Abstract
The final step of denitrification is the reduction of nitrous oxide (N(2) O) to N(2) , mediated by Cu-dependent nitrous oxide reductase (N(2) OR). Its metal centers, Cu(A) and Cu(Z) , are assembled through sequential provision of twelve Cu(I) ions by a metallochaperone that forms part of a nos gene cluster encoding the enzyme and its accessory factors. The chaperone is the nosL gene product, an 18 kDa lipoprotein predicted to reside in the outer membrane of Gram-negative bacteria. In order to better understand the assembly of N(2) OR, we have produced NosL from Shewanella denitrificans and determined the structure of the metal-loaded chaperone by X-ray crystallography. The protein assembled a heterodinuclear metal site consisting of Zn(II) and Cu(I) , as evidenced by anomalous X-ray scattering. While only Cu(I) is delivered to the enzyme, the stabilizing presence of Zn(II) is essential for the functionality and structural integrity of the chaperone.