3.D.4.5.1
Quinol oxidase (CyoABCD). This cytochrome bo₃ ubiquinol oxidase is a transmembrane protein of 663 aas and 14 or 15 TMSs, which oxidizes ubiquinone and reduces oxygen, while pumping protons (Marušič et al. 2020). The cytochrome bo₃ complex is the component of the aerobic respiratory chain of E. coli that predominates when cells are grown under high aeration conditions. It has proton pumping activity, pumping 2 protons/electron. Protons are probably pumped via D- and K-channels found in the cyoB subunit (Abramson et al. 2000). Two independent structures of the proton-pumping, respiratory cytochrome bo 3 ubiquinol oxidase (cytbo₃) have been determined by 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 (Li et al. 2021). 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 TMS0, 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 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 TMS0. The authors proposed that H98(I) dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TMS0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98(I) (Li et al. 2021). Jose et al. 2021 investigated the key P_M intermediate, which forms after O–O bond cleavage and precedes proton pumping, using magnetic circular dichroism spectroscopy. The authors observed features demonstrating that P_M is a three-spin system,  consistent with a consensus model including an iron(IV)-oxo species, a copper(II) ion, and a tyrosyl radical. These results provide validation of the O–O cleavage mechanism and open the door to understanding the proton pumping step (Jose et al. 2021).