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

Abstract

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.