Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa(3) oxidase supercomplex from spores of Streptomyces coelicolor A3(2).
Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa(3) oxidase supercomplex from spores of Streptomyces coelicolor A3(2).
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DOI：
10.1002/2211-5463.13086
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发表时间：
2021-03
期刊：
影响因子：
2.6
通讯作者：
Sawers G
中科院分区：
文献类型：
--
作者：
Falke D;Fischer M;Ihling C;Hammerschmidt C;Sinz A;Sawers G
为了在体内还原硝酸盐，天蓝色链霉菌孢子特异的呼吸硝酸还原酶NAR1依赖于活性细胞色素BCC-Aa3氧化酶超复合体(BCC-Aa3超复合体)。这表明，由NarG1、NarH1和NarI1亚基组成的膜相关Nar1可能不是经典的菜单喹醇氧化酶，但可以接受来自BCC-Aa3超复合体的电子，或者需要超复合体稳定膜上的还原酶以使其发挥作用。为了解决这种对BCC-Aa3超复合体的依赖的生化基础，我们纯化了两个不同的Strep标记的Nar1变体，并使用不同的层析和电泳程序从孢子提取液中富集了天然酶复合体。用质谱仪鉴定了与分离的Nar1复合体相关的多肽，并包括BCC-Aa3超复合体的成分，以及另一种孢子特异的细胞色素b成分QcrB3。令人惊讶的是，我们还与天蓝色链球菌野生型菌株的Nar1共富集了Nar3酶。经过透明的天然聚丙烯酰胺凝胶电泳，可以鉴定出两个不同迁移活性的Nar1复合体；它们的质量约为450和250 kDa。细胞色素BD氧化酶和QcrB3的存在影响了活性的Nar1在这些复合体中的分布；后者的存在使Nar1进入了更大的复合体。综上所述，这些数据表明，几个呼吸复合体可以在孢子膜上结合，包括Nar1，Nar3和BCC-Aa3超复合体。此外，这些发现为Nar1和BCC-Aa3超复合体在物理上相关的假设提供了初步支持。孢子特异性呼吸硝酸还原酶(Nar1)在氧气受限时利用硝酸盐作为电子受体。Nar1的活性完全依赖于细胞色素bcc-aa3氧化酶超复合体。一种可能性是，如红色虚线箭头所示，超络合物将电子从菜籽酚(MK)重定向到Nar1。这项研究揭示了Nar1与BCC-Aa3超复合体的亚单位交配，支持这一假说。
In order to reduce nitrate in vivo, the spore‐specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc‐aa3 oxidase supercomplex (bcc‐aa3 supercomplex). This suggests that membrane‐associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc‐aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc‐aa3 supercomplex, we purified two different Strep‐tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc‐aa3 supercomplex, along with an alternative, spore‐specific cytochrome b component, QcrB3. Surprisingly, we also co‐enriched the Nar3 enzyme with Nar1 from the wild‐type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc‐aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc‐aa3 supercomplex physically associate. Spore‐specific respiratory nitrate reductase (Nar1) uses nitrate as an electron acceptor when oxygen is limiting. Nar1 activity is totally reliant on the cytochrome bcc‐aa3 oxidase supercomplex. One possibility is that the supercomplex redirects electrons to Nar1 from menaquinol (MK), as indicated by the red dotted arrow. This study reveals Nar1 copurifies with subunits of the bcc‐aa3 supercomplex, supporting this hypothesis.
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影响因子：
2.8
作者：
Fischer, Marco;Alderson, Jesse;Sawers, R. Gary
通讯作者：
Sawers, R. Gary
影响因子：
4.8
作者：
Bertero, MG;Rothery, RA;Strynadka, NCJ
通讯作者：
Strynadka, NCJ
影响因子：
3.2
作者：
Fischer, Marco;Falke, Doerte;Sawers, R. Gary
通讯作者：
Sawers, R. Gary
影响因子：
3.2
作者：
Falke, Doerte;Biefel, Bianca;Sawers, R. Gary
通讯作者：
Sawers, R. Gary
影响因子：
1.2
作者：
Falke, Doerte;Fischer, Marco;Sawers, R. Gary
通讯作者：
Sawers, R. Gary