Extended Data Fig. 8: Variant studies on the interactions of NosD.

a, Residue C48(NosL) bridges the Zn²⁺- and Cu⁺-binding sites of the chaperone. Its removal renders the chaperone inactive, as evidenced by the failure to mature either Cu site of N₂OR at low external Cu. b, Residue P393(NosD) is in the NosD–NosY interface, at the N-terminus of helix hIII. Its replacement for a bulky Trp largely, but not fully impairs the assembly of Cu_Z. Note that the maturation assay was carried out at high Cu concentrations to have direct Cu_A assembly by Cu²⁺ as a positive control. c, Residue V407(NosD) marks the C-terminal end of helix hIII. Disturbing this interaction renders NosDFY fully non-functional. d, The lid loop of NosD with residue M279(NosD) is not highly conserved among NosD proteins. Accordingly, its deletion impairs, but not prevents Cu_Z maturation at low copper concentration (middle). At high Cu concentrations, N₂OR maturation is intact, pointing towards a role of the lid loop in stabilizing Cu bound to NosD.