A cyanate-bridged binuclear center in bovine cytochrome c oxidase

Cytochrome [Formula: see text] oxidase (C[Formula: see text]O) catalyzes the four-electron reduction of oxygen to water while coupling this reaction to proton translocation across biological membranes. The catalytic chemistry occurs at the binuclear center (BNC), composed of heme [Formula: see text] and Cu[Formula: see text]. Cyanate has long been recognized as a ligand of C[Formula: see text]O, but its binding mode and structural consequences have remained unknown. Here, we report the crystal structure of the C[Formula: see text]O-cyanate complex at 2.1 [Formula: see text] resolution. The structure reveals that cyanate adopts a [Formula: see text]-bridging configuration between heme [Formula: see text] and Cu[Formula: see text], with its nitrogen atom coordinated to the heme iron and its oxygen atom coordinated to Cu[Formula: see text]. Ligand orientation was established using a complementary thiocyanate complex, in which sulfur anomalous scattering identifies the chalcogen terminus on the Cu[Formula: see text] side of the BNC. Geometric analysis indicates that the bound ligand predominantly adopts the [Formula: see text] resonance form. Despite replacing the native heme [Formula: see text]-bound hydroxide and Cu[Formula: see text]-bound water ligands with a single linear bridge, cyanate preserves the active-site architecture of the resting oxidized enzyme, including the Fe-Cu[Formula: see text] separation and the absence of the water molecule associated with deprotonated Y244 observed in several catalytic intermediates. In addition, we observe a previously unidentified water molecule hydrogen-bonded to the oxygen terminus of cyanate. Together, these findings establish cyanate as a close structural mimic of the resting oxidized active site and provide a framework for future studies of ligand binding, electronic structure, and proton-coupled processes in the C[Formula: see text]O binuclear center.