The copper enzyme nitrous oxide reductase catalyzes the two-electron reduction of nitrous oxide N₂O to dinitrogen N₂. Its maturation largely occurs in the periplasm and includes the insertion of at least one Ca²⁺ ion per monomer. Here we have investigated the role of this structural cation in recombinantly produced apo-N₂OR from Shewanella denitrificans and have determined the three-dimensional structure of the protein by X-ray crystallography. In the absence of Ca²⁺, substantial parts of the enzyme surrounding the binding sites for the copper ions show structural disorder. Reconstitution of the binuclear CuA site was possible in vitro but required the presence of Ca²⁺ ions for a stable insertion of the center. In contrast, an excess of Ca²⁺ prevented copper insertion, and the structural analysis of the Ca²⁺apo form revealed that the cation is sufficient to structure the disordered regions of the protein even in the absence of Cu ions, indicating that the geometry of the two noncanonical copper centers is largely predetermined by the protein structure.