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) Falke D. Fischer M. Biefel B. Ihling C. Hammerschmidt C. Reinefeld K. Haase A. Sinz A. Sawers R.G. 10.6084/m9.figshare.7830800.v1 https://karger.figshare.com/articles/dataset/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_/7830800 <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. 2019-03-12 10:47:28 Actinobacteria Oxygen respiration Cytochrome oxidase Menaquinol:cytochrome bcc oxidoreductase Respiratory supercomplex