Revisiting the metal sites of nitrous oxide reductase in a low-dose structure from Marinobacter nauticus.

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas NO to yield N. It contains two metal centers, the binuclear electron transfer site Cu, and the unique, tetranuclear Cu 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 NOR 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 Cu site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the Cu 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 Ca, 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] Cu site, a mechanistic model is presented, with a structurally flexible Cu center that does not require the complete dissociation of a sulfide prior to NO binding.