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

Abstract

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

Activity of Spore-Specific Respiratory Nitrate Reductase 1 of Streptomyces coelicolor A3(2) Requires a Functional Cytochrome bcc-aa 3 Oxidase Supercomplex

“…Only low levels of nitrate-reducing activity could be determined for the enzyme in mycelium. This would be consistent with the Nar1 enzyme being unable to accept electrons directly from menaquinol but would imply at the same time that the bcc-aa 3 complexes are not identical in spores and mycelium, a suggestion made in another recent study (9). While further studies will be required to determine what makes the Nar1 enzyme dependent on the bcc-aa 3 supercomplex in order to be able to reduce nitrate, it will also be important to determine whether the composition of the bcc-aa 3 supercomplex is different in these cellular compartments.…”

“…Manifestation of Nar1 activity requires the complete cytochrome bcc-aa 3 supercomplex. The results of a recent study have revealed that the bcc complex and the cytochrome aa 3 oxidase function as a supercomplex in S. coelicolor in both spores and mycelium during aerobic growth (9). The lack of Nar1 enzyme activity in spores of strain COE502A (Table 2), where only the CtaD subunit of the aa 3 oxidase is missing, suggested that the cytochrome bcc complex alone is insufficient to support Nar1 activity and that the complete bcc-aa 3 supercomplex is required to support Nar1dependent nitrate reduction in spores.…”

“…During oxidation of the reduced electron donor(s), the electrons are passed via one of two routes to O 2 . When the O 2 concentration is low, they oxidize menaquinol via a high-affinity cytochrome bd oxidase, while at higher O 2 concentrations the electrons flow through a large proton-translocating supercomplex comprising a menaquinol-cytochrome c oxidoreductase (bcc complex) and an aa 3 -type cytochrome oxidase, referred to henceforth as the bcc-aa 3 supercomplex (8,9). Such a bcc-aa 3 supercomplex has been identified in all actinobacteria studied so far (10)(11)(12)(13), and the structure of the supercomplex from Mycobacterium smegmatis has been resolved recently using cryo-electron microscopy (14,15).…”

Co‐purification of nitrate reductase 1 with components of the cytochrome bcc‐aa3 oxidase supercomplex from spores of Streptomyces coelicolor A3(2)

“…Recent studies have resolved the structure of the bcc-aa 3 supercomplex from Mycobacterium smegmatis [8,9], revealing it to have a linear supramolecular organization comprising a complex III (bcc complex) dimer flanked at each end by an aa 3 oxidase (complex IV). A similar structure is also proposed for the Corynebacterium glutamicum supercomplex [10], and our own studies have revealed that a bcc-aa 3 supercomplex also exists in S. coelicolor [11]. However, the bcc-aa 3 in S. coelicolor appears to have a different composition in spores compared with mycelium [11].…”

“…A similar structure is also proposed for the Corynebacterium glutamicum supercomplex [10], and our own studies have revealed that a bcc-aa 3 supercomplex also exists in S. coelicolor [11]. However, the bcc-aa 3 in S. coelicolor appears to have a different composition in spores compared with mycelium [11]. It is currently unclear whether this compositional difference in S. coelicolor is perhaps due to altered physical stability, that is, disassembly, of the streptomycete supercomplex.…”