Supplementary Material for: Cytochrome <b><i>bcc-aa3</i></b> Oxidase Supercomplexes in the Aerobic Respiratory Chain of <b><i>Streptomyces coelicolor</i></b> A3(2)

<i>Streptomyces coelicolor</i> A3(2), an obligately aerobic, oxidase-positive, and filamentous soil bacterium, lacks a soluble cytochrome <i>c</i> in its respiratory chain, having instead a membrane-associated diheme <i>c</i>-type cytochrome, QcrC. This necessitates complex formation to allow electron transfer between the cytochrome <i>bcc</i> and <i>aa3</i> oxidase respiratory complexes. Combining genetic complementation studies with in-gel cytochrome oxidase activity staining, we demonstrate that the complete <i>qcrCAB-ctaCDFE</i> gene locus on the chromosome, encoding, respectively, the <i>bcc</i> and <i>aa3</i> complexes, is required to manifest a cytochrome oxidase enzyme activity in both spores and mycelium of a <i>qcr-cta</i> deletion mutant. Blue-native-PAGE identified a cytochrome <i>aa3</i> oxidase complex of approximately 270 kDa, which catalyzed oxygen-dependent diaminobenzidine oxidation without the requirement for exogenously supplied cytochrome <i>c</i>, indicating association with QcrC. Furthermore, higher molecular mass complexes were identified upon addition of soluble cytochrome <i>c</i>, suggesting the supercomplex is unstable and readily dissociates into subcomplexes lacking QcrC. Immunological and mass spectrometric analyses of active, high-molecular mass oxidase-containing complexes separated by clear-native PAGE identified key<i></i> subunits of both the <i>bcc</i> complex and the <i>aa3</i> oxidase, supporting supercomplex formation. Our data also indicate that the cytochrome <i>b</i> QcrB of the <i>bcc</i> complex is less abundant in spores compared with mycelium.