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Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl–. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding. The [4Cu:2S] CuZ site in M. nauticus N 2O reductase. The electron density map shown is contoured at the 5 σ level, highlighting the presence of two sulfide ligands. 705x677mm (72 x 72 DPI) [ABSTRACT FROM AUTHOR]

Nitrous oxide reductase (N[sub2]OR) catalyzes the two-electron reduction of N[sub2]O to N[sub2] and H[sub2]O in the last step of the bacterial denitrification process. It is a dimeric protein. The recently solved crystal structure of N[sub2]OR indicates that in each subunit there is a Cu[subA] center, which is the electron-transfer site, and a Cu[subZ] center, which is the catalytic site. The neighboring Cu[subA] and Cu[subZ] centers are from different subunits. The whole Cu[subZ] cluster is coordinated by seven His ligands with a weakly bound water at the Cu[subI]/Cu[subIV] edge, which is the substrate access site.