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

腐殖土壤细菌天蓝色链霉菌经历了一个复杂的发育周期，包括作为底物菌丝体的生长和代谢活性降低的孢子的产生。菌丝体的生长需要氧气（O2），需氧呼吸链包括铜-aa 3型细胞色素c氧化酶。由于放线菌只有一个与细胞膜结合的二血红素细胞色素c，从甲基萘醌：细胞色素bcc氧化还原酶（bcc复合物）到aa 3氧化酶的电子转移需要蛋白质-蛋白质相互作用和形成细胞色素bcc-aa 3氧化酶超复合物。孢子也利用这种超复合体来呼吸氧气。菌丝体和孢子均为S.当氧气变得有限时，腔棘鱼也可以用硝酸盐呼吸。虽然硝酸盐呼吸不支持菌丝体的生长，但它有助于维持菌丝体和孢子中的质子动力，从而有助于持久性。在S.在腔棘鱼中，Nar 1酶仅存在于孢子中并具有活性; Nar 2在指数生长的菌丝体中具有活性，Nar 3在静止期菌丝体中具有活性。我们的研究结果表明，Nar 1的活性，但不是合成，是绝对依赖于bcc-aa 3超复合物。从孢子中纯化Strep标记的Nar 1揭示了Rieske铁硫亚基（QcrA）和bcc-aa 3超复合物的电子转移亚基（CtaC）的共纯化，表明这些酶复合物之间的直接相互作用。这导致的假设，Nar 1，与典型的Nar型还原酶，通过bcc-aa 3超复合物接收电子。此外，这表明硝酸盐还原可能与超复合物的Q循环相耦合，使孢子能够比直接将硝酸盐还原与甲萘醌氧化相耦合节省更多的能量。因此，本提案的目的是确定：1）Nar 1如何使用生物化学和化学交联/质谱方法与孢子中的bcc-aa 3超复合物相互作用;以及2）硝酸盐还原是否确实与Q循环相结合。通过从菌丝体中纯化bcc-aa 3超复合物，我们有证据表明它也与Nar 2相互作用。nar 2的活性也部分依赖于bcc-aa 3超复合物。bcc-aa 3超复合物的组成在菌丝体和孢子中也似乎不同。通过使用化学交联/MS方法纯化和分析这种复合物，我们的目标是确定这些超分子复合物在孢子和菌丝体之间的差异。最后，我们将分析Nar 3是否依赖于静止期菌丝体中的bcc-aa 3超复合物。这些研究将为链霉菌呼吸O2-硝酸盐界面的生物能量学提供新的见解，并将揭示当O2不可用时能量受限孢子的生存策略。

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.