A [3Cu:2S] cluster provides insight into the assembly and function of the Cu(Z) site of nitrous oxide reductase

Nitrous oxide reductase (N(2)OR) is the only known enzyme reducing environmentally critical nitrous oxide (N(2)O) to dinitrogen (N(2)) as the final step of bacterial denitrification. The assembly process of its unique catalytic [4Cu:2S] cluster Cu(Z) remains scarcely understood. Here we report on a mutagenesis study of all seven histidine ligands coordinating this copper center, followed by spectroscopic and structural characterization and based on an established, functional expression system for Pseudomonas stutzeri N(2)OR in Escherichia coli. While no copper ion was found in the Cu(Z) binding site of variants H129A, H130A, H178A, H326A, H433A and H494A, the H382A variant carried a catalytically inactive [3Cu:2S] center, in which one sulfur ligand, S(Z2), had relocated to form a weak hydrogen bond to the sidechain of the nearby lysine residue K454. This link provides sufficient stability to avoid the loss of the sulfide anion. The UV-vis spectra of this cluster are strikingly similar to those of the active enzyme, implying that the flexibility of S(Z2) may have been observed before, but not recognized. The sulfide shift changes the metal coordination in Cu(Z) and is thus of high mechanistic interest.