Characterisation of [Cu₄S], the catalytic site in nitrous oxide reductase, by EPR spectroscopy

The enzyme nitrous oxide reductase (N₂OR) has a unique tetranuclear copper centre [Cu₄S], called Cu_Z, at the catalytic site for the two-electron reduction of N₂O to N₂. The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N₂OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu_Z, that undergoes a one-electron redox cycle and Cu_Z*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu_Z showing a rich hyperfine splitting in the g _ˈ-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu_Z and to calculate EPR parameters. The results for the oxidation state [Cu⁺¹₃Cu⁺²₁S]³⁺ are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu_Z. The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu_I 20.1%, Cu_II 9.5%, Cu_III 4.8% and Cu_IV 9.2%, the µ₄-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu_Z, allowing electron redistribution within the plane during interaction with the substrate, N₂O.