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.

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Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the β-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment.