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 CuZ°, 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 CuZ° is a 1-hole (i.e., 3CuICuII) state with spin density delocalized evenly over CuI and CuIV. 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 CuZ° has a terminal hydroxide ligand coordinated to CuIV, stabilized by a hydrogen bond to a nearby lysine residue. CuZ° can be reduced via electron transfer from CuA 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 CuZ° is the catalytically relevant oxidized form of N2OR. Terminal hydroxide coordination to CuIV in the CuZ° 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 μ4S2− bridged tetranuclear copper cluster to N2O via a single Cu atom to accomplish N–O bond cleavage.

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N2O production

“…The intramolecular electron transfer between CuA and CuZ appears to be rate limiting for N 2 O reduction in the range of 4 < pH < 8, when the electron transfer rate to CuZ is hampered due to conformational changes of CuA (Gorelsky et al, 2006). However, at more alkaline pH (pH > 8.9) a loss of catalytic activity was also observed, likely due to the deprotonation of a lysine residue (pI 5 9.7) in CuZ, which in its deprotonated form cannot stabilize the Cu Z 8 intermediate anymore (Johnston et al, 2017). Therefore, the protonation of the lys397 residue appears to be essential for a successful reduction of N 2 O via Cu Z 8.…”

“…Therefore, the protonation of the lys397 residue appears to be essential for a successful reduction of N 2 O via Cu Z 8. The balance between both effects of pH, either the effect of pH on the electron transfer from CuA to CuZ or the protonation state of lys397, may contribute to an observed pH optimum of pH 7-8 (Fujita and Dooley, 2007;Johnston et al, 2017).…”

N2O Reductase Activity of a [Cu4S] Cluster in the 4CuI Redox State Modulated by Hydrogen Bond Donors and Proton Relays in the Secondary Coordination Sphere

“…A large body of enzymological, spectroscopic, and computational work from Solomon, Moura, and co‐workers has led to a recently updated mechanistic proposal for N 2 O reduction at Cu Z . In this proposal (Figure a), the fully reduced 4Cu I state of Cu Z binds N 2 O along the Cu I ‐Cu IV edge of the cluster with assistance from a second‐sphere LysH + residue (Lys397).…”

Influence of Copper Coordination Spheres on Nitrous Oxide Reductase (N2Or) Activity of a Mixed-Valent Copper Complex Containing a {Cu2S} Core

“…The involvement of a Cuz° intermediate with a µ,1-3 coordination mode for N2O at the CuI-(µ,S)-CuIV edge, together with the participation of a hydrogen bond with a neighboring lysine residue to favor the N-O bond cleavage, has been proposed. 9 In our group, we aim at developing bio-inspired Cu2S-containing complexes representative of the truncated active Cu4S native cluster to propose structure/activity correlations for N2O activation/reduction. In the literature, few examples of Cux-Sy (x = 2-4 and y = 1, 2) architectures have been reported for stoichiometric N2O reduction at low temperature (-80°C).…”