Co‐purification of nitrate reductase 1 with components of the cytochrome bcc‐aa3 oxidase supercomplex from spores of Streptomyces coelicolor A3(2). Issue 3 (14th February 2021)

• Record Type: Journal Article
• Authors: Falke, Dörte; Fischer, Marco; Ihling, Christian; Hammerschmidt, Claudia; Sinz, Andrea; Sawers, Gary
• Is Part Of: FEBS open bio. Volume 11:Issue 3(2021)
• Page Start: 652
• Page End: 669
• Publication Date: 2021-02-14
• Subjects: actinobacteria -- nitrate respiration -- O2 respiration -- protein–protein interaction -- respiratory supercomplex -- spores; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1002/2211-5463.13086
• Languages: English
• ISSNs: 2211-5463

Abstract

Abstract : 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. Abstract : 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.