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 (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.