Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies

AbstractAerobic phototrophic bacterium Roseobacter denitrificans has a nitric oxide reductase (NOR) homologue with cytochrome c oxidase (CcO) activity. It is composed of two subunits that are homologous with NorC and NorB, and contains heme c, heme b, and copper in a 1:2:1 stoichiometry. This enzyme has virtually no NOR activity. Electron paramagnetic resonance (EPR) spectra of the air-oxidized enzyme showed signals of two low-spin hemes at 15 K. The high-spin heme species having relatively low signal intensity indicated that major part of heme b3 is EPR-silent due to an antiferromagnetic coupling to an adjacent CuB forming a Fe–Cu binuclear center. Resonance Raman (RR) spectrum of the oxidized enzyme suggested that heme b3 is six-coordinate high-spin species and the other hemes are six-coordinate low-spin species. The RR spectrum of the reduced enzyme showed that all the ferrous hemes are six-coordinate low-spin species. ν(Fe–CO) and ν(C–O) stretching modes were observed at 523 and 1969 cm−1, respectively, for CO-bound enzyme. In spite of the similarity to NOR in the primary structure, the frequency of ν(Fe–CO) mode is close to those of aa3- and bo3-type oxidases rather than that of NOR