Assembly ofCuZandCuAin Nitrous Oxide Reductase

Abstract

AbstractNitrous oxide (N2O) reductase is a copper enzyme that catalyzes the reduction of N2O 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 cytochromecoxidase, 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 N2O, 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 thenosgenes 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‐N2OR 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, pCuAC, 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 bynosDFYgenes. The other accessory proteins NosR and NosX have been assigned broader functions. NosR has been implicated innosZexpression, as well as in maintaining the activity of N2OR, a function that has been shared by NosX.In this chapter the main biochemical properties of N2OR, as well as the biogenesis of its two copper centers, will be reviewed.