Cryo-EM structures of Escherichia coli cytochrome bo₃ reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 118; no. 34; pp. 1 - 10
• Main Authors: Li, Jiao, Han, Long, Vallese, Francesca, Ding, Ziqiao, Choi, Sylvia K., Hong, Sangjin, Luo, Yanmei, Liu, Bin, Chan, Chun Kit, Tajkhorshid, Emad, Zhu, Jiapeng, Clarke, Oliver, Zhang, Kai, Gennis, Robert
• Format: Journal Article
• Language: English
• Published: Washington National Academy of Sciences 24-08-2021
• Subjects: BASIC BIOLOGICAL SCIENCES; Binding sites; bioenergetics; Biological Sciences; Carbonyl compounds; Carbonyls; Chains; Coenzyme Q8; Computer applications; Copolymers; Cytochrome; Cytochrome c; Cytochromes; E coli; Electron microscopy; electron transport; Grooves; Heme; heme–copper oxidoreductase; Histidine; Hydrogen; Hydrogen bonds; Hydrophobicity; Hydroquinone; Imidazole; Internal water; Isoprene; Maleic acid; Membrane proteins; Oxidation; Phospholipids; Physical Sciences; proton pump; Protons; science & technology; Styrene; Substrates; Tyrosine; Ubiquinol; Ubiquinol oxidase; Ubiquinone; Water chemistry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2106750118

Two independent structures of the proton-pumping, respiratory cytochrome bo₃ ubiquinol oxidase (cyt bo₃) have been determined by cryogenic electron microscopy (cryo-EM) in styrene–maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o₃, and CuB), the redox-active cross-linked histidine–tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I. In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.