The Copper Chaperone NosL Forms a Heterometal Site for Cu Delivery to Nitrous Oxide Reductase.

The final step of denitrification is the reduction of nitrous oxide (N2 O) to N2 , mediated by Cu-dependent nitrous oxide reductase (N2 OR). Its metal centers, CuA and CuZ , are assembled through sequential provision of twelve CuI 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 N2 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 ZnII and CuI , as evidenced by anomalous X-ray scattering. While only CuI is delivered to the enzyme, the stabilizing presence of ZnII is essential for the functionality and structural integrity of the chaperone.