Structure of the Membrane-intrinsic Nitric Oxide Reductase from <i>Roseobacter denitrificans</i>

Abstract

Membrane-intrinsic nitric oxide reductases (NORs) are key components of bacterial denitrification pathways with a close evolutionary relationship to the cytochrome oxidase (COX) complex found in aerobic respiratory chains. A key distinction between COX and NOR is the identity of the metal directly opposite heme <i>b</i>₃ within the active site. In NOR, this metal is iron (Fe_B), whereas in COX, it is copper (Cu_B). The purified NOR of <i>Roseobacter denitrificans</i> contains copper and has modest oxidase activity, raising the possibility that a COX-like active site might have independently arisen within the context of a NOR-like protein scaffold. Here we present the crystal structure of the <i>Roseobacter denitrificans</i> NorBC complex and anomalous scattering experiments probing the identity of each metal center. Our results refute the hypothesis that copper occupies the active site and instead reveal a new metal center in the small subunit not seen in any other NOR or COX