Electron-transfer within the supercomplexes of cytochrome bcc-aa3 oxidase and respiratory nitrate reductase in spores of Streptomyces coelicolor.

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The saprophytic soil bacterium Streptomyces coelicolor undergoes a complex developmental cycle, including growth as substrate mycelium and production of spores with reduced metabolic activity. Growth of the mycelium requires oxygen (O2) and the aerobic respiratory chain includes a cytochrome c oxidase of the copper-aa3-type. Due to the fact that actinobacteria have only a membrane-associated diheme cytochrome c, electron transfer from the menaquinol:cytochrome bcc oxidoreductase (bcc complex) to the aa3 oxidase necessitates protein-protein interaction and formation of a cytochrome bcc-aa3 oxidase supercomplex. Spores also use this supercomplex to respire with O2. Both mycelium and spores of S. coelicolor can also respire with nitrate when O2 becomes limiting. Although nitrate respiration does not support growth of mycelium, it helps maintain a proton motive force in mycelium and spores, thus aiding persistence. Of the three respiratory nitrate reductases (Nar) present in S. coelicolor, the Nar1 enzyme is exclusively present and active in spores; Nar2 is active in exponentially growing mycelium and Nar3 is active in stationary-phase mycelium. Our results have shown that activity, but not synthesis, of Nar1 is absolutely dependent on the bcc-aa3 supercomplex. Purification of Strep-tagged Nar1 from spores has revealed co-purification of the Rieske iron-sulphur subunit (QcrA) and the electron-transfer subunit (CtaC) of the bcc-aa3 supercomplex, suggesting a direct interaction between these enzyme complexes. This leads to the hypothesis that Nar1, in contrast to typical Nar-type reductases, receives electrons via the bcc-aa3 supercomplex. Moreover, this suggests that nitrate reduction might be coupled to the Q-cycle of the supercomplex, enabling spores to conserve more energy than by directly coupling nitrate reduction to oxidation of menaquinol. Therefore, the aim of this proposal is to determine: 1) how Nar1 interacts with the bcc-aa3 supercomplex in spores using both biochemical and chemical cross-linking/mass spectrometry approaches; and 2) whether nitrate reduction is indeed coupled with the Q-cycle. By purifying the bcc-aa3 supercomplex from mycelium we have evidence that it also interacts with Nar2. Nar2 activity is also partially dependent on the bcc-aa3 supercomplex. The composition of the bcc-aa3 supercomplex also appears to differ in mycelium and spores. By purifying and analyzing this complex using chemical cross-linking/MS approaches we aim to determine how these supramolecular complexes differ between spores and mycelium. Finally, we will analyze whether Nar3 is dependent on the bcc-aa3 supercomplex in stationary-phase mycelium. These studies will provide new insights into the bioenergetics of the respiratory O2-nitrate interface in streptomycetes and will uncover the survival strategy of energy-limited spores when O2 becomes unavailable.