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

Nitrous oxide reductase (N₂OR) is the only known enzyme reducing environmentally critical nitrous oxide (N₂O) to dinitrogen (N₂) 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 N₂OR 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.