Structure Of The Alternative Complex Iii From Flavobacterium Johnsoniae In A Supercomplex With Cytochrome C Oxidase

Alternative Complex III (ACIII), like Complex III, catalyzes the oxidation of membrane‐bound quinol and reduction of cytochrome c , but is structurally unrelated to Complex III. We solubilized the ACIII directly from membranes of Flavobacterium johnsoniae using styrene maleic acid (SMA) copolymer in the absence of traditional detergents. The ACIII was isolated as a functional 1:1 supercomplex with an aa 3 ‐type cytochrome c oxidase (cyt aa 3 ) within SMA copolymer nanodiscs. We determined the structure of the ACIII component of the supercomplex to 3.4 Å resolution by cryo‐EM and constructed an atomic model for its six subunits, two of which are anchored to the lipid bilayer with N‐terminal triacylated cysteine residues, resolved here for the first time. The structure also contains a [3Fe‐4S] cluster, a [4Fe‐4S] cluster, and six hemes c along with 11 phospholipid molecules. The ACIII is in direct contact with subunit III of the cyt aa 3 component of the supercomplex. The structure revealed that this subunit is structurally modified from the canonical form of subunit III to facilitate association with ACIII, suggesting a specific role of the supercomplex in the respiratory system of this bacterium. Support or Funding Information This work was supported by funds from the National Institutes of Health (R01‐HL16101 to R.B.G., P41‐GM104601, U54‐GM087519, and R01‐GM123455 to E.T.) and Canadian Institutes of Health Research (MOP‐81294 to J.L.R.); J.L.R. was supported by the Canada Research Chairs program. Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy, supported by grants from the Simons Foundation (349247), and the National Institute of General Medical Sciences (GM103310, OD019994). Molecular dynamics simulations were performed at Blue Waters (ACI‐1713784 to E.T.) and XSEDE (MCA06N060 to E.T.). Blue Waters is supported by the National Science Foundation (OCI‐0725070 and ACI‐1238993) and the state of Illinois. XSEDE is supported by the National Science Foundation (ACI‐1548562). We thank Mark Mcbride for kindly providing us with the Flavobacterium johnsoniae UW101 strain. We thank Bridget Carragher and Clint Potter for facilitating access to the Titan Krios and Zhening Zhang for collecting data. We also thank Mohammad Mazhab‐Jafari for advice on building atomic models.