Assembly ofCuZandCuAin Nitrous Oxide Reductase

Abstract

Nitrous oxide (N 2 O) reductase is a copper enzyme that catalyzes the reduction of N 2 O to dinitrogen, the last step of the denitrification pathway. This enzyme has two copper centers: a CuA center that is the electron transfer center and the “CuZ center” where the catalysis occurs. This enzyme has been the center of several studies over the last 20 years, and many of its spectroscopic and catalytic properties have been determined, as well as its structure in different oxidation states. These studies have also revealed that the CuA center is similar to the one present in cytochrome c oxidase, being a binuclear copper center, while the CuZ center is unique in biology, being a tetranuclear copper center bridged by a sulfur atom. Moreover, these studies have also identified that the CuZ center can exist in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S). The first has a high turnover number in the fully reduced state, while the CuZ(4Cu2S) form of CuZ center has a very small turnover number and cannot explain the high capacity of the whole cells in reducing N 2 O, from which the enzyme is isolated with CuZ center mainly in that form. This fact envisages an activation mechanism still to be unraveled that might involve one or more enzymes/proteins encoded by the nos genes and a putative sulfur‐displacement mechanism. The biogenesis of these two centers has not been extensively studied, and at least with respect to the copper insertion, it has been postulated that there are alternative routes. The apo‐N 2 OR is synthesized in the cytoplasm in the apo form and is transported to the periplasmic space by either the Sec (unfolded state) or Tat system (dimer folded state), where the centers are assembled. Thus, CuA assembly has been proposed to be dependent on a copper chaperone, pCu A C, and Sco, a thiol‐disulfide reductase that reduces the disulfide bridge between the two cysteines in the apo‐CuA center. Relative to the CuZ center, the delivery of copper atoms is by NosL, an outer‐membrane protein, while the sulfur atoms are transported by the ABC transporter encoded by nosDFY genes. The other accessory proteins NosR and NosX have been assigned broader functions. NosR has been implicated in nosZ expression, as well as in maintaining the activity of N 2 OR, a function that has been shared by NosX. In this chapter the main biochemical properties of N 2 OR, as well as the biogenesis of its two copper centers, will be reviewed.

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase

“…Another major difference between TcDH and N 2 OR is that TcDH activity, that is its active site, can be recovered just by adding copper ions, while it is well known that N 2 OR activity cannot be reconstituted in vitro, and incorporation of CuZ cluster requires participation of a set of genes organized in a special nos operon (35,36). Tv.…”

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis

“…On the other hand, it is known that NosZ possesses copper centers in its structure, and these centers need a maturation process to be active ( 96 ). One of the steps of this maturation process is the reduction of disulfide bonds present in the chaperone of the CuA center of NosZ and this reduction is carried out by an SCO protein ( 97 , 98 ). There is no evidence that this SCO protein is the protein in charge of this maturation of NosZ in H. mediterranei , but its gene is the only one encoding an SCO protein with high expression under denitrifying conditions.…”

Microbial Small RNAs – The Missing Link in the Nitrogen Cycle?

“…Copper has long been recognized as an important factor in the regulation of NosZ activity (Figure 2) (Sullivan et al, 2013). Around 20% of Europe's arable lands are biologically copper deficient and as NosZ requires the unique multi-copper-sulphide centres, Cu Z and Cu A to bind and activate N 2 O, it places a high Cu demand on the bacterium (Sinclair and Edwards, 2008;Pauleta and Moura, 2017). Other enzymes in bacterial enzymes require Cu for activity, such as haem Cu-oxidases or superoxide dismutases but for all of these enzymes there are non-Cu alternatives that can perform the same function in the absence of Cu (Zumft, 2005).…”