Spectroscopic Definition of the Cu_Z° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into...

Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of Cu_Z°, an intermediate form of the Cu₄S active site of nitrous oxide reductase (N₂OR) that is observed in single turnover of fully reduced N₂OR with N₂O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that Cu_Z° is a 1-hole (i.e., 3Cu^ICu^II) state with spin density delocalized evenly over Cu_I and Cu_IV. Resonance Raman spectroscopy shows two Cu-S vibrations at 425 and 413 cm^-1, the latter with a -3 cm^-1 O¹⁸ solvent isotope shift. DFT calculations correlated to these spectral features show that Cu_Z° has a terminal hydroxide ligand coordinated to Cu_IV, stabilized by a hydrogen bond to a nearby lysine residue. Cu_Z° can be reduced via electron transfer from Cu_A using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >10⁴ faster than the unobserved IET in the resting state, showing that Cu_Z° is the catalytically relevant oxidized form of N₂OR. Terminal hydroxide coordination to Cu_IV in the Cu_Z° intermediate yields insight into the nature of N₂O binding and reduction, specifying a molecular mechanism in which N₂O coordinates in a μ-1,3 fashion to the fully reduced state, with hydrogen bonding from Lys397, and two electrons are transferred from the fully reduced μ₄S^2- bridged tetranuclear copper cluster to N₂O via a single Cu atom to accomplish N-O bond cleavage.