Cryo-EM structures of Escherichia coli cytochrome bo 3 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
• 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: United States 24-08-2021
• Subjects: Binding Sites; Cryoelectron Microscopy; Cytochrome b Group - chemistry; Cytochrome b Group - metabolism; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Heme - chemistry; Heme - metabolism; Oxidation-Reduction; Phospholipids - metabolism; Protein Conformation; Proton Pumps; Ubiquinone - metabolism; ubiquinone; electron transport; bioenergetics; proton pump; heme–copper oxidoreductase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2106750118

Two independent structures of the proton-pumping, respiratory cytochrome
ubiquinol oxidase (cyt
) 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
, heme
, and Cu
), 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
oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75
and R71
In both structures, residue H98
occupies two conformations. In conformation 1, H98
forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98
has flipped to form a hydrogen bond with E14
at the N-terminal end of TM0. We propose that H98
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 H98
.