These are (I) assimilatory NiRs that function in biosynthetic nitrate assimilation of higher plants, algae, and fungi, (2) ammonia-forming dissimilatory NiRs involved in anaerobic nitrate respiration of diverse bacteria, and (3) assimilatory and dissimilatory sulfite reductases... 

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