Nitrous oxide reductase is the terminal component of a respiratory chain utilizing N2O in lieu of oxygen. It is a homodimer carrying in each subunit the electron transfer site, CuA, and the substrate-reducing catalytic center, CuZ. Spectroscopic data have provided robust evidence for CuA as a binuclear, mixed-valence metal site. To provide further structural information on the CuA center of N2O reductase, site-directed mutagenesis and Cu K-edge X-ray absorption spectroscopic investigation have been undertaken. Candidate amino acids as ligands for the CuA center of the enzyme from Pseudomonas stutzeri ATCC14405 were substituted by evolutionary conserved residues or amino acids similar to the wild-type residues. The mutations identified the amino acids H583, C618, C622 and M629 as ligands of Cu1, and C618, C622 and H626 as the minimal set of ligands for Cu2 of the CuA center. Other amino acid substitutions indicated H494 as a likely ligand of CuZ, and an indirect role for D580, compatible with a docking function for the electron donor. Cu binding and spectroscopic properties of recombinant N2O reductase proteins point at intersubunit or interdomain interaction of CuA and CuZ. Cu K-edge X-ray absorption spectra have been recorded to investigate the local environment of the Cu centers in N2O reductase. Cu K-edge Extended X-ray Absorption Fine Structure (EXAFS) for binuclear Cu chemical systems show clear evidence for Cu backscattering at ca. 2.5 Å. The Cu K-edge EXAFS of the CuA center of N2O reductase is very similar to that of the CuA center of cytochrome c oxidase and the optimum simulation of the experimental data involves backscattering from a histidine group with Cu-N of 1.92 Å, two sulfur atoms at 2.24 Å and a Cu atom at 2.43 Å, and allows for the presence of a further light atom (oxygen or nitrogen) at 2.05 Å. The interpretation of the CuA EXAFS is in line with ligands assigned by site-directed mutagenesis. By a difference spectrum approach, using the Cu K-edge EXAFS of the holoenzyme and that of the CuA-only form, histidine was identified as a major contributor to the backscattering. A structural model for the CuA center of N2O reductase has been generated on the basis of the atomic coordinates for the homologous domain of cytochrome c oxidase and incorporating our current results and previous spectroscopic data.