Mechanism of N₂O Reduction by the μ₄-S Tetranuclear Cu_Z Cluster of Nitrous Oxide Reductase

Reaction thermodynamics and potential energy surfaces are calculated using density functional theory to investigate the mechanism of the reductive cleavage of the N−O bond by the μ₄-sulfide-bridged tetranuclear Cu_Z site of nitrous oxide reductase. The Cu_Z cluster provides an exogenous ligand-binding site, and, in its fully reduced 4Cu^I state, the cluster turns off binding of stronger donor ligands while enabling the formation of the Cu_Z−N₂O complex through enhanced Cu_Z → N₂O back-donation. The two copper atoms (Cu_I and Cu_IV) at the ligand-binding site of the cluster play a crucial role in the enzymatic function, as these atoms are directly involved in bridged N₂O binding, bending the ligand to a configuration that resembles the transition state (TS) and contributing the two electrons for N₂O reduction. The other atoms of the Cu_Z cluster are required for extensive back-bonding with minimal σ ligand-to-metal donation for the N₂O activation. The low reaction barrier (18 kcal mol^-1) of the direct cleavage of the N−O bond in the Cu_Z−N₂O complex is due to the stabilization of the TS by a strong Cu_IV²⁺−O^- bond. Due to the charge transfer from the Cu_Z cluster to the N₂O ligand, noncovalent interactions with the protein environment stabilize the polar TS and reduce the activation energy to an extent dependent on the strength of proton donor. After the N−O bond cleavage, the catalytic cycle consists of a sequence of alternating protonation/one-electron reduction steps which return the Cu_Z cluster to the fully reduced (4Cu^I) state for future turnover.