Cryo-EM structures of <i>Escherichia coli</i> cytochrome <i>bo</i> _<i>3</i> reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

Abstract

Two independent structures of the proton-pumping, respiratory cytochrome <i>bo</i>_<i>3</i> ubiquinol oxidase (cyt <i>bo</i>_<i>3</i> ) 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 <i>b</i>, heme <i>o</i>_<i>3</i> , and Cu_B), 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 <i>c</i> oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75^I and R71^I In both structures, residue H98^I occupies two conformations. In conformation 1, H98^I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98^I has flipped to form a hydrogen bond with E14^I at the N-terminal end of TM0. We propose that H98^I 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^I.